Note: Descriptions are shown in the official language in which they were submitted.
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.1
NOVEL c-mpl RECEP~OR AGONISTS
The present application claims priority under 35 USC
119(e) of United States provisional application Serial No.
60/004,384 filed October 05, 1995.
Field of the Invention
The present invention relates to human c-mpl receptor
agonists (thrombopoietin) with activity on hematopoietic
differentiation and expansion.
B~ck~round of the Invention
Megakaryocyte (MK) maturation and platelet production
has been long thought to be regulated by lineage specific
humoral growth factors in a manner similar to cytokines that
induce erythrocyte (erythropoietin) and granulocyte (G-CSF)
expansion and maturation. Platelets are responsible for the
prevention of bleeding in response to vascular injury.
Therefore, platelet production is a vital component of
hematopoietic regulation. Patients undergoing chemotherapy
or bone marrow transplantation usually experience severely
depressed platelet levels (thrombocytopenia) which may result
in life threatening bleeding episodes. Several known growth
factors and cytokines have been found to stimulate
megakaryocytes and platelet production but most are
pleiotropic both in vitro and in vivo (IL-3, IL-6, IL-ll,
SCF). Plasma, serum and urine from thrombocytopenic dogs and
hllm~n~ have been found to contain growth factors that have
specific megakarypoietic and thrombopoietic activities
distinct from all known cytokines. These factors have been
termed Meg-CSF, MK-CSF, megakaryocyte growth and development
factor (MGDF), megakaryopoietin, and thrombopoietin but the
molecular structure has not been identified until recently.
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The identification of the thrombopoietlc cytokine, c-mpl
ligand, originated with the identification of a
myeloproliferative leukemia virus (MPLV, Wending et al.,
Virology 149:242-246, 1986). Mice infected with this virus
gave rise to multi-lineage myeloproliferation. Subsequent
studies (Souyri et al., Cell 63:1137-1147, 1990) demonstrated
that the retrovirus encoded an oncogene (v-mpl) that when
fused with viral envelope gene gave rise to a membrane
anchored protein that resembles the cytoplasmic domain of the
hematopoietic growth factor receptor family. V-mpl was used
to probe both human and murine RNA libraries for homologous
genes. Clones were identified in both species and termed c-
mpl (Vigon et al., PNAS USA 89:5640-5644, 1992; Vigon et al.,
Oncogene 8:2607-2615, 1993). C-mpl is a member of the
cytokine receptor super-family with regions of homology to
mIL-5rc, IL3rc, IL4rc, mEPOrc and mGCSFrc. A chimera of the
intracellular domain of c-mpl and the extracellular domain of
hIL4rc was transfected into a growth factor dependent cell
line (BaF3). Once transfected, the cells proliferated in
response to hIL4 indicating that the c-mpl cytoplasmic domain
was fully sufficient to transduce a proliferative signal
(Skoda et al., EMBO J. 12(7):2645-2653, 1993).
Message for c-mpl was found in a number of hematopoietic
2S cell lines using reverse transcriptase polymerase chain
reaction (RT PCR) including the pluripotential cell lines TF-
1, Mo-7E, UT-7 and KU812; and erythro/megakaryocytic cell
lines HEL, DAMI and K153. Transcripts were also identified
in bone marrow, fetal liver, megakaryocytes, platelets and
CD34+ enriched cells (Methia et al., Blood 82(5):1395-1401,
1993).
The identification of a putative receptor triggered
several investigative teams to search for a naturally
occurring ligand for c-mpl. In June of 1994 several
CA 022340~9 1998-04-06
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simultaneous publications reported on a ligand that bound to
c-mpl and had megakaryocytopoietic properties tde Sauvage et
~ al., Nature 369:533-539, 1994; Lok et al., Nature 369:565-
568, 1994; Wendling et al., Nature 369:571 574, 1994) and
sartley et al., Cell 77 :1117-1124, 1994). The ligand named
c-mpl ligand or thrombopoietin is a peptide with a predicted
molecular mass of 35,000 kDa. The protein has a two domain
structure with an amino-terminal domain (153 amino acids)
with homology to erythropoietin and a carboxy-terminus rich
in serine, threonine and proline residues which also contains
several glycosylation sites. There are two potential
arginine cleavage sites resulting in two shorter peptides of
25 kDa and 31 kDA forms both of which are biologically
active. There is high inter species homology between human,
murine, porcine, canine, rat and rabbit c-mpl ligand and most
forms are active on all species tested.
C-mpl ligand has been shown to stimulate the
differentiation of CD34+ cells into cells megakaryocyte
characteristics. CD34+ cells in the presence of c-mpl ligand
underwent endomitosis (Kaushansky et al., Nature 369:568-571,
1994), expressed the megakaryocyte lineage specific cell
surface antigen CD41a and had morphology characteristic of
megakaryocytes. In vivo administration of c-mpl ligand have
given rise to increased circulating platelets in normal mice
(Lok et al., Nature 369:565-568, 1994). C-mpl deficient mice
generated by gene targeting demonstrated a 85% decrease in
circulating platelets and megakaryocytes but had normal
amounts of other hematopoietic lineages (Gurney et al.,
Science a6s:l44s-l447, 1994). Absolute thrombocytopenia was
not observed in these ~n;m~l S indicating that other cytokines
may have some activity in expansion of the MK lineage.
Studies to date show that c-mpl ligand is a cytokine
with specific activity on the maturation of megakaryocytes
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WO 97/12978 PCTAJS96/15938
and in platelet production. Other cytokines have been shown
to have activity on megakaryocyte expansion and
differentiation, including IL-3, IL-6, IL-11 and c-kit
ligand. Recent studies have demonstrated that these
cytokines (with the exception of IL-3) act by stimulating the
production of c-mpl ligand and do not have megakaryocyte
stimulating activity by themselves (Kaushansky et al., PNAS
USA 92:3234-3236, 1995).
The ability of c-mpl ligand to stimulate the proliferation
and maturation of megakaryocytes and production of platelets
indicates that c-mpl ligand may have therapeutic use in
restoring circulating platelets to normal amounts in those
cases where the number of platelets have been reduced due to
diseases or therapeutic treatments such as radiation and/or
chemotherapy.
EP 675,201 Al relates to the c-mpl ligand (Megakaryocyte
growth and development factor or 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.
Wo 95/27732 discloses circularly permuted GM-CSF, G-
CSF, IL-2 and IL-4 and fusions with Pseudomonas exotoxin.
A previously published abstract (Eaton et al., Blood
84(10) Suppl. abstract 948, 1994) reported c-DNA for an
alternative splice form of c-mpl ligand identified in man,
dog and mouse. The encoded protein has 4 amino deletion at
position aall2-115. Although this molecule showed no activity
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in their bioassays, mRNA for this variant was found to be
abundant in all three specles indicating that it may be a
~ naturally occurring alternative form of c-mpl ligand.
Rearran~ement of Protein Seouences
In evolution, rearrangements of DNA sequences serve an
important role in generating a diversity of protein structure
and function. Gene duplication and exon shuffling provide an
important mechanism to rapidly generate diversity and thereby
provide organisms with a competitive advantage, especially
since the basal mutation rate is low (Doolittle, Protein
Science 1:191-200, 1992).
The development of recombinant DNA methods has made it
possible to study the effects of sequence transposition on
protein folding, structure and function. The approach used
in creating new sequences resembles that of naturally
occurring pairs of proteins that are related by linear
reorganization of their amino acid sequences (Cunningham, et
al., Proc. Natl. Acad. sci. U.S.A. 76:3218-3222, 1979;
Teather & Erfle, J. Bacteriol. 172: 3837-3841, 1990;
S~himming et al., Eur. J. Biochem. 204: 13-19, 1992; Yamiuchi
and ~in~mikawa, FEBS Lett. 260:127-130, 1991, MacGregor et
al., FEBS Lett. 378:263-266, 1996). The first in vitro
application of this type of rearrangement to proteins was
described by Goldenberg and Creighton (J. Mol. Biol. 16S:407-
413, 1983). A new N-terminus is selected at an internal site
tbreakpoint) of the original sequence, the new sequence
having the same order of amino acids as the original from the
breakpoint until it reaches an amino acid that is at or near
the original C-terminus. At this point the new se~uence is
- ~ joined, either directly or through an additional portion of
sequence (linker), to an amino acid that is at or near the
original N-terminus, and the new sequence continues with the
same sequence as the original until it reaches a point that
CA 022340~9 1998-04-06
W O 97/12978 PCTAJS96/15938
is at or near the amino acid that was N-terminal to the
breakpoint site of the original sequence, this residue
forming the new C-terminus of the chain.
This approach has been applied to proteins which range
in size from 58 to 462 amino acids (Goldenberg & Creighton,
. Mol. Biol. 165:407-413, 1983; Li & Coffino, Mol. Cell.
Biol . 13:2377-2383, 1993). The proteins examined have
represented a broad range of structural classes, including
proteins that contain predominantly a -helix (interleukin-4;
Kreitman et dl., Cytokine 7 :311-318, 1995), ~ -sheet
(interleukin-1; Horlick et al., Protein Eng. 5:427-431,
1992), or mixtures of the two (yeast phosphoribosyl
anthranilate isomerase; Luger et al., Science 243:206-210,
1989). Broad categories of protein function are represented
in these sequence reorganization studies:
Enzymes
T4 lysozyme Zhang et al., Biochemistry
32:12311-12318 (1993); Zhang et
al., Nature Struct. Biol . 1:434-438
(1995)
dihydrofolate Buchwalder et al., siochemistry
25 reductase 31:1621-1630 (1994); Protasova et
al., Prot. Eng. 7:1373-1377 (1995)
ribonuclease T1 Mullins et al., J. Am. Chem. Soc.
116:5529-5533 (1994); Garrett et al.,
Protein Science 5:204-211 (1996)
Bacillus ~-glucanse Hahn et al., Proc. Natl. Acad. Sci.
U.S.A. 91:10417-10421 (1994)
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aspartate Yang & Schachman, Proc. Natl. Acad.
transcarbamoylase Sci. U.S.A. 90:11980-11984 (1993)
.,
phosphoribosyl Luger et al., Science 243:206-210
5 anthranilate (1989); Luger et al., Prot . Eng.
isomerase 3:249-258 (1990)
pepsin/pepsinogen Lin et al., Protein Science 4:159-
166 (1995)
glyceraldehyde-3- Vignais et al., Protein Science
phosphate dehydro- 4:994-1000 (1995)
genase
15 ornithine Li & Coffino, Mol . Cell . Biol .
decarboxylase 13:2377-2383 (1993)
yeast Ritco-Vonsovici et al., Biochemistry
phosphoglycerate 34:16543-16551 (1995)
20 dehydrogenase
Enzyme Inhibitor
basic pancreatic Goldenberg & Creighton, J. Mol.
25 trypsin inhibitor Biol . 165:407-413 (1983)
Cytokines
interleukin-1~ Horlick et al., Protein Eng. 5:427-
431 (1992)
interleukin-4 Kreitman et al., Cytokine 7:311-
318 (1995)
Tyrosine Rinase
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Recognition Domain
~-spectrin SH3 Viguera, et al., J.
domain Mol. Biol. 247:670-681 (1995)
Transmembrane
Protein
omp A Koebnik ~ Kramer, J. Mol. Biol.
250:617-626 (1995)
Chimeric Protein
interleukin-4- Kreitman et al., Proc. Natl. Acad.
Pseudomonas Sci. U.S.A. 91:6889-6893 (1994).
exotoxin
fusion molecule
The results of these studies have been highly variable.
In many cases substantially lower activity, solubility or
thermodynamic stability were observed (E. coli dihydrofolate
reductase, aspartate transcarbamoylase, phosphoribosyl
anthranilate isomerase, glyceraldehyde-3-phosphate
dehydrogenase, ornithine decarboxylase, omp A, yeast
phosphoglycerate dehydrogenase~. In other cases, the se~uence
rearranged protein appeared to have many nearly identical
properties as its natural counterpart (basic pancreatic
trypsin inhibitor, T4 lysozyme, ribonuclease Tl, Bacillus
-glucanase, interleukin-1~, a-spectrin SH3 domain,
pepsinogen, interleukin-4). In exceptional cases, an
unexpected improvement over some properties of the natural
sequence was observed, e.g., the solubility and refolding
rate for rearranged a-spectrin SH3 ~m~in sequences, and the
receptor affinity and anti-tumor activity of transposed
interleukin-4- Pseudomonas exotoxin fusion molecule (Kreitman
CA 022340~9 1998-04-06
W O 97/12978 PCTrUS96/15938
g
et al., Proc. Natl. Acad. Sci. U.S.A. 91:6889-6893, 1994;
Kreitman et al., Cancer Res. 55:3357-3363, 1995).
The primary motivation for these types of studies has
been to study the role of short-range and long-range
interactions in protein folding and stability. Sequence
rearrangements of this type convert a subset of interactions
that are long-range in the original se~uence into short-range
interactions in the new se~uence, and vice versa. The fact
that many of these sequence rearrangements are able to attain
a conformation with at least some activity is persuasive
evidence that protein folding occurs by multiple folding
pathways (Viguera, et al., J. Mol . Biol . 247: 670-681, 1995).
In the case of the SH3 domain of a-spectrin, choosing new
termini at locations that corresponded to ~-hairpin turns
resulted in proteins with slightly less stability, but which
were nevertheless able to fold.
The positions of the internal breakpoints used in the
studies cited here are found exclusively on the surface of
proteins, and are distributed throughout the linear sequence
without any obvious bias towards the ends or the middle (the
variation in the relative distance from the original N-
terminus to the breakpoint is ca. 10 to 80% of the total
se~Iuence length). The linkers connecting the original N- and
C-termini in these studies have ranged from 0 to 9 residues.
In one case (Yang & Schachman, Proc. Natl . Acad . Sci . U. S.A.
90:11980-11984, 1993), a portion of se~uence has been deleted
from the original C-terminal segment, and the connection made
from the truncated C-terminus to the original N-terminus.
Flexible hydrophilic residues such as Gly and Ser are
fre~uently used in the linkers. Viguera, et al. ~J. Mol. Biol.
247:670-681, 1995) compared joining the original N- and C-
termini with 3- or 4-residue linkers; the 3-residue linker
was less thermodynamically stable. Protasova et al. (Protein
Eng. 7:1373-1377, 1994) used 3- or 5-residue linkers in
connecting the original N-termini of E. coli dihydrofolate
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reductase; only the 3-residue linker produced protein in good
yield. More systematic studies o~ linker length and
composition have not been reported.
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11
SUM~RY OF THE INVF.NTION
The present invention relates to novel c-mpl receptor
agonists of the ~ollowing formulas:
1. A c-mpl receptor agonist o~ the Formula:
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer
1 5 10 15
HisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrPro
ValLeuLeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGlu
40 45 50 55
ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla
60 65 70 75
AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly
80 85 90 95
GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnXaaXaaXaa
25100 105 110
XaaGlyArgThrThrAlaHisLysAspProAsnAlaIlePheLeuSerPheGlnHis
115 120 125 130
LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal
135 140 145 150
ArgArgAlaProProThrThrAlaValProSerArgThrSerLeuValLeuThrLeu
155 160 165 170
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. 12
AsnGluLeuProAsnArgThrSerGlyLeuLeuGluThrAsnPheThrAlaSerAla
175 180 185 190
ArgThrThrGlySerGlyLeuLeuLysTrpGlnGlnGlyPheArgAlaLysIlePro
5195 200 205
GlyLeuLeuAsnGlnThrSerArgSerLeuAspGlnIleProGlyTyrLeuAsnArg
210 215 220 225
IleHisGluLeuLeuAsnGlyThrArgGlyLeuPheProGlyProSerArgArgThr
230 235 240 245
LeuGlyAlaProAspIleSerSerGlyThrSerAspThrGlySerLeuProProAsn
250 255 260 265
LeuGlnProGlyTyrSerProSerProThrHisProProThrGlyGlnTyrThrLeu
270 275 280 285
PheProLeuProProThrLeuProThrProValValGlnLeuHisProLeuLeuPro
290 295 300
AspProSerAlaProThrProThrProThrSerProLeuLeuAsnThrSerTyrThr
305 310 315 320
HisSerGlnAsnLeuSerGlnGluGly (SEQ ID NO:1)
325 330 332
wherein;
Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro,
Phe, Trp, or Met;
Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu,
Ile, Trp, or Met;
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13
Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu,
Ile, Trp, or Met;
Xaa at position 115 is deleted or Gln, Gly, Ser, Thr, Tyr, or
Asn;
S
wherein from 1 to 179 amino acids can be deleted from the C-
terminus;
wherein the N-terminus is joined to the C-terminus directly
or through a linker capable of joining the N-terminus to the
C-terminus and having new c- and N-termini at amino acids;
26-27 51-52 108-109
27-28 52-53 109-110
28-29 53-54 110-111
29-30 54-55 111-112
30-31 55-56 112-113
32-33 56-57 113-114
33-34 57-58 114-115
34-35 58-59 115-116
36-37 59-60 116-117
37-38 78-79 117-118
38-39 79-80 118-119
40-41 80-81 119-120
41-42 81-82 120-121
42-43 82-83 121-122
43-44 83-84 122-123
44-45 84-85 123-124
46-47 85-86 124-125
47-48 86-87 125-126
48-49 87-88 126-127
50-51 88-89 or 127-128; and
additionally said c-mpl receptor agonist can be immediately
preceded by (methionine~1), (~l~n;ne~1) or (methionine~2,
alanine~l).
2. A c-mpl receptor agonist of the Formula:
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer
1 5 10 15
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14
HisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrPro
20 25 30 35
ValLeuLeuProAlaValAspSerLeuGlyGluTrpLysThrGlnMetGluGlu
40 45 50 55
ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla
AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly
80 85 90 95
GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnXaaXaaXaa
100 105 110
XaaGlyArgThrThrAlaHisLysAspProAsnAlaIlePheLeuSerPheGlnHis
115 120 125 130
LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal
135 140 145 150
Arg (SEQ ID NO: 2)
153
wherein;
Xaa at position 112 iS deleted or Leu, Ala, Val, Ile, Pro,
Phe, Trp, or Met;
Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu,
Ile, Trp, or Met;
Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu,
Ile, Trp, or Met;
Xaa at position 115 is deleted or Gln, Gly, Ser, Thr, Tyr,
or Asn;
wherein the N-terminus is joined to the C-terminus directly
or through a linker capable of joining the N-terminus to the
C-terminus and having new C- and N-termini at amino acids;
26-27 51-52 108-109
27-28 52-53 109-110
28-29 53-54 110-111
29-30 54-55 111-112
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~ 15
30-31 55-56 112-113
32-33 56-57 113-114
33-34 57-58 114-115
- 34-35 58-59 115-116
36-37 59-60 116-117
37-38 78-79 117-118
38-39 79-80 118-119
40-41 80-81 119-120
41-42 81-82 120-121
42-43 82-83 121-122
43-44 83-84 122-123
44-45 84-85 123-124
46-47 85-86 124-125
47-48 86-87 125-126
48-49 87-88 126-127
50-51 88-89 or 127-128; and
additionally said c-mpl receptor agonist can be immediately
preceded by (methionine~l), (alanine~l) or (methionine~2,
alanine~l).
The more preferred breakpoints at which new C-terminus
and N-terminus can be made in the amino acid sequence of (SEQ
ID NO:2) above are; 80-81, 81-82, 82-83, 83-84, 84-85, 85-86,
86-87, 108-109, 109-110, 110-111, 111-112, 112-113, 113-114,
114-115, 115-116, 116-117, 117-118, 118-119, 119-120, 120-
121, 121-122, 122-123, 123-124, 124-125, 125-126 and 126-127.
The most preferred breakpoints at which new C-terminus
and N-terminus can be made in the amino acid se~uence of (SEQ
ID NO:2) above are; 81-82, 108-109, 115-116, 119-120, 122-123
and 125-126.
These human c-mpl receptor agonists may contain amino
acid substitutions, deletions and/or insertions and may also
have amino acid deletions at either/or both the N- and C-
termini.
The modified human c-mpl receptor agonists of the
present invention can be represented by the Formula:
16
X1-(L)a-X2
wherein;
a is 0 or 1;
X1 is a peptide comprising an amino acid sequence
corresponding to the sequence of residues n+1 through J;
X2 is a peptide comprising an amino acid sequence
corresponding to the sequence of residues 1 through n;
n is an integer ranging from 1 to J-1; and
L is a linker.
In the formula above the constituent amino acids
residues of Human c-mpl ligand are numbered sequentially 1
through J from the amino to the carboxy terminus. A pair of
adjacent amino acids within this protein may be numbered n
and n+1 respectively where n is an integer ranging from 1 to
J-1. The residue n+1 becomes the new N-terminus of the new c-
mpl receptor agonist and the residue n becomes the new C-
terminus of the the new c-mpl recepter agonist.
In a preferred embodiment of the present invention the
linker (L) joining the N-terminus to the C-terminus is a
polypeptide selected from the group consisting of:
GlyGlyGlySer (SEQ ID NO:73);
GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:74);
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:75);
SerGlyGlySerGlyGlySer (SEQ ID NO:76);
GluPheGlyAsnMetAla (SEQ ID NO:77);
GluPheGlyGlyAsnMetAla (SEQ ID NO:78);
GluPheGlyGlyAsnGlyGlYAsnMetAla (SEQ ID NO:79); and
GlyGlyGlySerAspMetAlaGly (SEQ ID NO:80).
The present invention also encompasses recombinant human
c-mpl receptor agonists co-administrated with one or more
additional colony stimulating factors (CSF) including,
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, 17
cytokines, lymphokines, interleukins, hematopoietic growth
factors (herein collectively referred to as "colony
stimulating factorsn) each of which may act through a
different and specific cell receptor to initiate
complementary biological activities.
These co-administrated molecules may be characterized by
having the usual activity of both of the peptides or it may
be further characterized by having a biological or
physiological activity greater than simply the additive
function of the presence of human c-mpl receptor agonist or
the second colony stimulating factor alone. The co-
administration may also unexpectedly provide an enhanced
effect on the activity or an activity different from that
expected by the presence of human c-mpl receptor agonist or
the second colony stimulating factor or human c-mpl ligand
variant. The co-administration may also have an improved
activity profile which may include reduction of undesirable
biological activities associated with native human c-mpl
ligand or native cytokine.
In addition to the use of co-administration of the
present invention in vivo, it is envisioned that in vitro
uses would include the ability to stimulate bone marrow and
blood cell activation and growth before infusion into
patients.
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. 18
BRTFF DF~CRIPTION OF THF FIGURFS
Figure 1 schematically illustrates the sequence
rearrangement of a protein. The N-terminus (N) and the C-
terminus (C) of the native protein are joined through a
linker, or joined directly. The protein is opened at a
breakpoint creating a new N-terminus (new N) and a new C-
terminus (new-C) resulting in a protein with a new linear
amino acid sequence. A rearranged molecule may be synthesized
de novo as linear molecule and not go through the steps of
joining the original N-terminus and the C-terminus and
opening of the protein at the breakpoint.
Figure 2 shows a schematic of Method I, for creating new
proteins in which the original N-terminus and C-terminus of
the native protein are joined with a linker and different N-
terminus and C-terminus of the protein are created. In the
example shown the sequence rearrangement results in a new
gene encoding a protein with a new N-terminus created at
amino acid 97 of the original protein, the original C-
terminus (a~a. 174) joined to the amino acid 11 (a.a. 1- 10
are deleted) through a linker region and a new C-terminus
created at amino acid 96 of the original sequence.
Figure 3 shows a schematic of Method II, for creating
new proteins in which the original N-terminus and C-terminus
of the native protein are joined without a linker and
different N-terminus and C-terminus of the protein are
created. In the example shown the sequence rearrangement
results in a new gene encoding a protein with a new N-
terminus created at amino acid 97 of the original protein,
the original C-terminus (a.a. 174) joined to the original N-
terminus and a new C-terminus created at amino acid 96 of the
original se~uence.
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~ 19
Figure 4 shows a schematic of Method III, for creating
new proteins in which the original N-terminus and C-terminus
of the native protein are joined with a linker and different
N-terminus and C-terminus of the protein are created. In the
example shown the sequence rearrangement results in a new
gene encoding a protein with a new N-terminus created at
amino acid 97 of the original protein, the original C-
terminus (a.a. 17~) joined to amino acid 1 through a linker
region and a new C-terminus created at amino acid 96 of the
original sequence.
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/15938
. 20
D~TATTl~n DFSCRIPTION OF TH~ INVF.~TION
Receptor agonists of the present invention may be useful
in the treatment of diseases characterized by a decreased
levels of megakaryocyte cells of the hematopoietic system.
A c-mpl receptor agonist may be useful in the treatment
or prevention of thrombocytopenia. Currently the only therapy
for thrombocytopenia is platelet transfusions which are
costly and carry the significant risks of infection (HIV,
HBV) and alloimunization. A c-mpl receptor agonist may
alleviate or ~;m;n;sh the need for platelet transfusions.
Severe thrombocytopenia may result from genetic defects such
as Fanconi's Anemia, Wiscott-Aldrich, or May-Hegglin
syndromes. Ac~uired thrombocytopenia may result from auto- or
allo-antibodies as in Immune Thrombocytopenida 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 c-mpl receptor agonists of the present
invention may be useful in the mobilization of hematopoietic
progenitors and stem cells into 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
cell collection and dramatically decreased the cost of the
procedure by decreasing the number of plasmaphereses
CA 022340~9 1998-04-06
WO97/12978 PCT~S96/15938
21
required. The c-mpl receptor agonists may be useful in
mobilization of stem cells and further enhance the efficacy
of peripheral stem cell transplantation.
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 as meprobamate, analgesics
such as aminopyrine and dipyrone, anti convulsants such as
phenytoin or carbamazepine, antithyroids such as
propylthiouracil and methimazole and diuretics. The c-mpl
receptor agonists 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.
c-mpl ligand may be useful in treating such hematopoietic
deficiency.
As another aspect of the present invention, there is
provided a novel method for producing the novel family of
human c-mpl 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
se~uence coding for expression of a novel c-mpl receptor
agonist polypeptide. Suitable cells or cell lines may
include various strains of E. coli, yeast, mAmmAlian cells,
or insect cells may be utilized as host cells in the method
of the present invention.
Another aspect of the present invention provides plasmid
DNA vectors for use in the method of expression of these
novel c-mpl receptor agonists. These vectors contain the
novel DNA sequences described above which code for the novel
polypeptides of the invention. Appropriate vectors which can
CA 022340~9 1998-04-06
W O 97/12978 PCTAUS96/15938
. 22
transform microorganisms capable of expressing the c-mpl
receptor agonists include expression vectors comprising
nucleotide sequences coding for the c-mpl 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 c-mpl receptor agonist polypeptides.
The vector employed in the method also contains selected
regulatory sequences in operative association with the DNA
coding sequences of the invention and capable of directing
the replication and expression thereof in selected host
cells.
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 c-mpl 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 c-mpl receptor agonists of the present invention may
be useful in the mobilization of multipotential hematopoietic
progenitors 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
:
CA 022340~9 1998-04-06
WO97/12978 PCT~S96/15938
2~
and stem cel's in the peripheral blood. This has simplified
the procedure for peripheral stem cell collection and
dramatically decreased the cost of the procedure by
decreasing the number of pheresis required. The c-mpl
receptor agonists may be useful in mobilization of
multipotential hematopoietic cells and further enhance the
efficacy of peripheral blood cell transplantation.
The c-mpl receptor agonists of the present invention may
also be useful in the ex vivo expansion of multipotential
hematopoietic cells. Colony stimulating factors (CSFs), such
as hIL-3, have been ~m; n; stered alone, co-administered with
other colony stimulating factors, or in combination with bone
marrow transplants subse~uent to high dose chemotherapy to
treat the neutropenia and thrombocytopenia which are often
the result o' 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: l) the number of stem cells
in bone marrow or other is limited, 2) Graft Versus Host
Disease, 3) graft rejection and 4) possible cont~min~tion
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 exits such
that a greater number of stem cells will enhance
hematopoietic recovery. Therefore, the in vitro expansion of
CA 022340~9 1998-04-06
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. 24
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 autologous 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 selected based on the presence of specific surface
antigen 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.
5,199,942 describes a method for autologous hematopoietic
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
CA 022340~9 l998-04-06
W O 97/12978 PCTAUS96/1~938
of IL-3, flk3 ligand, c-kit ligand, GM CSF, IL-l, GM-CSF/IL-3
fusion 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
m~mm~l ian cells derived from peripheral blood.
30 WO 93/18648 relates to a composition comprising human
neutrophil precursor cells with a high content of myeloblasts
and promyelocytes for treating genetic or acquired
neutropenia.
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/15938
26
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.
WO 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 ~. 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 multipotential hematopoietic cells as
well as early progenitor and precursors 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 separated stem cells with a
selected medium which contains a c-mpl receptor agonist and
optionally a colony stimulating factor; and (c) harvesting
said stems cells. Stem cells as well as committed progenitor
cells destined to become neutrophils, erythrocytes,
CA 022340~9 1998-04-06
WO97/12978 PCT~S96/15938
27
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, flt-3 ligand or combinations
thereof. The proliferation of the stem cells can be monitored
by ~nllmerating the number of stem cells and other cells, by
standard techni~ues (e.g. hemacytometer, CFU, LTCIC) or by
flow cytometry prior and subse~uent to incubation.
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/15938
. 28
Several methods for ex-vivo expanslon of stem cells have
been reported utilizing a number of selection methods and
expansion using various colony stimulating factors including
c-kit ligana (Brandt et al., Blood 83:1507-1514, 1994;
McKenna et al., Blood 86:3413-3420, 1995); IL-3 (srandt et
al., Blood ~3:1507-1514, 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., Exp. Hem. 21:1668-
1672, 1993; Sato et al., Blood 82:3600-3609, 1993), fIt-3
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., B~ood 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; ~ith 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., Blooa
73:1836-1841, 1989). However, no single factor has been shown
to be as effective as the combination of multiple factors.
The present invention provides methods for ex vivo expansion
that utilize c-mpl receptor agonists that are more effective.
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 ~ stromal cell line such as HS-5 (WO 96/02662,
35 Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has
CA 022340~9 1998-04-06
W O 97/12978 PCTrUS96/lS938
. 29
been supplemented with a c-mpl 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
;mmllnodifiencies (Kay and Woo, Trends Genet. 10:253-257,
20 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 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 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,
BioTechni~ues 6:616-629, 1988; Berkner, Current Top.
Microbiol. Immunol. 158:39-66, 1992; Brody and Crystal,
CA 022340~9 1998-04-06
W O 97/1~978 PCTAUS96/15938
Annal . 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 c-mpl receptor agonists that have improved
biological activity, including an activity not seen by any
single colony stimulation factor and/or physical properties.
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 c r 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 ~g/kg of non-
glycosylated c-mpl receptor agonists protein per kilogram of
body weight. Dosages would be adjusted relative to the
activity of a given receptor agonist 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 c-mpl 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 CSF or growth factors;
co-administration with chemotherapeutic drugs and/or
radiation; the use of glycosylated c-mpl receptor agonists;
and various patient-related issues mentioned earlier in this
section. As indicated above, the therapeutic method and
CA 022340~9 1998-04-06
WO97/12978 PCT~S96/15938
31
compositions may also include co-~ministration with other
human factors. A non-exclusive list of other appropriate
~ hematopoietins, CSFs and interleukins for simultaneous or
serial co-administration with the polypeptides of the present
invention includes GM-CS~, G-CSF, 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-ll, IL-12, IL-13, IL-15, LIF, flt3iflk2 ligand,
human growth hormone, s-cell growth factor, s-cell
differentiation factor, eosinophil differentiation factor and
stem cell factor (SCF) also known as steel factor or c-kit
ligand, (herein collectively referred to as ~colony
stimulating factorsn), or combinations thereof. In addition
to the list above, IL-3 variants taught in WO 94/12639 and WO
94/12638 can be co-administered with the polypeptides of the
present invention. The c-mpl receptor agonists of the present
invention can be co-administered as with another "colony
stimulating factor~ as discussed above in a fashion taught in
WO 95/20976 and Wo 95/20977. The dosage recited 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 the J,inker.
The length of the amino acid sequence of the linker can
be selected empirically or with guidance from structural
information, or by using a combination of the two approaches.
When no structural information is available, a small
series of linkers can be prepared for testing using a design
whose length is varied in order to span a range from 0 to 50
A and whose sequence is chosen in order to be consistent with
surface exposure (hydrophilicity, Hopp & Woods, Mol. Immunol.
20: 483-489, 1983; Kyte & Doolittle, ~. Mol . Biol . 157:105-
132, 1992; solvent exposed surface area, Lee & Richards, J.
.
CA 022340~9 1998-04-06
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. 32
Mol. Biol. 55:379-400, 1971) and the ability to adopt the
necessary conformation without deranging the con~ormation of
the c-mpl receptor agonist (con~ormationally flexible;
Karplus & Schulz, Naturwissenschaften 72:212-213, 1985).
Assuming an average of translation of 2.0 to 3.8 A per
residue, this would mean the length to test would be between
0 to 30 residues, with 0 to 15 residues being the pre~erred
range. Exemplary of such an empirical series would be to
construct linkers using a cassette sequence such as Gly-Gly-
Gly-Ser (SEQ ID NO:3) repeated n times, where n is 1, 2, 3 or
4. Those skilled in the art will recognize that there are
many such se~uences that vary in length or composition that
can serve as linkers with the primary consideration being
that they be neither excessively long nor short (cf., Sandhu,
Critical Re~r. Biotech. 12: 437-462, 1992); if they are too
long, entropy effects will likely destabilize the three-
dimensional fold, and may also make folding kinetically
impractical, and if they are too short, they will likely
destabilize the molecule because of torsional or steric
strain.
Those skilled in the analysis of protein structural
information will recognize that using the distance between
the chain ends, defined as the distance between the c-alpha
carbons, can be used to define the length of the sequence to
be used, or at least to limit the number o~ possibilities
that must be tested in an empirical selection of linkers.
They will also recognize that it is sometimes the case that
the positions of the ends of the polypeptide chain are ill-
defined in structural models derived from x-ray diffraction
or nuclear magnetic resonance spectroscopy data, and that
when true, this situation will therefore need to be taken
into account in order to properly estimate the length of the
linker required. From those residues whose positions are
well defined are selected two residues that are close in
CA 022340~9 1998-04-06
W O 97/12978 PCTrUS96/15938
. 33
sequence to the chain ends, and the distance between their c-
alpha carbons is used to calculate an approximate length for
a linker between them. Using the calculated length as a
guide, linkers with a range of number of residues (calculated
using 2 to 3.8A per residue) are then selected. These
linkers may be composed of the original sequence, shortened
or lengthened as necessary, and when lengthened the
additional residues may be chosen to be flexible and
hydrophilic as described above; or optionally the original
seauence may be substituted for using a series of linkers,
one example being the Gly-Gly-Gly-Ser (SEQ ID NO:3) cassette
approach mentioned above; or optionally a combination of the
original sequence and new sequence having the appropriate
total length may be used.
Determin~tion of the Amino and C~rboxYl ~ermini of c-~nl
lia~nd
Sequences of c-mpl ligand of folding to biologically
active states can be prepared by appropriate selection of the
beginning (amino terminus) and ending (carboxyl terminus)
positions from within the original polypeptide chain while
using the linker sequence as described above. Amino and
2S carboxyl termini are selected from within a common stretch of
sequence, referred to as a breakpoint region, using the
guidelines described below. A novel amino acid sequence is
thus generated by selecting amino and carboxyl termini from
within the same breakpoint region. In many cases the
selection of the new termini will be such that the original
position of the carboxyl terminus immediately preceded that
of the amino terminus. However, those skilled in the art
will recognize that selections of termini anywhere within the
region may function, and that these will effectively lead to
CA 022340~9 1998-04-06
W O 97/12978 PCTAUS96/15938
34
either deletions or additions to the amino or carboxyl
portions of the new sequence.
It is a central tenet of molecular biology that the
primary amino acid sequence of a protein dictates folding to
the three-~;m~n~ional structure necessary for expression of
its biological function. Methods are known to those skilled
in the art to obtain and interpret three-~im~n~ional
structural information using x-ray diffraction of single
protein crystals or nuclear magnetic resonance spectroscopy
of protein solutions. Examples of structural information
that are relevant to the identification of breakpoint regions
include the location and type of protein secondary structure
(alpha and 3-10 helices, parallel and anti-parallel beta
sheets, chain reversals and turns, and loops; Kabsch &
15 Sander, Biopolymers 22: 2577-2637, 1983), the degree of
solvent exposure of amino acid residues, the extent and type
of interactions of residues with one another (Chothia, Ann.
Rev. Biochem. 53:537-572, 1984) and the static and dynamic
distribution of conformations along the polypeptide chain
20 (Alber & Mathews, Methods Enzymol. 154: 511-533, 1987). In
some cases additional information is known about solvent
exposure of residues; one example is a site of post-
translational attachment of carbohydrate which is necessarily
on the surface of the protein. When experimental structural
information is not available, or is not feasible to obtain,
methods are also available to analyze the primary amino acid
sequence in order to make predictions of protein tertiary and
secondary structure, solvent accessibility and the occurrence
of turns and loops. Biochemical methods are also sometimes
applicable for empirically determining surface exposure when
direct structural methods are not feasible; for example,
using the identification of sites of chain scission following
limited proteolysis in order to infer surface exposure
(Gentile ~ Salvatore, Eur. J. Biochem. 218: 603-621, 1993)
CA 022340~9 1998-04-06
.
W097/12978 PCT~S96/15938
~5
Thus using either the experimentally derived structural
information or predictive methods (e.g., Srinivisan ~ Rose
Proteins: Struct., Funct. & Genetics, 22: 81-99, 1995) the
parental amino acid sequence is inspected to classify regions
according to whether or not they are integral to the
maintenance of secondary and tertiary structure. The
occurrence of sequences within regions that are known to be
involved in periodic secondary structure (alpha and 3-lO
helices, parallel and anti-parallel beta sheets) are regions
that should be avoided. Similarly, regions of amino acid
sequence that are observed or predicted to have a low degree
of solvent exposure are more likely to be part of the so-
called hydrophobic core of the protein and should also be
avoided for selection of amino and carboxyl termini. In
contrast, those regions that are known or predicted to be in
surface turns or loops, and especially those regions that are
known not to be required for biological acti~ity, are the
preferred sites for location of the extremes of the
polypeptide chain. Continuous stretches of amino acid
sequence tha~ are preferred based on the above criteria are
referred to as a breakpoint region.
All references, patents or applications cited herein are
incorporated by reference in their entirety.
M~terials and Methods
Unless noted otherwise, all specialty chemicals were
obtained from Sigma Co., (St. Louis, MO). Restriction
endonucleases and T4 DNA ligase were obtained from New
England Biolabs (Beverly, MA).
Metho~ for creation of ~enes with new N-terminus/C-terminus
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96tl5938
~ 36
Method I. Creation of genes with new N-terminus/C-terminus
which contain a linker region.
Genes with new N-terminus/C-terminus which contain a
linker region separating the original C-terminus and N-
terminus can be made essentially following the method
described in L. S. Mullins, et al (.J. Am. Chem. Soc. 116,
5529-5533, 1994). Multiple steps of polymerase chain
reaction ( PCR) amplifications are used to rearrange the DNA
10 sequence encoding the primary amino acid sequence of the
protein. The steps are illustrated in Figure 2.
n the first step, the first primer set ("new start~ and
IlLinker start") is used to create and amplify, from the gene
15 sequence, the DNA fragment ("Fragment Start~) that contains
the sequence encoding the new N-terminal portion of the new
protein followed by the linker that connects the C-terminal
and N-terminal ends of the original protein. In the second
step, the second primer set (~new stop'~ and '~linker stop~) is
20 used to create and amplify, from the gene sequence, the DNA
fragment ("Fragment Stop") that encodes the same linker as
used above, followed by the new C-terminal portion of the new
protein. The "new start~ and "new stop" primers are designed
to include the appropriate restriction sites which allow
25 cloning of the new gene into expression plasmids. Typical PCR
conditions are one cycle 95~C melting for two minutes; 25
cycles 94~C denaturation for one minute, 50~C annealing for
one minute and 72~C extension for one minute; plus one cycle
72~C extension for seven minutes. A Perkin Elmer GeneAmp PCR
30 Core Reagents kit is used. A 100 ul reaction contains 100
pmole of each primer and one ug of template DNA; and lx PCR
buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP,
2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2. PCR
reactions are performed in a Model 480 DNA thermal cycler
(Perkin Elmer Corporation, Norwalk, CT).
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/1~938
37
Fragment Start" and "Fragment Stop", which have
complementary se~uence in the linker region and the coding
se~uence fo~ the two amino acids on both sides o~ the linker,
are joined together in a third PCR step to make the full-
length gene encoding the new protein. The DNA fragments
IlFragment Start~ and ~Fragment stop~ are resolved on a 196 TAE
gel, stained with ethidium bromide and isolated using a Qiaex
Gel Extraction kit (Qiagen). These fragments are combined in
e~uimolar quantities, heated at 70~C for ten minutes and slow
cooled to allow annealing through their shared sequence in
"Linker start" and Illinker stop". In the third PCR step,
primers "new start" and "new stop" are added to the annealed
fragments to create and amplify the full-length new N-
terminus/C-terminus gene. Typical PCR conditions are one
cycle 95~C melting for two minutes; 25 cycles 94~C
denaturation for one minute, 60~C annealing for one minute
and 72~C extension for one minute; plus one cycle 72~C
extension fo- seven minutes. A Perkin Elmer GeneAmp PCR Core
Reagents kit is used. A 100 ul reaction contains 100 pmole
of each primer and approximately 0.5 ug of DNA; and lx PCR
buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP,
2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2. PCR
reactions are purified using a Wizard PCR Preps kit
(Promega).
Method II. Creation of genes with new N-terminus/C-terminus
without a linker region.
New N-terminus/C-terminus genes without a linker joining
the original N-terminus and C-terminus can be made using two
steps of PCR amplification and a blunt end ligation. The
steps are illustrated in Figure 3. In the first step, the
first primer set ("new start" and ~P-bl start~) is used to
create and amplify, from the original gene se~uence, the DNA
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/15938
38
fragment ("Fragment Start") that contains the sequence
encoding the new N-terminal portion of the new protein. In
the second step, the second primer set ("new stop" and "P-bl
stop") is used to create and amplify, from gene se~uence, the
DNA fragment ("Fragment Stop'~) that contains the se~uence
encoding the new C-terminal portion of the new protein. The
~new start" and "new stop" primers are designed to include
appropriate restriction sites which allow cloning of the new
gene into expression vectors. Typical PCR cGnditions are one
cycle 95~C melting for two minutes; 25 cycles 94~C
denaturation for one minute, 50~C annealing for 45 seconds
and 72~C extension for 45 seconds. Deep Vent polymerase (New
England Biolabs) is used to reduce the occurrence of
overhangs in conditions recommended by the manufacturer. The
"P-bl start" and "P-bl stop" primers are phosphorylated at
the 5' end to aid in the subsequent blunt end ligation of
~Fragment Start~ and ~Fragment Stop~ to each other. A 100 ul
reaction contained 150 pmole of each primer and one ug of
template DNA; and lx Vent buffer (New England siolabs), 300
uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit
Deep vent polymerase. PCR reactions are performed in a Model
480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk,
CT). PCR reaction products are purified using a Wizard PCR
Preps kit (Promega).
The primers are designed to include appropriate restriction
sites which allow for the cloning of the new gene into
expression vectors. Typically ~Fragment Start'~ is designed to
create NcOI restriction site , and "Fragment Stop" is
designed to create a HindIII restriction site. Restriction
digest reactions are purified using a Magic DNA Clean-up
System kit (Promega). Fragments Start and Stop are resolved
on a 1% TAE gel, stained with ethidium bromide and isolated
using a Qiaex Gel Extraction kit (Qiagen). These fragments
are combined with and annealed to the ends of the ~ 3800 base
CA 022340~9 1998-04-06
WO97/12978 PCT~S96/15938
39
pair NcoI/HindIII vector fragment of pMON3934 by heating at
50~C for ten minutes and allowed to slow cool. The three
fragments are ligated together using T~ DNA ligase
(Boehringer ~annheim). The result is a plasmid cont~ining the
full-length new N-terminus/C-terminus gene. A portion of the
ligation reaction is used to transform E. coli strain DH5a
cells (Life Technologies, Gaithersburg, MD). Plasmid DNA is
purified and se~uence confirmed as below.
Method III. Creation of new N-terminus/C-terminus genes b~
tandem-duplication method
New terminus/c-terminus genes can be made based on the
method described in R. A. Horlick, et al Protein Eng. 5:427-
431, 1992). Polymerase chain reaction (PCR) amplification ofthe new N-terminus/C-terminus genes is performed using a
tandemly duplicated template DNA. The steps are illustrated
in Figure 4.
The tandemly-duplicated template DNA is created b~
cloning and contains two similar, but not necessarily
identical, copies of the yene separated by DNA se~uence
encoding a linker connecting the original C- and N-terminal
ends of the two copies of the gene. Specific primer sets are
used to create and amplify a full-length new N terminus/C-
terminus gene from the tandemly-duplicated template DNA.
These primers are designed to include appropriate restriction
sites which allow for the cloning of the new gene into
expression vectors. Typical PCR conditions are one cycle 95~C
melting for two minutes; 25 cycles 94~C denaturation for one
minute, 50~C annealing for one minute and 72C' extension for
one minute; plus one cycle 72~C extension for seven minutes.
A Perkin Elmer GeneAmp PCR Core Reagents kit (Perkin Elmer
Corporation, Norwalk, CT) is used. A 100 ul reaction
contains 100 pmole of each primer and one ug of template DNA;
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/15938
. 40
and lx PCR buf~er, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200
uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgC12.
PCR reactions are performed in a Model 480 DNA thermal cycler r
(Perkin Elmer Corporation, Norwalk, CT). PCR reactions are
purified using a Wizard PCR Preps kit (Promega).
The construction o~ the PCR templates involves the steps
outlined below;
1. Generation of the c-mpl ligand gene with and without
amino acid codons 112-115 via reverse
transcriptase/polymerase chain reaction (RT/PCR). Human
liver contains c-mpl ligand mRNA with and without a deletion
of amino acids 112-115.
2. Subcloning the PCR products into an mammalian
expression vector.
3. Assembly of dimer templates as geneI/linker/geneII
from; i) a gene encoding c-mpl ligand amino acids 1-153 for
the geneI position; ii) uni~ue synthetic linkers for
separation of the two genes; and iii) a gene encoding c-mpl
ligand amino acids 1-153, with or without amino acids 112-115
for the geneII position.
Step 1: Reverse tr~nscri~tase/~olvmerase chain reaction
The two forms of c-mpl ligand, one with a deletion of
amino acids 112-115 and one without the deletion, can be
isolated RT/PCR technology. Synthetic primers are designed so
that they would anneal to either c-mpl ligand DNA or mRNA (c-
mpl ligand sequence based on Genebank accession #L33410 or deSauvage et al., Nature 369, 1994, pp.533 538) for priming
first-strand complementary DNA (cDNA) synthesis. The
resulting cDNA is used as a template in PCR (Saiki, 1985) to
generate double-stranded DNA (dsDNA or DNA) which can be used
in additional PCR or digested with appropriate restriction
CA 022340~9 1998-04-06
WO97/12978 PCT~S96/15938
41
enzymes for transfer to E. coli or m~mmAlian expression
plasmids.
For the revelse transcriptase (RT) reaction, human fetal (lot
5 ~38130) and adult liver (lot #46018) A+ RNA can be obtained
from Clontech (Palo Alto, CA). The RT reactions are carried
out using a cDNA Cycle~ Kit obtained from Invitrogen (San
Diego, CA). One microgram (ug) of each RNA sample is
combined and denatured at 65~C for 10 min. in the presence of
either random primers, oligo dT primer or a speci~ic 3' anti-
sense primer. Following denaturation, the samples are cooled
for 2 min. on ice and spun down for 10 sec. at 10,000 x g.
RNAse inhibitor, reverse transcriptase buffer,
deoxynucleotides, sodium pyrophosphate and reverse
transcriptase are added as described by manufacturer, and the
20 microliter reaction is incubated at 42~C for 1 hr.
For PCR a specific 5~ sense primer and 3~ anti-sense primers
are added to the RT reactions and the PCR is carried out
using reagents from Boehringer Mannheim (Indianapolis, IN) or
Perkin-Elmer (Norwalk, CT) as described by the manufacturers
using Tag polymerase. The PCR reactions are subjec~ed to 30
cycles of the following; 1 min. @ 94~C, l min. @ 58~C, 90
sec. @ 72~C. An equal volume of loading dye (0.01% each
bromophenol blue and xylene cyanole blue) is added to 10
microliters of the final product for electrophoresis through
a 1% SeaKemR LE agarose (FMC, Rockland, ME) gel in the
presence of lx TBE/EtBr (Tris-borate-EDTA plus ethidium
bromide;Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold
Spring Horbor, NY, 1989). For molecular weight stAn~Ards, 1
microgram of phiX174 phage DNA digested with HaeIII
restriction enzyme (New England Biolabs, Beverly, MA) is
loaded onto the gel. The product (about 1090 base pairs) is
3 5 visualized using a short-wave W light source. The reactions
CA 022340~9 1998-04-06
W O 97/12978 PCTAJS96/15938
42
are purified using a WizardTM PCR Preps kit from Promega
(Madison, WI). Briefly, the PCR reactions are added to 100
microliters of Direct Purification buffer, and 1 milliliter
(mL) of PCR Preps DNA Purification Resin is added to this
5 mixture. After 1 minute incubation at 24~C, the supernatant
is removed by vacuum filtration through a filtration column.
Two mLs of 80% isopropanol is used to wash the resin via
vacuum filtration. The column containing the resin is then
subjected to centrifugation at 10,000 x g for 30 seconds to
10 remove residual isopropanol. The PCR product is eluted with
50 microliters of 10 mM Tris-Cl, 1 mM EDTA, pH7.4, via
centrifugation at 10,000 x g for 30 seconds followed by
transfer of supernatant to a new tube.
Step 2: Subcloning the PCR products into a m~3mm~3 lian
expression vector
The c-mpl ligand PCR products are digested with the
appropriate restriction enzymes for ligation to a mammalian
20 expression vector. The m~mm~ 1 ian expression vector is a
derivative of pMON3359 which is a pUC18-based vector
containing a mammalian expression cassette. The cassette
includes a herpes simplex viral promoter IE110 (-800 to
+120), an IL-3 secretion signal sequence and a SV40 late
25 poly-adenylation (poly-A) signal which has been subcloned
into the pUC18 polylinker (Hippenmeyer et al.,
Bio/Technology, 1037-1041, 1993). Restriction enzyme
digestions are incubated for 1 hour at 37~C as described by
the manufacturer prior to electrophoresis through a 19~
30 agarose/lx TBE/EtBr gel. Fragments are first visualized by
long-wave W and gel-purified using a Qiaex DNA Extraction
kit (Qiagen, Chatsworth, CA). The DNA fragments are purified
from the resin by agarose solublization, addition of a DNA-
binding resin, and extensive washing of the resin prior to
35 elution with water. The purified DNA products are combined at
CA 022340~9 1998-04-06
WO97/12978 PCT~S96/15938
~ 43
a molar excess of PCR product to vector fragment and the
ligation reactions are carried out according to the
manufacturer's recommended conditions for T4 DNA ligase. An
E. coli strain is then transformed with the ligation, plated
out onto LB-agar plus ampicillin (100 ug/ml). The colonies
are screened for presence of a c-mpl ligand gene and DNA is
isolated for DNA sequencing to identify both forms c-mpl
ligand, one with amino acids 112-115 deleted and one with
them present.
3. Assembly of dimer PCR templates
The linkers that join c-mpl (1-153) ligand genes are created
by annealing 200 picomoles each of a pair of complementary
synthetic oligonucleotides in 5 microliters of ligation
buffer (Boehringer-Mannheim #1243 292). Each linker, which
has flankins ~coRI and AflIII sites, is ligated overnight
with a 3.7 Kbp EcoRI/sstXI fragment from a form of c-mpl
ligand with amino acids 1-153 (step 2), and a 1 Kbp
NcoI/BstXI fragment from the either of the two types of
clones in step 2 above, one with and one without a deletion
of amino acids 112-115. The resulting DNA is used to
transform E. coli DH5~~ cells. Transformed cells are selected
on LB agar plates containing ampicillin (100 ug~ml). Plasmid
DNA is obtained from single colonies of several clones and
sequenced to verify correct assembly of the dimer via the
linker. The resulting plasmid DNA template can be used in
making novel c-mpl ligand molecules via the Horlick method
(Prot. Eng. 5:427-433, 1992).
B. Horlick Method
5~ sense ana 3' anti-sense primers are combined with a dimer
template for PCR using reagents from Boehringer Mannheim
(Indianapolis, IN) or Perkin-Elmer (Norwalk, CT) as described
by the manufacturersusing Taq polymerase. The PCR reactions
are subjected to 30 cycles of the following; 1 min. @ 94~C, 1
CA 022340~9 1998-04-06
W O 97/12978 PCTAUS96/15938
44
min. @ 58~C, 90 sec. ~ 72~C. The product (about 480 base
pairs) is visualized using a short-wave W light source. The
reactions are purified using a Wizard~ PCR Preps kit from
Promega (Madison, WI). Briefly, the PCR reactions are added
to 100 microliters of Direct Purification buffer, and 1
milliliter (mL) of PCR Preps DNA Purification Resin is added
to this mixture. After 1 minute incubation at 24~C, the
supernatant is removed by vacuum filtration through a
filtration column. Two mLs of 80% isopropanol is used to wash
the resin via vacuum filtration. The column containing the
resin is then subjected to centrifugation at 10,000 x g for
30 seconds to remove residual isopropanol. The PCR product
is eluted with 50 microliters of 10 mM Tris-Cl, 1 mM EDTA,
pH7.4, via centrifugation at 10,000 x g for 30 seconds
followed by transfer of supernatant to a new tube.
Subcloning novel c-mpl receptor agonists into expression
vectors
The novel c-mpl receptor agonists PCR products are digested
with the appropriate restriction enzymes for ligation to
either mammalian or E. col i expression vectors.
Mammalian Expression Vectors
The mammalian expression vectors are derivatives of pMON3359
which is a pUC18-based vector containing a m~mm~l ian
expression cassette. The cassette includes a herpes simplex
viral promoter IE110 (-800 to +120), an IL-3 signal peptide
se~uence and a SV40 late poly-adenylation (poly-A) signal
which has been subcloned into the pUC18 polylinker [See
Hippenmeyer et al., Bio/Technology, 1993, pp.1037-1041].
Restriction enzyme digestions are incubated for 1 hour at
37~C as described by the manufacturer prior to
electrophoresis through a 1% agarose/lx TsE/EtBr gel.
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/15938
Fragments ar~ first visualized by long-wave W and gel-
purified using a Qiaex DNA Extraction kit (Qiagen,
Chatsworth, CA). The DNA fragments are purified from the
resin by agarose solublization, addition of a DNA-binding
resin, and extensive washing of the resin prior to elution
with water. The purified DNA products are combined at a molar
excess of PCR product to vector and the ligation reactions
are carried out according to the manufacturer~s recommended
conditions for T4 DNA ligase.
E. coli Expression Vectors
The E. coli expression vectors that direct high-level
production o~ heterologous proteins in the cytoplasm are
derivatives of that described elsewhere (Olins et al.,
Methods Enzym., 185:115-119, 1988 and Rangwala et al.,
Gene, 122: 2~3-269, 1992). The expression cassette
consists of the recA promoter and T7 gene 10 ribosome binding
site (RBS) as well as an Ml3 origin of replication or a
tandem inverted repeat of a phage P22 gene which acts as a
transcription terminator. These cassettes are on a plasmid
with the pBR327 origin of replication and encode a gene
either for spectinomycin or ampicillin resistance.
Transformation of E. coli strains
E. coli strains DH5~~ (Life Technologies, Gaithersburg,
MD) and TG1 (Amersham Corp., Arlington Heights, IL) are used
for all transformation of ligation reactions and are the
source of plasmid DNA for transfecting m~mm~l ian cells. E.
coli strain MON105 can be obtained from the American Type
Culture Col~ection (ATCC, Rockville, MD) and is the host for
expressing alternate forms of c-mpl ligand in the cytoplasm
or periplasmic space, respectively, of E. coli.
MON105 ATCC~55204: F-, lamda-,IN(rrnD, rrE)1, rpoD+, rpoH358
CA 022340~9 1998-04-06
W O 97/12978 PCTAJS96/15938
46
DH5a~: F-, phi80dlacZdeltaM15, delta~lacZYA-argF)U169, deoR,
recA1, endA1, hsdR17(rk-,mk+), phoA, supE441amda-, thi-1,
gyrA96, relA1
TG1: delta(lac-pro), supE, thi-1, hsdD5/F'(traD36, proA+B+,
lacIq, lacZdeltaM15)
DH5~ Subcloning efficiency cells are purchased as
competent cells and are ready for transformation using the
manufacturer's protocol, while both E. coli strains TG1 and
MON105 are rendered competent to take up DNA using a CaC12
method. Typically, 20 to 50 mLs of cells are grown in Ls
medium (1% bacto-tryptone, 0.5% bacto-yeast extract, 150
millimolar NaCl) to a density of approximately 1.0 optical
density units at 600 nanometers (OD600) as measured by a
saush & Lomb Spectronic spectrophotometer (Rochester, NY).
The cells are collected by centrifugation and resuspended in
one-fifth culture volume of CaC12 solution (50 millimolar
CaC12, 10 millimolar Tris-Cl, pH7.4) and are held at 4 C for
30 minutes. The cells are again collected by centrifugation
and resuspended in one tenth culture volume of CaC12
solution. Ligated DNA is added to 0.2 mL of these cells, and
the samples are held at 4 C for 1 hour. The samples are
shifted to 42 C for two minutes and 1.0 mL of Ls is added
prior to shaking the samples at 37 C for one hour. Cells
from these samples are spread on plates (Ls medium plus 1.5%
bacto-agar) containing either ampicillin (100 micrograms/mL,
ug/mL) when selecting for ampicillin-resistant transformants,
or spectinomycin (75 ug/mL) when selecting for spectinomycin-
resistant transformants. The plates are incubated overnightat 37 C. Single colonies are picked, grown in LB
supplemented with appropriate antibiotic for 6-16 hours at
37 C with shaking.
Colonies are picked and inoculated into LB plus
appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL
CA 022340~9 1998-04-06
W O 97/12978 PCTrUS96/15938
. 47
spectinomycin) and are grown at 37~C while shaking. Before
harvesting the cultures, 1 ul of cells are analyzed by PCR
for the presence of a c-mpl ligand gene. The PCR is carried
out using a combination of primers that anneal to the c-mpl
ligand gene and/or vector. After the PCR is complete,
loading dye is added to the sample followed by
electrophoresis as described earlier. A gene has been
ligated to the vector when a PCR product of the expected size
is observed.
DNA isol~tion and characterization
Plasmid DNA is isolated using the Promega Wizard~
Miniprep kit (Madison, WI) or the Qiagen QIAwell Plasmid
isolation kits (Chatsworth, CA). Both kits follow the same
general procedure for plasmid DNA isolation. Briefly, cells
are pelleted by centrifugation (5000 x g), plasmid DNA
released with sequential NaOH & acid treatment, and cellular
debris is removed by centrifugation (10000 x g). The
supernatant (containing the plasmid DNA) is loaded onto a
column containing a DNA-binding resin, the column is washed,
and plasmid DNA eluted with TE. After screening for the
colonies with the plasmid of interest, the E. coli cells are
inoculated into 100 mls of LB plus appropriate antibiotic for
overnight growth at 37~C in an air incubator while shaking.
Plasmid DNA is isolated using the Qiagen Plasmid Midi kit
(Chatsworth, CA) which is a scaled-up version of the Qiagen
QIAwell Plasmid isolation kit described earlier. The DNA is
used for DNA sequencing, further restriction enzyme
digestion, additional subcloning of DNA fragments and
transfection into m~mm~l ian or E. coli cells.
Purified recombinant double-stranded DNA is sequenced
using the Applied Biosystems Inc. (ABI, Foster City, CA)
PRISM~ Ready Reaction DyeDeoxy~ Terminator Sequencing
system. The ABI system relies on incorporation of four
fluorescence labelled dideoxy nucleotides into single-
CA 022340~9 1998-04-06
W O 97/12978 PCTAUS96/15938
. 48
stranded DNA during multiple rounds of amplification.
Plasmid DNA and a sequencing primer are added to the reaction
mixture (including Taq DNA polmerase, buffer and
nucleotides), which is subjected to 25 cycles of
S amplification (30 seconds at 96~C, 15 seconds at 50~C, 4
minutes at 60~C). Following amplification, unincorporated
nucleotides are removed using Centri-Sep spin columns
(equilibrated in water) as described by Princeton
Separations, Inc. (Adelphia, NJ). Briefly, the samples are
loaded onto a column that has excess water removed by
centrifugation (700 x g) for 2 minutes, and the purified
sequencing product is eluted by centrifugation (700 x g) for
4 minutes. The samples are then dried down in a Speed Vac
(Savant, Hicksville, NY) prior to addition of loading
solution. The samples are electrophoresed through a 4.75%
polyacrylamide sequencing gel containing 7M urea in lX TsE at
70 watts constant power. The ABI system uses a detector that
recognizes each differntially labelled PCR product as they
are being subjected to electrophoresis.
Pro~uction of novel c-mDl rece~tor a~onists
Mammalian Cell Transfection/Production of Conditioned Media
The BHK-21 cell line can be obtained from the ATCC
(Rockville, MD). The cells are cultured in Dulbecco's
modified Eagle media (DMEM/high-glucose), supplemented to 2
millimolar (mM) L-glutamine and 10% fetal bovine serum (EBS).
This formulation is designated BHK growth media.- Selective
media is BHK growth media supplemented with 453 units/ml
hygromycin B (Calbiochem, San Diego, CA). The sHK-21 cell
line was previously stably transfected with the HSV
transactivating protein VP16, which transactlvates the IE110
promoter found on the plasmid pMON3359 (Hippenmeyer et
al., Bio/Technology, 1037-1041, 1993). The VP16 protein
drives expression of genes inserted behind the IE110
CA 022340~9 1998-04-06
WO97/12978 PCT~S96/15938
49
promoter. BHK-21 cells expressing the transactivating protein
VP16 is designated BHK-VP16. The plasmid pMON1118 (Highkin et
al., Poultry Sci., 70:970-981, 1991)
expresses the hygromycin resistance gene from the SV40
promoter. A similar plasmid is available from ATCC, pSV2-
hph.
BHK-VP16 cells are seeded into a 60 millimeter (mm)
tissue culture dish at 3 X 105 cells per dish 24 hours prior
to transfection. Cells are transfected for 16 hours in 3 mL
of '1OPTIMEM'VTM (Gibco-sRL, Gaithersburg, MD) containing 10 ug
of plasmid DNA containing the gene of interest, 3 ug
hygromycin resistance plasmid, pMON1118, and 80 ug of Gibco-
sRL ~LIPOFECT~MINE~ per dish. The media is subse~uently
aspirated and replaced with 3 mL of growth media. At 48 hours
post-transfection, media from each dish is collected and
assayed for activity (transient conditioned media). The cells
are removed from the dish by trypsin-EDTA, diluted 1:10 and
transferred to 100 mm tissue culture dishes containing 10 mL
of selective media. After approximately 7 days in selective
media, resistant cells grow into colonies several millimeters
in diameter. The colonies are removed from the dish with
filter paper (cut to approximately the same size as the
colonies and soaked in trypsin/EDTA) and transferred to
individual wells of a 24 well plate containing 1 mL of
selective media. After the clones are grown to confluency,
the conditioned media is reassayed, and positive clones are
expanded into growth media.
Fxnression and ~urification of recombinant ~rotein from ~.
coli
E. coli strain MON105 harboring the plasmid of interest
are grown at 37~C in M9 plus casamino acids medium with
shaking in a air incubator Model G25 from New Brunswick
Scientific (Edison, New Jersey). Growth is monitored at
CA 022340~9 1998-04-06
W O 97/12978 PCTAUS96/lS938
oD600 until it reaches a value of 1.0 at which time Nalidixic
acid (10 milligrams/mL) in 0.1 N NaOH is added to a final
concentration of 50 ~g/mL. The cultures are then shaken at
37~C for thr-e to four additional hours. A high degree of
aeration is maintained throughout culture period in order to
achieve maximal production of the desired gene product. The
cells are examined under a light microscope for the presence
of inclusion bodies (IB). One mL ali~uots of the culture are
removed for analysis of protein content by boiling the
pelleted cells, treating them with reducing buffer and
electrophoresis via SDS-PAGE (Maniatis et al. Molecular
Cloning: A Laboratory Manual, 1982). After centrifugation
(5000 x g) to pellet the cells, the first step in
purification of the protein is either sonication or
homogenization of the cells. For sonication, the cells are
resuspended in one-tenth volume (based on culture size)
sonication buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA). These
resuspended cells are subjected to several repeated
sonication blrsts using the microtip from a Sonicator cell
disrupter, Model W-375 obtained from Heat Systems-Ultrasonics
Inc. (Farmingdale, New York). The extent of sonication is
monitored by e~mi~ing the homogenates under a light
microscope. After all of the cells are disrupted, the
homogenates are fractionated by centrifugation at 10000 x g
for 20 minutes at 4~C in a JA-20 rotor and J2-21 centrifuge
(Beckman, Fullerton, CA). Alternatively, the IBs are
released from the cells by lysing the cells in sonication
buffer with a Manton-Gaulin homogenizer (Holland) followed by
centrifugation as above. The IB pellets, which are highly
enriched for the recombinant protein, are then subjected to
another round of sonication and centrifugation as described
above. The recombinant protein is purified by a variety of
standard methods. The most common methods involve
solublization of the IBs with 4-6 molar urea or guanidine-HCl
buffers at pH 9-12, and air oxidation/folding in the presence
CA 022340~9 1998-04-06
W O 97/12978 PCTrUS96/15938
51
of cataytic concentrations of cysteine, beta-mercaptoethanol
or dithiothreitol for 24 to 72 hours. The protein is
purified from E. coli cont~min~nts using ion-exhange
chomotography, such as Q-sepharose (anion) and S-sepharose
(cation), gel filtration, hydrophobic chromatography or
reversed phase HPLC. After dialysis against a low ionic
strength buffer, the purified protein is stored frozen or
lyophilized.
Withou~ further elaboration, it is believed that one
skilled in the art can, using the preceding description,
utilize the present invention to its fullest extent. The
following preferred specific embodiments are, therefore, to
be construed as merely illustrative, and not limitative of
the remainder of the disclosure in any way whatsoever.
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 co-filed United
States Patent Applications WO 94/12639, WO 94/12638, WO
95/20976, WO 95/21197, WO 95/20977, WO 95/21254, and US
Serial No. 08/383,035 which are hereby incorporated by
reference ir. their entirety.
Further details known to those skilled in the art may be
found in T. Maniatis, et al., Molecular Clonina, A T~horatorv
M~nu~l, Cold Spring Harbor Laboratory (1982) and references
; 30 cited therein, incorporated herein by reference; and in J.
Sambrook, et al., Molecul~r Clonina, A TaboratorY Manl~l, 2nd
edition, Cold Spring Harbor Laboratory (1989) and references
cited therein, incorporated herein by reference.
CA 02234059 1998-04-06
W O 97/12978 PCTrUS96/15938
- 52
All references, patents or applications cited herein are
incorporated by re~erence in their entirety.
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/15938
. 53
TARR~ 1 OTIGONUCT.~OTIDF..S
c-mplNcoI ACGTCCATGGCNTCNCCNGCNCCNCCTGCTTGTGCACTCCGAGTC
(SEQ ID NO:4)
N=A,C,G or T
Ecompl ATGCACGAATTCCCTGACGCAGAGGGTGGA (SEQ ID NO:5)
c-mplHindIII TGACAAGCTTACCTGACGCAGAGGGTGGACCCT
(SEQ ID NO:6)
4L-5' AATTCGGCAA (SEQ ID NO:7)
15 4L-3' CATGTTGCCG (SEQ ID NO:8)
5L-5' AATTCGGCGGCAA (SEQ ID NO:9)
5L-3' CATGTTGCCGCCG (SEQ ID NO:10)
8L-5' AATTCGGCGGCAACGGCGGCAA (SEQ ID NO:11)
8L-3' CATGTTGCCGCCGTTGCCGCCG (SEQ ID NO:12)
25 31-5' CGATCCATGGAGGTTCACCCTTTGCCT (SEQ ID NO:13)
31-3' GATCAAGCTTATGGGCACTGGCTCAGTCT (SEQ ID NO:14)
35-5' CGATACATGTTGCCTACACCTGTCCTG (SEQ ID NO:15)
35-3' GATCAAGCTTAAGGGTGAACCTCTGGGCA (SEQ ID NO:16)
39-5' CGATCCATGGTCCTGCTGCCTGCTGTG (SEQ ID NO:17)
35 39-3' GATCAAGCTTAAGGTGTAGGCAAAGGGTG (SEQ ID NO:18)
43-5' CGATCCATGGCTGTGGACTTTAGCTTGGGA (SEQ ID NO:19)
43-3' GATCAAGCTTAAGGCAGCAGGACAGGTGT (SEQ ID No:20)
45-5' CGATCCATGGACTTTAGCTTGGGAGAA (SEQ ID NO:21)
45-3' GATCAAGCTTACACAGCAGGCAGCAGGAC (SEQ ID NO:22)
45 49-5' CGATCCATGGGAGAATGGAAAACCCAG (SEQ ID NO:23)
49-3' GATCAAGCTTACAAGCTAAAGTCCACAGC (SEQ ID NO:24)
82-5' CGATCCATGGGACCCACTTGCCTCTCA (SEQ ID NO:25)
82-3' GATCAAGCTTACAGTTGTCCCCGTGCTGC (SEQ ID NO:26)
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/15938
. 54
109-5' CAGTCCATGGGAACCCAGCTTCCTCCA ~SEQ ID NO:27)
109-3' GATCAAGCTTAAAGGAGGCTCTGCAGGGC (SEQ ID NO:28)
116-5' CGATCCATGGGCAGGACCACAGCTCAC (SEQ ID No:29)
116-3' GATCAAGCTTACTGTGGAGGAAGCTGGGTT (SEQ ID NO:30)
120-5' CGATCCATGGCTCACAAGGATCCCAATGCC (SEQ ID NO:31)
lZ0-3' GATCAAGCTTATGTGGTCCTGCCCTGTGG (SEQ ID NO:32)
123-5' CGATCCATGGATCCCAATGCCATCTTCCTG (SEQ ID NO:33)
15 123-3' GATCAAGCTTACTTGTGAGCTGTGGTCCT (SEQ ID NO:34)
126-5' CGATCCATGGCCATCTTCCTGAGCTTCCAA (SEQ ID NO:35)
126-3' GATCAAGCTTAATTGGGATCCTTGTGAGCTGT (SEQ ID
NO:36)
CA 022340~9 1998-04-06
W O 97/12978 PCTrUS96/15938
TARRF 2
'~ GF.l~F. S EouF~l~c F~ c;
PMON26458
TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAG~AGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC (SEQ ID NO:37);
PMON28548
TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGC
TCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCAC
AGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTG
TGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCT
GGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT
TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGC
AGAGCCTCCTTGGAACCCAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCT
GAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACC
CTCTGCGTCAGG (SEQ ID NO:38);
PMON32132
TCTCCCGCTCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO:82)
PMON32133
TCTCCCGCTCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
CA 022340~9 1998-04-06
W O 97/lZ978 PCT~US96/15938
56
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGC-TCCTTGGAACCCAGGGCAGGACCACAGCTCACAAGGATCCCAATGCC
ATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAG
GGTCCACCCTCTGCGTCAGG (SEQ ID NO:83)
pMON28500
TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCAACATGGCGTCTCCCGCTCC
GCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC
AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGG
ACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGG
AGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC
CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGA
GCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGC
CATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGA
GGGTCCACCCTCTGCGTCAGG (SEQ ID NO:39);
PMON28501
TCCCCAGCTCCACCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGC
TCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCAC
AGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTG
TGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCT
GGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT
TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGC
AGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAA
TGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA
GGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO:40);
PMON32136
TCCCCAGCGCCgCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATG
TCCTTCACAGCA-,ACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCT
GCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGG
GACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGG
GGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGA
CA 022340~9 1998-04-06
W O 97/12978 PCTAUS96/15938
57
TGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACGGCGGCAACATGGC
GTCCCCAGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCAT
GTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGC
TGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACA
GGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG
GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTG
GGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAA
GGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG
ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGG (SEQ ID NO:41);
PMON30373
GAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAA
TGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTG
CTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTG
GGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACC
CAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGC
TTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTC
TGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGA
GTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCA
(SEQ ID NO:61);
PMON30374
TTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAG
ATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTG
ATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCT
GGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCA
CAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTG
CTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAA
TTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAA
CTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCT
(SEQ ID NO:62);
PMoN30375
GTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACC
AAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGG
GGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGT
CTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACC
ACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAG
GTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAAC
ATGGCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGAC
TCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCT
(SEQ ID NO:63);
PMON30376
GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC
ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA
CA 022340~9 1998-04-06
W O 97/12978 PCTrUS96/15938
. 58
CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG
GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG
ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG
GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT
CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT
(SEQ ID NO:64);
PMON30377
GACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTG
GGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACT
TGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTG
CAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCC
AATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTT
GTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCG
CCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGC
AGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTG
(SEQ ID NO:65);
PMON30378
GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC
CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC
CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT
GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC
CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC
ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCNTCTCCGGCGCCGCCTGCTTGTGAC
CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG
TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG
(SEQ ID NO:66);
PMoN30379
GGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTT
GGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCAC
AAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTC
CTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCT
CCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTC
CTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTG
CCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAG
GACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTG
(SEQ ID NO:6/);
PMON30380
GGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTC
CTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCC
ACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCGGCGCCGCCTGCTTGTGAC
CTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAG
TGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTG
CA 022340~9 1998-04-06
WO97/12978 PCT~S96/15938
. 59
GGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACC
CTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCC
CTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTT
(SEQ ID NO:68);
PMON3 0 3 81
GGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTC
CGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTC
10 GGCGGCAACATG'J-GTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTG
CTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTG
CCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATG
GAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATG
GCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGA
CAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAG
(SEQ ID NO:69)
PMON30382
GCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTG
CGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATG
GCGTCTCCGGCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCC
CATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTC
CTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAG
GCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGA
CAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTC
CTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACA
(SEQ ID NO:70);
30 PMON30383
GATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTG
ATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCG
GCGCCGCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTT
3 5 CACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCT
GCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGAC
ATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGA
CCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGG
GCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAG
40 (SEQ ID NO:71);
PMON30384
GCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTA
GGAGGGTCCACCCTCTGCGTCAGGGAATTCGGCGGCAACATGGCGTCTCCCGCTCCGCCT
GCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGA
CTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGAC
TTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGA
GCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGC
CTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAG
AGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAAT
(SEQ ID NO:72).
CA 022340~9 l998-04-06
W O 97/12978 PCT~US96/15938
TARRF 3
PROTF.TN SF.OUF.~C~.s
PMON26458pep
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis
ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu
LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla
GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln
LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu
LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla
HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg
PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPhe (SEQ ID No:42);
PMON28548pep
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis
ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu
LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla
GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyvalMetAlaAlaArgGlyGln
LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu
LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla
HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg
PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyGlyAsnMetAla
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis
ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu
LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla
GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln
LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu
LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnGlyArgThrThrAlaHisLysAspPro
AsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArgPheLeuMetLeu
ValGlyGlySerThrLeuCysValArg (SEQ ID NO:43);
PMON32132
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis
ValLeuHisSe~ArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu
LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla
GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln
LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu
LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla
HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg
PheLeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID No : 44);
PMON32133
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis
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~ 61
ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu
LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla
GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln
LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu
LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnGlyArgThrThrAlaHisLysAspPro
AsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArgPheLeuMetLeu
ValGlyGlySerThrLeuCysValArg (SEQ ID NO:45);
PMON28500
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis
ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu
LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla
GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln
LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu
LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla
HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg
PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyAsnMetAlaSer
ProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHisVal
LeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeuLeu
ProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAlaGln
AspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGlnLeu
GlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeuLeu
GlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAlaHis
LysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArgPhe
LeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:46);
PMON28501
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis
ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu
LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla
GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln
LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu
LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla
HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg
PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyGlyAsnMetAla
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis
ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu
LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla
GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln
LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu
LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla
HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg
PheLeuMetLeuValGlyGlySerThrLeuCysValArg (SEQ ID NO:47);
pMON32136
SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSerHis
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ValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThrProValLeu
LeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGluThrLysAla
GlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAlaArgGlyGln
LeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnValArgLeuLeu
LeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLeuProProGlnGlyArgThrThrAla
HisLysAspProAsnAlaIlePheLeuSerPheGlnHisLeuLeuArgGlyLysValArg
PheLeuMetLeuValGlyGlySerThrLeuCysValArgGluPheGlyGlyAsnGlyGly
AsnMetAlaSerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArg
AspSerHisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProThr
ProValLeuLeuProAlaValAspPheSerLeuGlyGluTrpLysThrGlnMetGluGlu
ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAlaAla
ArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGlyGlnVal
ArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnLel;ProProGlnGlyArg
ThrThrAlaHisLysAspproAsnAlaIlepheLeuserpheGlnHisLeuLeuArgGly
LysValArgPheLeuMetLeuValGlyGlySerThrLeuCysValArg
(SEQ ID NO:48);
pMON30373
GluValHisProLeuProThrProValLeuLeuProAlaValAspPheSerLeuGlyGlu
TrpLysThrGlnMetGluGluThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeu
LeuGluGlyValMetAlaAlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeu
GlyGlnLeuSerGlyGlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThr
GlnLeuproproGlnGlyArgThrThrAlaHisLysAspproAsnAlaIlepheLeuser
PheGlnHisLeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeu
CysValArgGluPheGlyGlyAsnMetAlaSerProAlaProProAlaCysAspLeuArg
ValLeuSerLysLeuLeuArgAspSerHisValLeuHisSerArgLeuSerGlnCysPro
(SEQ ID NO:49);
pMON31074
Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly
Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly
Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser
Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly
Thr Gln Leu .ro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro
Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val
Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu
Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu
Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser
Arg Leu Ser Gln Cys Pro Glu Val His Pro (SEQ ID NO:50);
pMON30375
Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr
Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr
Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro
Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg
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. 63
Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro
Pro Gln Gly Arg Thr Thr Ala HiS Lys Asp Pro Asn Ala Ile Phe
Leu Ser Phe Gln HiS Leu Leu Arg Gly Lys Val Arg Phe Leu Met
Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn
5 Met Ala Ser -ro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser
Lys Leu Leu Arg Asp Ser His Val Leu HiS Ser Arg Leu Ser Gln
Cys Pro Glu Val His Pro Leu Pro Thr Pro ( SEQ ID NO:51);
,.
10 pMON30376
Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu
Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu
Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser
15 Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly
Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg
Thr Thr Ala HiS Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln
HiS Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly
Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro
20 Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg
Asp Ser His Val Leu HiS Ser Arg Leu Ser Gln Cys Pro Glu Val
His Pro Leu Pro Thr Pro Val Leu Leu Pro (SEQ ID No:52);
25 pMON30377
Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys
Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val
Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu
3 0 Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu
Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr
Ala HiS Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln HiS Leu
Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr
Leu Cys val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro
3 5 Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser
His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val HiS Pro
Leu Pro Thr Pro Val Leu Leu Pro Ala Val ( SEQ ID NO: 53 );
40 pMON30378
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile
Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg
Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu
r 45 Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu
Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala HiS Lys Asp
Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys
- Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg
Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp
50 Leu Arg Val: eu Ser Lys Leu Leu Arg Asp Ser HiS Val Leu HiS
Ser Arg Leu Ser Gln Cys Pro Glu Val HiS Pro Leu Pro Thr Pro
Val Leu Leu Pro Ala Val Asp Phe Ser Leu ( SEQ ID NO: 5 4 );
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64
pMON30379
5 Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln
Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala
Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe
Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly
10 Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val
Leu Ser Lys Leu Leu Arg Asp Ser HiS Val Leu His Ser Arg Leu
Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu
Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu
Glu Thr Lys ~la Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu
15 Glu Gly Val Met Ala Ala Arg Gly Gln Leu (SEQ ID NO:55);
pMON30380
20 Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp
Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys
Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg
Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp
Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His
25 Ser Arg Leu Ser Gln Cys Pro Glu Val HiS Pro Leu Pro Thr Pro
Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr
Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr
Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro
Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg
30 Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu ( SEQ ID NO : 56);
pMON30381
35 Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser
Phe Gln HiS Leu Leu Arg Gly Lys val Arg Phe Leu Met Leu Val
Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu
Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro
40 Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp
Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala
Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met
Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu
Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln
~5 Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln ( SEQ ID NO : 57);
pMON30382
50 Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu
Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr
Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro
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Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser
His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro
Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly
Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly
Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser
Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly
Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr (SEQ ID No:58);
pMON30383
Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln HiS Leu Leu Arg Gly
Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val
Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys
Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu
His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr
Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys
Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val
Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly
Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val
Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu
Pro Pro Gln Gly Arg Thr Thr Ala His Lys (SEQ ID NO:59);
pMON30384
Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg
Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe
Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg
Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg
Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu
Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met
Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu
Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys
Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu
Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln
Gly Arg Thr Thr Ala His Lys Asp Pro Asn (SEQ ID No:6o);
The following examples will illustrate the invention in
greater detail although it will be understood that the
invention is not limited to these speci~ic examples.
F~AMPT.F~
Construction of the parental plasmid cont~; n; ng the first
gene of the dimer template
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In order to generate a plasmid DNA with the coding
sequence of c-mpl (1-153) ligand followed by a unique EcoRI
restriction site, the gene is isolated via reverse
transcriptase/polymerase chain reaction (RT/PCR). Human
fetal (lot ~38130) and adult liver (lot #46018) A+ RNA are
obtained from Clontech (Palo Alto, CA) for source of c-mpl
ligand messager RNA (mRNA). The first strand cDNA reactions
are carried out using a cDNA Cycle~ Kit obtained from
Invitrogen (San Diego, CA). In the RT reaction, random
primers and oligo dT primer are used to generate cDNA from a
combination of human and fetal liver mRNA. For amplification
of c-mpl ligand gene fragment encoding amino acids 1-153, the
RT product serves as the template for PCR with a combination
of the primers, Forward primer: c-mplNcoI (SEQ ID NO:4) and
Reverse primer: Ecompl (SEQ ID NO:5). The c-mplNcoI (SEQ ID
NO:4) primer anneals to the c-mpl ligand gene (bases #279-311
based on c-mpl ligand se~uence from Genebank accession
#L33410 or de Sauvage et al., Nature 369:533-538, 1994) and
encodes a NcoI restriction enzyme site just 5' to the first
mature codon (Serl). The NcoI restriction enzyme site codes
for methionine and alanine codons prior to ser-l and includes
codon degeneracy for the Ala codon and the first four c-mpl
ligand codons (Serl, Pro2, Ala3, & Pro4). The Ecompl (SEQ ID
NO:5) primer ~nneals to bases #720-737 of c-mpl ligand and
encodes a EcoRI recognition site in-frame with the c-mpl
ligand gene immediately following Argl53. The EcoRI site
creates Glul54 and Phel55 codons following Argl53. The ca.
480 bp PCR product is purified, digested with NcoI and EcoRI
and ligated to the NcoI-EcoRI vector fragment of pMON3993
(ca. 4550 bp.). pMON3993 is a derivative of pMON3359. The
expression cassette in pMON3359 includes a herpes simplex
viral promoter IE110 (-800 to +120), an IL-3 signal peptide
se~uence and a SV40 late poly-adenylation (poly-A) signal
which has been subcloned into the pUC18 polylinker (See
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Hippenmeyer et al., Bio/Technology, 1037-1041, 1993). The
human IL-3 signal peptide sequence, which had been subloned
as a BamHI f.~gment into the unique BamHI site between the
110 promoter and poly-A signal, contains an NcoI site at
its 3' end and is then followed by a unique EcoRI site. The
DNA sequence of the signal peptide is shown below
(restriction enzyme sites are indicated above). The ATG
(methionine) codon within the NcoI site is in-frame with the
initiator ATG of the signal peptide (underlined);
samHI
GGATCCACC~AGCCGCCTGCCCGTCCTGCTCCTGCTCCAACTCCTGGTCCGCCCCGC
NcoI
CATGG (SEQ ID NO: 81)
pMON26458, coding for c-mpl ligand amino acids 1-153, is a
result of this cloning.
F.~iMPT,F. 2
Construction of the parental plasmids containing the second
genes of the dimer templates
For amplification of c-mpl ligand gene fragments
starting at amino acid 1 (Ser) with a termination codon
following amino acid 153 (Arg), the RT reaction from Example
1 serves as the template for PCR with a combination of the
following primers; c-mplNcoI (SEQ ID NO:4) (forward primer)
and c-mplH;n~TII (SEQ ID NO:6) (reverse primer). The c-
mplNcoI (SEQ ID NO:4) primer is described in Example 1. The
c-mplHindIII (SEQ ID NO:6) primer, which an~als to bases
~716-737 of c-mpl ligand, adds both a termination codon and a
~;n~TTI restriction enzyme site immediately following the
final codon, Argl53.
Two types of PCR products are generated from the RT cDNA
samples, one with a deletion of the codons for amino acids
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112-115 and one without the deletion of these codons. The c-
mpl ligand PCR products (ca. 480 bp) are digested with NcoI
and HindIII restriction enzymes for transfer to a mammalian
expression vector, pMON3934, which is a derivative of
pMON3359 (see Example 1). pMoN3934 is digested with NcoI and
HindIII (ca. 3800 bp) and will accept the PCR products.
The plasmid, pMON32132, contians the DNA se~uence of (SEQ ID
NO:82) which encodes for amino acids 1-153 of c-mpl ligand
(SEQ ID NO:~) was a result of this cloning. The plasmid,
pMON32133, contains the DNA sequence of (SEQ ID NO:83) which
encodes amino acids 1-153 of c-mpl ligand with a deletion of
codons 112-115 (~112-115) (SEQ ID NO:45) was also a result of
this cloning.
EXAMPT .F. 3
Generation of PCR 5L dimer template containing ~112-115 in
the secondlgene
A PCR template for generating novel forms of c-mpl ligand is
constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of
pMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32133
(containing a deletion of amino acids 112-115) along with the
EcoRI/AflIII 5L synthetic oligonucleotide linker 5L-5' (SEQ
ID NO:9) and 5L-3' (SEQ ID NO:10).
The EcoRI end of the linker will ligate to the EcoRI end of
pMON26458. The AflIII end of the linker will ligate to the
NcoI site of pMON32133, and neither restriction site will be
retained upon ligation. The BstXI sites of pMON26458 and
pMON32133 will ligate as well. Plasmid, pMON28548, is a
result of the cloning and contains the DNA se~uence of (SEQ
ID NO:38) which encodes amino acids 1-153 c-mpl ligand fused
via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker to amino
acids 1-153 c-mpl ligand that contains a deletion of amino
acids 112-115 (SEQ ID NO:43).
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F.XAMPT.F. 4
Generation of PCR 4L dimer template pMON28500
A PCR template for generating novel forms of c-mpl ligand is
constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of
pMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132
along with the EcoRI/AflIII 4L synthetic oligonucleotide
linker 4L-5~ (SEQ ID NO:7) and 4L-3~ (SEQ ID NO:8).
The EcoRI end of the linker will ligate to the EcoRI end of
pMoN26458. The AflIII end of the linker will ligate to the
NcoI site of pMON32132, and neither restriction site will be
retained upon ligation. The BstXI sites of-pMON26458 and
pMON32132 will ligate as well. Plasmid, pMON28500 is a
result of the cloning and contains the DNA sequence of (SEQ
ID NO:39) which encodes amino acids 1-153 c-mpl ligand fused
via a GluPheGlyAsnMetAla (SEQ ID NO:77) linker (4L) to amino
acids 1-153 c-mpl ligand (SEQ ID NO:46).
F.X.j~[PT.F. 5
Generation of PCR 5L dimer template pMON28501
A PCR template for generating novel forms of c-mpl ligand is
constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of
pMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132
along with the EcoRI/AflIII 5L synthetic oligonucleotide
linker 5L-5' (SEQ ID NO:9) and 5L-3' (SEQ ID NO:10).
The EcoRI end of the linker will ligate to the EcoRI end of
pMON26458. The AflIII end of the linker will ligate to the
NcoI site of pMON32132, and neither restriction site will be
retained upon ligation. The BstXI sites of pMoN26458 and
pMON32132 will ligate as well. PLasmid, pMON28501 is a result
of the cloning and contains the DNA se~uence of (SEQ ID NO:
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~ 70
40) which encodes amino acids 1-153 c-mpl ligand fused via a
GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker (5L) to amino
acids 1-153 c-mpl ligand (SEQ ID No:47)~
F.X~MPI.F. 6
Generation of PCR 8L dimer template pMON32136
A PCR template for generating novel forms of c-mpl
ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI
fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from
pMON32132 along with the EcoRI/AflIII 8L synthetic
oligonucleotide linker 8L-5~ (SEQ ID NO:ll) and 8L-3' (SEQ ID
NO:12).
The EcoRI end of the linker will ligate to the EcoRI end of
pMON26458. The AflIII end of the linker will ligate to the
NcoI site of pMON32132, and neither restriction site will be
retained upon ligation. The BstXI sites of pMON26458 and
pMON32132 will ligate as well. Plasmid, pMON32136 is a
result of the cloning which contains the DNA seauence of
(SEQ ID NO:41) and encodes amino acids 1-153 c-mpl ligand
fused via a GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79)
linker (8L) _o amino acids 1-153 c-mpl ligand (SEQ ID No:48).
F.XA~PT, F..S 7--18
Generation of novel c-mpl receptor agonists
A. PCR aener~tion of novel c-mnl rece~tor aaonists.
Novel c-mpl ligand genes are generated using the Horlick
method. The PCR reaction was carried out using dimer
template pMON28501 and one of the sets of synthetic primer
sets below (number refers to first amino acid of new
molecule).
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71
31-5' (SEQ ID NO:13) and 31-3' (SEQ ID NO:14), 35-5' (SEQ ID
NO:15) and 35-3' (SEQ ID NO:16), 39-5~ (SEQ ID NO:17) and 39-
- 3' (SEQ ID NO:18), 43-5' (SEQ ID NO:19) and 43-3' (SEQ ID
NO:20), 45-5' (SEQ ID NO:21) and 45-3' (SEQ ID NO:22), 49-5'
(SEQ ID NO:23) and 49-3' (SEQ ID NO:24), 82-5~ (SEQ ID NO:25)
and 82-3' (SEQ ID NO:26), 109-5' (SEQ ID NO:27) and 109-3'
(SEQ ID NO:28), 116-5' (SEQ ID NO:29) and 116-3' (SEQ ID
NO:30) ,120-5' (SEQ ID NO:31) and 120-3~ (SEQ ID NO:32), 123-
5' (SEQ ID NO:33) and 123-3~ (SEQ ID NO:34), 126-5~ (SEQ ID
NO:35) and 126-3' (SEQ ID NO:36)
.
The products that are generated are about 480 bp and are
purified via Magic PCR Clean up kits (Promega). Table 4 shows
the template, the primer set used in the PCR reaction and the
breakpoint for each Example.
B. Sl~hclonin~ of novel c-mnl receDtor a~onists into m~mm~ n
ex~ression vector
The c-mpl receptor agonist PCR products are digested with
NcoI and HindIII or AflIII and HindIII restriction enzymes
(ca. 470 bp) for transfer to a mammalian expression vector.
The expression vector, pMON3934, is digested with NcoI and
HindIII (ca. 3800 bp) and accepts the PCR products as NcoI-
HindIII or AflIII-HindIII fragments. Table 4 shows the
restriction digest of the PCR product and the resulting
expression plasmid pMON designation.
CA 022340~9 l998-04-06
W O 97/12978 PCTAUS96/15938
72
TABLE 4
Example PCR PCR PCR Product Resulting
# template Primer Breakpoint Linker Restriction Plasmid
~et Dige~t pMON
7 pMON28501 31 30-31 5LNcoI/HindIII pMON30373
8 pMON28501 35 34-35 5LAflIII/HindIII pMON30374
9 pMON28501 39 38-39 5LNcoI/HindIII pMON30375
pMON2;501 43 42-43 5LNcoI/HindIII pMoN3o376
11 pMON28501 45 44-45 5LNcoI/HindIII pMON30377
12 pMON28501 49 48-49 SLNcoI/HindIII pMON30378
13 pMON28501 82 81-82 SLNcoI/HindIII pMON30379
14 pMON28501 109 108-109 SLNcoI/HindIII pMON30380
lS pMON28501 116 115-116 5LNcoI/HindIII pMON30381
16 pMON28501 120 119-120 5LNcoI/HindIII pMoN30382
17 pMON28501 123 122-123 5LNcoI/HindIII pMON30383
18 pMON28501 126 125-126 SLNcoI/HindIII pMON30384
In a similar manner the dimer templates pMON28500,
pMON32136 and pMON28548 could be used in the PCR reaction as
described in Examples 7-18.
Bioactivity determination of c-mpl receptor agonits
Transfected cell lines: Cell lines such as BafJ3 cell line
can be transfected with a colony stimulating factor receptor,
such as the human IL-3 receptor or human c-mpl receptor,
which the cell line does not normally 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
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/15938
73
cells were transfected with the plasmid via electroporation.
The cells were grown under G418 selection in the presence of
- mouse IL-3 in Wehi conditioned media. Clones were established
through limited dilution.
The BHK expression levels and bioactivity data of some
of the c-mpl receptor agonists of the present invention are
shown in Table 5. The supernatant from the transfected BHK
cells was evaluated for expression of the c-mpl receptor
agonists by western analysis using an antobody raised against
c-mpl ligand. constructs that expressed at a ~++++~ level
were assayed in the Baf-3/c-mpl cell proliferation assay.
TABLE 5
Expres~ on and Bioactivity of c-mpl rece~tor aqonist
pMON# c-mpl receptor BHK expression Baf-3/c-mpl
agonist levels cell
prolife-ation
p~O~ / - C + n~
p V!O~ "_ _ / ,J / _ _ ~ . + n~
pvo\ __ / ~ n~
p~o~ ~ - / L/ -~ + n~
p~ON ' ~ -_ / _/ -~ ++ ne
p~ON ' ~-_ / _/ -~. ++ ne
pMON '~ - . + ne
pMON _ -. / L/ -_ ++++ +
pMON~ -_ / L/_-__ ++++ +
pMON _0-: / L/:- + nd
pMON _ _ - 3-_ / L/~ + nd
2. Bone marrow proliferation assay
a. CD34+ Cell Purification:
Between 15-20 mL bone marrow aspirates are obtained from
normal allogeneic marrow donors after informed consent. Cells
are diluted 1:3 in phosphate buffered saline (PBS, Gibco-
BRL), 30 mL are layered over 15 mL Histopa~ue-1077 (Sigma)
and centrifuged for 30 minutes at 300 RCF. The mononuclear
interface layer is collected and washed in PBS. CD34+ cells
CA 022340~9 1998-04-06
W O 97/12978 PCTrUS96/15938
. 74
are enriched from the mononuclear cell preparation using an
affinity column per manufacturers instructions (CellPro, Inc,
Bothell WA). After enrichment, the purity of CD34+ cells is
70% on average as determined by using flow cytometric
analysis using anti CD3A monoclonal antibody conjugated to
fluorescein and anti-CD38 conjugated to phycoerythrin
(Becton Dickinson, San Jose CA).
Cells are resuspended at 40,000 cells/mL in X-Vivo 10
media (Bio-Whittaker, Walkersville, MD) and 1 mL is plated in
12-well tissue culture plates (Costar). Human IL-3 variant,
pMON13288, is used at 10 ng/mL or 100 ng/mL. Conditioned
media from BHK cells transfected with plasmid encoding c-mpl
ligand are tested by addition of 100 ~l of supernatant added
to 1 mL cultures (approximately a 10% dilution). Cells are
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 is
obtained for each condition. For fluorescence analysis and
ploidy determination cells, are washed in megakaryocyte
buffer (MK buffer, 13.6 mM Sodium Citrate, 1 mM Theophylline,
2.2 ~m PGEl, 11 mM Glucose, 3% w/v BSA, in PBS, pH 7.4,)
(Tomer et al., Blood 70(6): 1735-42 [1987]) resuspended in
500 ~l of MK buffer cont~;n-ng anti-CD41a FITC antibody
(1:200, AMAC, Westbrook, ME) and washed in MK buffer. For
DNA analysis cells are made permeable in MK buffer containing
0.5% Tween 20 (Fisher, Fair Lawn NJ) for 20 minutes on ice
followed by fixation in 0.5% Tween-20 and 1% paraformaldehyde
(Fisher Chemical) for 30 minutes followed by incubation in
Propidium Iodide (Calbiochem , La Jolla Ca) (50 ~g/mL) with
RNAase (400 U/mL) in 55% v/v MK buffer (200 mOsm) for 1-2
hours on ice. Cells are analyzed on a FACScan or Vantage
flow cytometer (Becton Dickinson, San Jose, CA). Green
fluorescence (CD41a-FITC) is collected along with linear and
log signals for red fluorescence (PI) to determine DNA
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/15938
ploidy. All cells are collected to determine the percent of
cells that are CD41+. Data analysis is performed using LYSIS
- software (Becton Dickinson, San Jose, CA). Percent of cells
expressing the CD41 antigen is obtained from flow cytometry
analysis (Percent). Absolute (Abs) number of CD41+ cells/mL
is calculated by: (Abs)=(Cell Count)*(Percent)/100.
3. Megakaryocyte fibrin clot assay.
CD34+ enriched population are isolated as described
above. Cells are suspended at 25,000 cellsJmL with/without
cytokine(s) in a media consisting of a base Iscoves IMDM
media supplemented with 0.3% BSA, 0.4 mg/mL apo-transferrin,
6.67~M FeCl2, 25~g/mL CaCl2, 25 ~g/mL L asparagine, 500 ~g/mL
E-amino-n-caproic acid and Penicillin/Streptomycin. Prior to
plating into 35 mm plates, thrombin is added (0.25 units/mL)
to initiate ~-lot formation. Cells are incubated at 37~C for
13 days at 5% C02 in a 37~C humidified incubator. At the end
of the culture period plates are fixed with methanol:acetone
(1:3), air dried and stored at -200C until st~ining. A
peroxidase immunocytochemistry staining procedure is used
(Zymed, Histostain-SP. San Francisco, CA) using a cocktail of
primary monoclonal antibodies consisting of anti CD41a, CD42
and CD61. Colonies are 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).
Various other examples will be apparent to the person
skilled in t:l- 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.
CA 022340~9 l998-04-06
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76
~QuAb~Ah. LISTING
(1) GENERAL INFORNATION:
(i) APPLICANT:
(A) NAME: G. D. Searle
(B) STREET: P.O. Box 5110
(C) CITY: Chicago
(D) STATE: Illinois
(E) COUNAA~Y: United States of America
(F) POSTAL CODE (ZIP): 60680
(G) TELEPHONE: (708) 470-6501
(H) TELEFAAY: (708) 470-6881
(A) NAME: Mon~Anto Company
(B) STREET: 88 North Lindbergh Boulevard
(C) CITY: St. Louis
(D) STATE: Missouri
(E) COUNAA~Y: United States of America
(F) POSTAL CODE (ZIP): 63167
(G) TELEPHONE: (314) 694-3131
(H) TELEFAAY: (314) 694-5435
(ii) TITLE OF lNv~N~ oN: c-mpl ligand Receptor Agonists
(iii) NUMLER OF ~Ab.yuAb~S: 83
(iv) C~ UAA~;K A~EADABLE FOA~A
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Vercion #1.30 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMA~ER: US C-2908
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/004,824
(B) FILING DATE: 05-OCT-1995
(2) INFORNATION FOR SEQ ID NO: 1:
( i ) ~Ab~YUA~N~A~ CHARACTERISTICS:
(A) LENGTH: 332 amino acids
(B) TYPE: amino acid
(C) STR~-N~ N~ S: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Modified-site
CA 022340~9 1998-04-06
W O 97/12978 PCTAUS96/15938
(B) LOCATION:112
(D) OTHER INFORMATION:/note= "position 112 is deleted or
Leu, Ala, Val, Ile, Pro, Phe, Trp, or N..."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:113
(D) OTHER INFORMATION:/note= "position 113 is deleted or
Pro, Phe, Ala, Leu, Ile, Trp, or Met"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:114
(D) OTHER INFORMATION:/note= "position 114 is deleted or
Pro, Phe, Ala, Val, Leu, Ile, Trp or Met~
(ix) FEATURE:
(A) NANE/KEY: Modified-site
(B) LOCATION:115
(D) OTHER INFORMATION:/note= "position 115 is deleted or
Gln, Gly, Ser, Thr, Tyr or Asn"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Xaa
100 105 110
Xaa Xaa Xaa Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
115 120 125
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Net Leu
130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala
145 150 155 160
CA 022340~9 l998-04-06
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78
Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn
165 170 175
Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr
180 185 190
Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile
195 200 205
Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly
210 215 220
Tyr Leu Asn Arg Ile His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe
225 230 235 240
Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly
245 250 255
Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser
260 265 270
Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu
275 280 285
Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro
290 295 300
Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr
305 310 315 320
Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly
325 330
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 153 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:112
(D) OTHER INFORMATION:/note= ''position 112 is deleted or
Leu, Ala,VAl, Ile, Pro, Phe, Trp or Met"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:113
CA 022340~9 l998-04-06
W O 97/12978 PCTAJS96/15938
79
(D) OTHER INFORMATION:/note= "positoin 113 is deleted or
Pro, Phe, Ala, Val, Leu, Ile, Trp or Met"
_ (ix) FEATURE:
tA) NAME/KEY: Modified-site
(B) LOCATION:114
(D) OTHER INFORMATION:/note= "position 114 is deleted or
Pro, Phe, Ala, Val, Leu, Ile, Trp or Met"
(ix) FEATURE:
(A) NAME/REY: Modified-site
(B) LOCATION:115
(D) OTHER INFORMATION:/note= "positon 115 is deleted or
Gln, Gly, Ser, Thr, Tyr, or Asn"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Xaa
100 105 110
Xaa Xaa Xaa Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
115 120 125
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu
130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg
145 150
(2) INFORMATION FOR SEQ ID NO: 3:
(i) ~:QU~NL~ CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nN~.~s: unknown
CA 022340~9 1998-04-06
WO 97/12978 PCT~US96/15938
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) ~Q~ DESCRIPTION: SEQ ID NO: 3:
Gly Gly Gly Ser
(2) INFORMATION FOR SEQ ID NO: 4:
(i) ~:QU~N~'~ CHARACTERISTICS:
(A) LENGTH: 4S base pairs
(B) TYPE: nucleic acid
.(C) STR~ S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) n
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
ACGTCCATGG ~Nl-N-~N~C N~NC~l~CT TGTGCACTCC GAGTC
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)~
(xi) ~QU~N~: DESCRIPTION: SEQ ID NO: 5:
ATG~AC~-AA~ TCCCTGACGC AGAGGGTGGA
(2) INFORMATION FOR SEQ ID NO: 6:
(i) ~QU~N~r: CHARACTERISTICS:
(A) LENGTH: 33 base pairs
CA 022340~9 1998-04-06
W O 97/12978 PCT~US96/1~938
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) ~:yU~:N~ DESCRIPTION: SEQ ID NO: 6:
TGACAAGCTT ACCTGACGCA GAGGGTGGAC CCT
33
(2) INFORMATION FOR SEQ ID NO: 7:
~N~ CHARACTERISTICS:
(A) LENGTH: l0 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = ~DNA (synthetic)"
(xi) ~:Qu~ DESCRIPTION: SEQ ID NO: 7:
AATTCGGCAA
(2) INFORMATION FOR SEQ ID NO: 8:
(i) ~:~u~N~ CHARACTERISTICS:
(A) LENGTH: l0 base pairs
(B) TYPE: nucleic acid
(C) sTRA~n~nN~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = ~DNA (synthetic)~
(xi) ~Qu~N~ DESCRIPTION: SEQ ID NO: 8:
CATGTTGCCG
(2) INFORMATION FOR SEQ ID NO: 9:
CA 022340~9 l998-04-06
WO 97/12978 PCTAJS96/15938
82
(i) ~:Q~N~ CHARACTERISTICS:
(A) LENGTH: 13 base pairs
~B) TYPE: nucleic acid
( C ) STR A NnEnNF~ C: S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
(xi) s~Q~N~: DESCRIPTION: SEQ ID NO: 9:
AAll~CGGCGG CAA
13
(2) INFORMATION FOR SEQ ID NO: lO:
(i) ~-QU~:N~ CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic) n
(xi) ~Q~ DESCRIPTION: SEQ ID NO: 10:
CA~ ~CCG CCG
13
(2) INFORMATION FOR SEQ ID NO: 11:
(i) ~UU~N~ CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRAN~N~:SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
(xi) s~Qu~: DESCRIPTION: SEQ ID NO: 11:
CA 022340~9 1998-04-06
W 0 97/12978 PCT~US96/15938
AATTCGGCGG CAACGGCGGC AA
22
_ (2) INFORMATION FOR SEQ ID NO: 12:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C~ STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CATGTTGCCG CCGTTGCCGC CG
22
(2) INFORMATION FOR SEQ ID NO: 13:
Q~N~ CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: other nucleic acid
(A) D~-CCRTPTION: /desc = ~DNA (synthetic)~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CGATCCATGG AGGTTCACCC TTTGCCT
27
(2) INFORMATION FOR SEQ ID NO: 14:
(i) ~Ou~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
CA 022340~9 l998-04-06
WO 97/l2978 PCT~US96/15938
84
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GATCAAGCTT ATGGGCACTG GCTCAGTCT
29
(2) INFORMATION FOR SEQ ID NO: 15:
( i ) ~QU N~ CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(Xi) ~U~N~'~ DESCRIPTION: SEQ ID NO: 15:
CGATACATGT TGCCTACACC l~lC~lG
27
(2) INFORMATION FOR SEQ ID NO: 16:
(i) s~Qu~N~r: CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANn~n~FCS single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = ~DNA (synthetic)"
(Xi) S~QU N~ DESCRIPTION: SEQ ID NO: 16:
CGATCCATGG TCCTGCTGCC ~lG~l~lG
27
(2) INFORMATION FOR SEQ ID NO: 17:
(i) ~QU~:N~ CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
CA 022340~9 1998-04-06
W O 97/12978 PCTAUS96/15938
(Xi) ~hUU~N~: DESCRIPTION: SEQ ID NO: 17:
CGATCCATGG ~l~C~l~G~l~GCC ~l~G~l~ G
27
(2) INFORMATION FOR SEQ ID NO: 18:
( i ) S~UU N~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GATCAAGCTT AAGGTGTAGG CAAAGGGTG
29
t2) INFORMATION FOR SEQ ID NO: 19:
QU~N~ CHARACTERISTICS:
(A) LENGTH: 3û base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic) n
(xi) ~UU~N~ DESCRIPTION: SEQ ID NO: 19:
CGATCCATGG CTGTGGACTT TAG~l~lGGGA
(2) INFORMATION FOR SEQ ID NO: 20:
( i ) ~QU~N - ~: CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 022340~9 1998-04-06
WO 97/12978 PCTAJS96/15938
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) ~:Qu~: DESCRIPTION: SEQ ID NO: 20:
GATCAAGCTT AAGGCAGCAG GACAGGTGT
29
(2) INFORMATION FOR SEQ ID NO: 2l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CGATCCATGG ACTTTAGCTT GGGAGAA
27
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GATCAAGCTT ACACAGCAGG CAGCAGGAC
29
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
CA 022340~9 1998-04-06
W O 97/12978 PCTrUS96/15938
87
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CGATCCATGG GAGAATGGAA AACCCAG
27
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B~ TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
GATCAAGCTT ACAAGCTAAA GTCCACAGC
29
(2) INFORMATION FOR SEQ ID NO: 25:
(i) ~Q~N~: CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) ~:Q~N~ DESCRIPTION: SEQ ID NO: 25:
CGATCCATGG GACCCACTTG C~l~l ~A
27
(2) INFORMATION FOR SEQ ID NO: 26:
CA 022340~9 1998-04-06
WO 97/12978 PCTrUS96/15938
(i) ~hQu~N~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(Xi) ~hQ~ DESCRIPTION: SEQ ID NO: 26:
GATCAAGCTT ACAGTTGTCC CC~lG~lGC
29
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
GATCAAGCTT AAAGGAGGCT CTGCAGGGC
29
(2) INFORMATION FOR SEQ ID NO: 28:
(i) ~Ou~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: s.ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
(xi) s~Q~N~ DESCRIPTION: SEQ ID NO: 28:
CA 022340~9 1998-04-06
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89
GATCAAGCTT AAAGGAGGCT CTGCAGGGC
29
- (2) INFORMATION FOR SEQ ID NO: 29:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
CGATCCATGG GCAGGACCAC AGCTCAC
27
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = ~DNA (synthetic)~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
GATCAAGCTT ACTGTGGAGG AAGCTGGGTT
(2) INFORMATION FOR SEQ ID NO: 3l:
(i) ~u~:~ CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetis)"
CA 022340~9 l998-04-06
WO 97/12978 PCT~US96/15938
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
GATCAAGCTT ACTGTGGAGG AAG~l~G~
(2) INFORMATION FOR SEQ ID NO: 32:
(i) ~Q~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) shQu~N~ DESCRIPTION: SEQ ID NO: 32:
GATCAAGCTT ATGTGGTCCT GCC~s~l~GG
29
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (syntheitc)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
CGATCCATGG ATCCCAATGC CAl~llC~
(2) INFORMATION FOR SEQ ID NO: 34:
(i) ~QU~l~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
CA 022340~9 1998-04-06
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91
(xi) ~QU~ : DESCRIPTION: SEQ ID NO: 34:
GATCAAGCTT ACTTGTGAGC ~l~l~lC~
29
(2) INFORMATION FOR SEQ ID NO: 35:
(i) ~QU~ CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
CGATCCATGG CCA~ C~l~ GAGCTTCCAA
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
(xi) ~uu~ DESCRIPTION: SEQ ID NO: 36:
GATCAAGCTT AATTGGGATC L~lu~lGAGCT GT
32
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 022340~9 1998-04-06
WO 97/12978 PCT~us96/15938
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AA~lG~-llCG TGACTCCCAT
C~ ACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG
120
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA
180
CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA
240
CTGGGACCCA CTTGCCTCTC A~l~CCC~l~C~l~ GGGCAGCTTT CTGGACAGGT CC~l~-l~C~l~C
300
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAG~llC~lC CACAGGGCAG GACCACAGCT
360
CACAAGGATC CCAATGCCAT ~~ lGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT
420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTC
465
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 927 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic)"
(xi) ~Q~ DESCRIPTION: SEQ ID NO: 38:
TCCC~CCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AA~ ~G TGACTCCCAT
~ ACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC AC~
120
CA 022340~9 l998-04-06
WO 97/12978 PCTAJS96/15938
CTGC~ ~ TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA
180
CAGGACATTC TGGGAGCAGT GA~C~ll~lG CTGGAGGGAG TGATGGCAGC ACGGGGACAA
240
CTGGGACCCA ~llGCC~ lC A~l~CC~lC'~lG GGGCAGCTTT CTGGACAGGT CC~l~ C~l~C300
~Nl~GGGCCC TGCAGAGCCT CCTTGGAACC CAG~llC~lC CACAGGGCAG GACCACAGCT
360
CACAAGGATC CCAATGCCAT ~llC~lGAGC TTCCAACACC TGCTCCGAGG AAAG~l~GC~
420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCGG CAACATGGCG
480
l~lCCCG~lC CGC~l~-~l~ TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT
540
~lC~llCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC ~ GC~l~AC ACCTGTCCTG
600
CTG~l~lG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA
660
CAGGACATTC TGGGAGCAGT GACC~ll~l~ CTGGAGGGAG TGATGGCAGC ACGGGGACAA
720
CTGGGACCCA Cll~C-l~l~ AlCC~CuG GGGCAGCTTT CTGGACAGGT CC~ L ~1 C~ . C
780
~ll~GGGCCC TGCAGAGCCT CCTTGGAACC CAGGGCAGGA CCACAGCTCA CAAGGATCCC
840
AATGCCATCT TCCTGAGCTT CCAACACCTG CTCCGAGGAA AG~l~GC~ CCTGATGCTT
900
GTAGGAGGGT CCACC~l~lG CGTCAGG
927
~2) INFORMATION FOR SEQ ID NO 39
~QU~N~ CHARACTERISTICS
tA) LENGTH 936 base pairs
(B) TYPE nucleic acid
(C) STRAN~ ~SS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = nDNA (synthetic)"
CA 022340~9 1998-04-06
WO 97/12978 PCTAUS96/15938
94
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
TCCCCAGCTC CACCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT
G~ lCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC AC~ lC~lG
120
CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA
180
CAGGACATTC TGGGAGCAGT GACC~ G CTGGAGGGAG TGATGGCAGC ACGGGGACAA
240
CTGGGACCCA CTTGCCTCTC ATCC~l~C~l~G GGGCAGCTTT CTGGACAGGT CCGTCTCCTC
300
~ll~GGGC~C TGCAGAGCCT CCTTGGAACC CAG~llC~lC CACAGGGCAG GACCACAGCT
360
CACAAGGATC CCAATGCCAT ~l~l~Cl~AGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT
420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCAA CATGGCGTCT
480
CCCG~lCCGC CTGCTTGTGA CCTCCGAGTC CTCAGTAAAC lG~ll~C~lGA CTCCCATGTC
540
CTTCACAGCA GACTGAGCCA GTGCCCAGAG GTTCACCCTT TGCCTACACC l~lC~-lGCTG
600
C~-l~l~lGG ACTTTAGCTT GGGAGAATGG AAAACCCAGA TGGAGGAGAC CAAGGCACAG
660
GACATTCTGG GAGCAGTGAC C~ll~lGCTG GAGGGAGTGA TGGCAGCACG GGGACAACTG
720
GGACCCACTT GCCTCTCATC CCTCCTGGGG CAG~lll~lG GACAGGTCCG 'l~lC~lC~
780
GGGGCC~lGC AGAGCCTCCT TGGAACCCAG ~ll~ lCCAC AGGGCAGGAC CACAGCTCAC
840
AAGGATCCCA ATGCCATCTT CCTGAGCTTC CAACACCTGC TCCGAGGAAA GGTGCGTTTC
900
CTGATGCTTG TAGGAGGGTC CACC~-~l~lGC GTCAGG
936
(2) INFORMATION FOR SEQ ID NO: 40:
CA 022340~9 1998-04-06
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 939 base pairs
- (B) TYPE: nucleic acid
(C) STR~Nn~nNFss: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) ~Qu~N~ DESCRIPTION: SEQ ID NO: 40:
TCCCCAGCTC CAC~l~G~ G TGACCTCCGA GTCCTCAGTA AA~lG~luC~ TGACTCCCAT
C~ll~CACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC AC~l~iC~
120
GC~l~CTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA
180
CAGGACATTC TGGGAGCAGT GACC~~ lG CTGGAGGGAG TGATGGCAGC ACGGGGACAA
240
CTGGGACCCA CTTGCCTCTC Al~CC~lC~l~ GGGCAGCTTT CTGGACAGGT ~ ~lC~lC
300
~ GGGCCC TGCAGAGCCT CCTTGGAACC CAG~ll-CC~C CACAGGGCAG GACCACAGCT
360
CACAAGGATC CCAATGCCAT ~ll~C'lGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT
420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AATTCGGCGG CAACATGGCG
480
TCTCCCGCTC CGCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT
540
C~ ACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC AC~ C~l~G
600
GC~l~GCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA
660
CAGGACATTC TGGGAGCAGT GACC~lc~l~G CTGGAGGGAG TGATGGCAGC ACGGGGACAA
720
CTGGGACCCA CTTGCCTCTC A~l~CC~lC~-~l~G GGGCAGCTTT CTGGACAGGT CC~l~l-~lC
780
CA 022340~9 l998-04-06
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96
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT
840
CACAAGGATC CCAATGCCAT ~l~ C~lGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT
900
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG
939
(2) INFORMATION FOR SEQ ID NO 41
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 948 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = nDNA (synthetic)"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 41
TCCCCAGCGC CGC~lG~ G TGACCTCCGA GTCCTCAGTA AA ~l~G~l~lCG TGACTCCCAT
~ LlcAcA GCAGACTGAG CCAGTGCCCA GAGGTTCACC ~-lllGC~lAC A~-~l~lC~-lG
120
~lGC~l~l~ TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA
180
CAGGACATTC TGGGAGCAGT GACCCll~lG CTGGAGGGAG TGATGGCAGC ACGGGGACAA
240
CTGGGACCCA CTTGCCTCTC ATCC~ G GGGCAGCTTT CTGGACAGGT CC~l~lC~lC
300
~ GGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT
360
CACAAGGATC CCAATGCCAT ~ C~lGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT
420
ll~l~ATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGG AA~ CGGCGG CAACGGCGGC
480
AACATGGCGT CCCCAGCGCC ~C~l~G~ GACCTCCGAG TCCTCAGTAA ACTGCTTCGT
540
GACTCCCATG ~l~C~ CACAG CAGACTGAGC CAGTGCCCAG AGGTTCACCC TTTGCCTACA
600
CA 022340~9 1998-04-06
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C~l~lC~l~C TGCCTGCTGT GGACTTTAGC TTGGGAGAAT GGAAAACCCA GATGGAGGAG
660
ACCAAGGCAC AGGACATTCT GGGAGCAGTG ACCCTTCTGC TGGAGGGAGT GATGGCAGCA
720
CGGGGACAAC TGGGACCCAC TTGCCTCTCA TCCCTCCTGG GGCAGCTTTC TGGACAGGTC
780
C~ C~lCC ~ GGGGCCCT GCAGAGCCTC CTTGGAACCC AG~lC~lCC ACAGGGCAGG
840
~ CTC ACAAGGATCC CAATGCCATC TTCCTGAGCT TCCAACACCT GCTCCGAGGA
900
AAG~l~C~ TCCTGATGCT TGTAGGAGGG TCCACCCTCT GCGTCAGG
948
(2) INFORMATION FOR SEQ ID NO: 42:
(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: l55 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) s~:~u~--~ DESCRIPTION: SEQ ID NO: 42:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Ar~ Val Leu Ser Lys Leu Leu
l S l0 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
= Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu
l00 105 ll0
CA 022340~9 l998-04-06
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98
Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
115 120 125
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu
130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe
145 150 155
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) ~:Qu~ DESCRIPTION: SEQ ID NO: 43:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu
100 105 110
Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
115 12û 125
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu
130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala
145 150 155 160
CA 022340~9 1998-04-06
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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 Ary Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Gly
260 265 270
Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln
275 280 285
His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser
290 295 300
Thr Leu Cys Val Arg
305
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 153 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) ~OLECULE TYPE: protein
r (xi) ~Q~N~: DESCRIPTION: SEQ ID NO: 44:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
CA 022340~9 1998-04-06
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100
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu
100 105 110
Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
115 120 125
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu
130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg
145 150
(2) INFORMATION FOR SEQ ID NO: 45:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 149 amino acids
(B) TYPE: amino acid
(C) STRA-N~N~:SS: llnknl, "
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
CA 022340~9 l998-04-06
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101
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Gly
100 105 110
Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln
115 120 125
His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser
130 135 140
Thr Leu Cys Val Arg
lg5
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 312 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) S~Q~ ~ DESCRIPTION: SEQ ID NO: 46:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His V~l Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
- 85 90 95
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu
100 105 110
Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
115 120 125
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu
130 135 140
CA 022340~9 l998-04-06
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Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Asn Met Ala Ser
145 150 15S 160
Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg
165 170 17S
Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His
180 18S 190
Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly
l9S 200 20S
Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly
210 21S 220
Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu
22S 230 23S 240
Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val
24S 2S0 255
Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro
260 26S 270
Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu
27S 280 28S
Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Lqu Val
290 29S 300
Gly Gly Ser Thr Leu Cys Val Arg
30S 310
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 313 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
2S 30
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His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
~eu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
~al Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu
100 105 110
Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
115 120 125
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu
130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala
145 150 155 160
~er Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
165 170 175
~rg 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
~eu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
245 250 255
~al 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
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(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 316 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
1 5 10 15
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu
100 105 110
Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
115 120 125
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu
130 135 140
Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Gly Gly
145 150 155 160
Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser
165 170 175
Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys
180 185 190
Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp
195 200 205
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Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln
210 215 220
Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala
225 230 235 240
Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu
245 250 255
Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly
260 265 270
Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn
275 280 285
Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe
290 295 300
Leu Net Leu Val Gly Gly Ser Thr Leu Cys Val Arg
305 310 315
(2) INFORNATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(Xi ) ~hUUhN~h DESCRIPTION: SEQ ID NO: 49:
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe
1 5 10 15
Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp
Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Net Ala Ala Arg
- 35 40 45
Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser
Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr
Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala
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106
~le Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu
100 105 110
Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn
115 120 125
Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys
130 135 140
Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro
145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRAWDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) ~UUk~: DESCRIPTION: SEQ ID NO: 50:
Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu
1 5 10 15
Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala
Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly
Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg
Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro
Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser
Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly
100 105 110
Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro
115 120 125
Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp
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130 135 140
Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro
- 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 51:
(i) ~Q~ ~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln
1 5 10 15
Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu
Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu
Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly
Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr
Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu
Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu
lOo 105 llO
Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala
115 120 125
Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu
130 135 140
His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro
145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 52:
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Qu~N~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr
l 5 10 15
Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val
Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu
Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser
Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys
Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys
Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu
100 105 110
Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg
115 120 125
Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu
130 135 140
Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro
145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 53:
~Qu~-~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
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- (xi) ~UU~N~ DESCRIPTION: SEQ ID NO: 53:
Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala
1 5 10 15
Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala
Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln
Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu
Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro
Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg
Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly
100 105 110
Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu
115 120 125
Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln
130 135 140
Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val
145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 54:
(i) ~uu~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B~ TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unkn~wn
(ii) MOLECULE TYPE: protein
(xi) ~Qu~N~ DESCRIPTION: SEQ ID NO: 54:
Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu
l 5 10 15
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~ly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln
Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu
Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu
Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala
100 105 110
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu
115 120 125
Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
130 13S 140
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 55:
(i) ~QukNC~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) ~u~ DESCRIPTION: SEQ ID NO: 55:
Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val
Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro
Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu
Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val
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Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser
Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg
Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His
100 105 110
Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly
115 120 125
Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly
130 135 140
Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu
145 150 155 160
(2~ INFORMATION FOR SEQ ID NO: 56:
(i) ~QU~N~: CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STR~NnF~nN~.'::S: llnkn~ ."
(D) TOPOLOGY: llnkn( ...1
(ii) MOLECULE TYPE: protein
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO: 56:
Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro
S 10 15
Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg
Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly
- Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu
Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln
Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val
Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala
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100 105 110
Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala
115 120 125
Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln
130 135 140
Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu
145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 57:
(i) ~hyu~N~: CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRA~n~nNF.~S: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) 8~QU~N~: DESCRIPTION: SEQ ID NO: 57:
Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe
1 5 10 15
Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly
Ser Thr Leu Cys Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala
Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser
His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu
Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp
Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val
100 105 110
Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro
115 120 125
Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu
130 135 140
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Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln
145 150 155 160
-
(2) INFORMATION FOR SEQ ID NO: 58:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY ~lnkn. .,
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu
1 5 10 15
Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys
Val Arg Glu Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys
Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His
Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val
Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met
Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu
100 105 110
Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser
115 120 125
Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala
130 135 140
Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr
145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 59:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids
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(B) TYPE: amino acid
(C) STRAN~E~N~SS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) ~:QU~N~: DESCRIPTION: SEQ ID NO: 59:
Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys
1 5 10 15
Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Ary Glu
Phe Gly Gly Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg
Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu
Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro
Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr
Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val
100 105 110
Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu
115 120 125
Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser
130 135 140
Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys
145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 60:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRAN~N~SS: unknown
tD) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
-
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(xi) ~;uu~ ; DESCRIPTION: SEQ ID NO: 60:
Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe
Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe Gly Gly
Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser
Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys
Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp
Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln
Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala
100 105 110
Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu
115 120 125
Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly
130 135 140
Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn
145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 61:
Qul~N~ ; CHARACTERISTICS:
( A ) LENGTH: 480 bas e pairs
(B) TYPE: nucleic acid
( C ) sTRp~l~nFnNF:~s s ingle
( D ) TOPOLOGY: 1 inear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic) "
(xi ) ~;uu~ ; DESCRIPTION: SEQ ID NO: 61:
GAGGTTCACC ~ GC~ L~AC AC~: l ~ l C~: l G ~:~l GC~l~G~; l G TGGACTTTAG CTTGGGAGAA
CA 022340~9 1998-04-06
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ll6
TGGAAAACCC AGATGGAGGA GACCAAGGCA CAGGACATTC TGGGAGCAGT GACC~~ G
120
CTGGAGGGAG TGATGGCAGC ACGGGGACAA CTGGGACCCA ~~ GC~l~lC AlCC~lC~lG
180
GGGCAGCTTT CTGGACAGGT CC~~ C CTTGGGGCCC TGCAGAGCCT CCTTGGAACC
240
CA~ l~lC CACAGGGCAG GACCACAGCT CACAAGGATC CCAATGCCAT ~llC~lGAGC
300
TTCCAACACC TGCTCCGAGG AAAG~l~l TTCCTGATGC TTGTAGGAGG GTCCACCCTC
360
TGCGTCAGGG AA~l~lCGGCGG CAACATGGCG l~lC'C~GCGC CGCCTGCTTG TGACCTCCGA
420
GTCCTCAGTA AACTGCTTCG TGACTCCCAT ~l~C~lCACA GCAGACTGAG CCAGTGCCCA
480
(2) INFOR~ATION FOR SEQ ID NO 62
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 480 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = rDNA (Synthetic)~
(xi) ~Q~N~ DESCRIPTION SEQ ID NO 62
TTGCCTACAC ~ C~lGCT GC~lG~l~sG GACTTTAGCT TGGGAGAATG GAAAACCCAG
ATGGAGGAGA CCAAGGCACA GGACATTCTG GGAGCAGTGA CC~ G~l~ GGAGGGAGTG
120
ATGGCAGCAC GGGGACAACT GGGACCCACT TGCCTCTCAT CC~lC~l~GGG GCAG~
180
GGACAGGTCC ~ CCl~C~-l~ TGGGGCCCTG CAGAGCCTCC TTGGAACCCA G~ C~l~CCA
240
CAGGGCAGGA CCACAGCTCA CAAGGATCCC AATGCCATCT TCCTGAGCTT CCAACACCTG
300
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CTCCGAGGAA AG~lGC~l~ CCTGATGCTT GTAGGAGGGT CCACC~l--lG CGTCAGGGAA
360
- TTCGGCGGCA ACAlGGC~lC lCCGGCGCCG CCTGCTTGTG AC~l~CCGAGT CCTCAGTAAA
420
C~lG ACTCCCATGT CCTTCACAGC AGACTGAGCC AGTGCCCAGA GGTTCACCCT
480
(2) INFORMATION FOR SEQ ID NO 63
(i) ~Qu~ CHARACTERISTICS
(A) LENGTH 480 base pairs
(B) TYPE nucleic acid
(C) STRANn~nN~ss single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = "DNA (synthetic) n
(xi) SEQUENCE DESCRIPTION SEQ ID NO 63
~ C ~lG~l~lGGA CTTTAGCTTG GGAGAATGGA AAACCCAGAT GGAGGAGACC
AAGGCACAGG ACA~ l~GG AGCAGTGACC ~11~1~1GG AGGGAGTGAT GGCAGCACGG
120
GGACAACTGG GACCCACTTG CCTCTCATCC ~lC~GGGGC AG~111~-lGG ACAGGTCCGT
180
C~lC~l-l~ GGGCC~lGCA GAGC~l~ll GGAACCCAGC llC~lCCACA GGGCAGGACC
240
ACAGCTCACA AGGATCCCAA TGCCATCTTC CTGAGCTTCC AACACCTGCT CCGAGGAAAG
300
~ C~lllCC TGATGCTTGT AGGAGGGTCC ACC~ GCG TCAGGGAATT CGGCGGCAAC
360
ATGGCGTCTC CGGCGCCGCC ~l~G~ll~l~AC CTCCGAGTCC TCAGTAAACT G~ll~C~l~GAC
420
TCCCATGTCC TTCACAGCAG ACTGAGCCAG TGCCCAGAGG TTCACCCTTT GCCTACACCT
480
(2) INFORMATION FOR SEQ ID NO 64
( i ) sh~U~N~: CHARACTERISTICS
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(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANnFnN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
GCTGTGGACT TTAGCTTGGG AGAATGGAAA ACCCAGATGG AGGAGACCAA GGCACAGGAC
ATTCTGGGAG CAGTGACCCT ~ lGGAG GGAGTGATGG CAGCACGGGG ACAACTGGGA
120
CCCACTTGCC TCTCATCCCT C~l~GGGCAG ~ GGAC AGGlCC~l~l~ C~lC~-l~lGGG
180
GCCCTGCAGA GC~lC~ll~G AACCCAGCTT CCTCCACAGG GCAGGACCAC AGCTCACAAG
240
GATCCCAATG CCATCTTCCT GAGCTTCCAA CACCTGCTCC GAGGAAAGGT GCGTTTCCTG
300
AlG~l~l~lAG GAGGGTCCAC C~~ GC~l~C AGGGAATTCG GCGGCAACAT GGC~l~lCCG
360
GCGCCGC~lG CTTGTGACCT CCGAGTCCTC AGTAAACTGC TTCGTGACTC CCAl~lC~
420
CACAGCAGAC TGAGCCAGTG CCCAGAGGTT CACC~l~llGC CTACACCTGT CCTGCTGCCT
480
(2) INFOR~ATION FOR SEQ ID NO: 65:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic) n
(xi) ~OU~N~ DESCRIPTION: SEQ ID NO: 65:
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119
GACTTTAGCT TGGGAGAATG GAAAACCCAG ATGGAGGAGA CCAAGGCACA GGACATTCTG
GGAGCAGTGA CC~ lGCT GGAGGGAGTG ATGGCAGCAC GGGGACAACT GGGACCCACT
120
GC~ -'AT CC~l~C~l~GGG GCAG~ GGACAGGTCC ~ lC~lC~l~ TGGGGCCCTG
180
CAGAGCCTCC TTGGAACCCA G~ C~lCCA CAGGGCAGGA CCACAGCTCA CAAGGATCCC
240
AATGCCATCT TCCTGAGCTT CCAACACCTG CTCCGAGGAA AGGTGCGTTT CCTGATGCTT
300
GTAGGAGGGT CCACCCTCTG CGTCAGGGAA TTCGGCGGCA ACA~l~GGC~l~C TCCGGCGCCG
360
C~ lG ACCTCCGAGT CCTCAGTAAA ~-~l~llCGTG ACTCCCATGT CCTTCACAGC
420
AGACTGAGCC AGTGCCCAGA GGTTCACCCT TTGCCTACAC ~l~lC~l~GCT GC~lG~l~lG
480
t2) INFORMATION FOR SEQ ID NO 66
(i) ~Qu~ CHARACTERISTICS
(A) LENGTH 479 base pairs
(B) TYPE nucleic acid
(C) STRANn~nN~S single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = ~DNA (synthetic)~
(xi) SEQUENCE DESCRIPTION SEQ ID NO 66
GGAGAATGGA AAACCCAGAT GGAGGAGACC AAGGCACAGG ACAll~lGGG AGCAGTGACC
C~ ~lGG AGGGAGTGAT GGr~r~rGG GGACAACTGG GACCCACTTG C~ ATCC
120
CTCCTGGGGC AG~lll~lGG ACAG~lCC~l~ C~lC~lG GGGCCCTGCA GAGC~lC~ll
180
GGAACCCAGC llC~l~CCACA GGGCAGGACC ACAGCTCACA AGGATCCCAA TGCCATCTTC
240
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120
CTGAGCTTCC AACACCTGCT CCGAGGAAAG ~lGC~ CC TGA'lG~ AGGAGGGTCC
300
ACC~L~lGCG TCAGGGAATT CGGCGGCAAC ATGGCTCTCC GGCGCCGCCT GCTTGTGACC
360
TCCGAGTCCT CAGTAAACTG ~ GACT CCCATGTCCT TCACAGCAGA CTGAGCCAGT
420
GCCCAGAGGT TCA~C~lllG CCTACACCTG TCCTGCTGCC TGCTGTGGAC TTTAGCTTG
479
(2) INFORMATION FOR SEQ ID NO 67
(i) s~Qu~N~ CHARACTERISTICS
(A) LENGTH 480 base pairs
(B) TYPE nucleic acid
(C) STRA~E~SS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = nDNA (synthetic) n
~Xi ) ~kQD~C~ DESCRIPTION SEQ ID NO 67
GGACCCACTT GCCTCTCATC CCTCCTGGGG CAG~ ~ GACAGGTCCG l~lC~lC~
GGGGCC~-l~C AGAGC-lC-l TGGAACCCAG ~lC~-lCCAC AGGGCAGGAC CACAGCTCAC
120
AAGGATCCCA ATGCCATCTT CCTGAGCTTC CAACACCTGC TCCGAGGAAA GGTGCGTTTC
180
CTGATGCTTG TAGGAGGGTC CACCCTCTGC GTCAGGGAAT ~l~CGGCGGCAA CATGGCGTCT
240
CCGGCGCCGC CTGCTTGTGA CCTCCGAGTC CTCAGTAAAC l~llC~lGA CTCCCATGTC
300
CTTCACAGCA GACTGAGCCA GTGCCCAGAG GTTCACCCTT TGCCTACACC ~l~lC~l~CTG
360
C~lG~l~lGG ACTTTAGCTT GG~-A~A~TGG AAAACCCAGA TGGAGGAGAC CAAGGCACAG
420
GACATTCTGG GAGCAGTGAC C~ll~lG~l~G GAGGGAGTGA TGGCAGCACG GGGACAACTG
480
(2) INFORMATION FOR SEQ ID NO 68
CA 022340~9 1998-04-06
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121
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic) n
(xi) ~ Qu~ DESCRIPTION: SEQ ID NO: 68:
GGAACCCAGC llC~lCCACA GGGCAGGACC ACAGCTCACA AGGATCCCAA TGCCATCTTC
CTGAGCTTCC AACACCTGCT CCGAGGAAAG GTGC~ll~lCC TGAlG~ll~ AGGAGGGTCC
120
AC~ ~CG TCAGGGAATT CGGCGGCAAC AlGGC~l~lC ~GGCGCCGC~ ~l~G~-~lu~l~AC
180
CTCCGAGTCC TCAGTAAACT G~lC~lGAC TCCCATGTCC TTCACAGCAG ACTGAGCCAG
240
TGCCCAGAGG TTCACCCTTT GCCTACACCT ~lC~l~-lGC ~l~l~l~GA CTTTAGCTTG
300
GGAGAATGGA AAACCCAGAT GGAGGAGACC AAGGCACAGG ACATTCTGGG AGCAGTGACC
360
~'1"1~ L G~'l'G~ AGGGAGTGAT GGCAGCACGG GGACAACTGG GACCCACTTG CCTCTCATCC
420
~lC~lGGGGC AG~~ lGG ACAG~lCC~l~ ~lC~lC~ll~ GGGCC~l~CA GAGC~lC~
480
(2) INFORMATION FOR SEQ ID NO: 69:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 480 base pairs
(8) TYPE: nucleic acid
(C) STRAN~:~N~:SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = nDNA (synthetic) n
CA 022340~9 1998-04-06
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122
(Xi) ~:QU~N~ DESCRIPTION SEQ ID NO 69
GGCAGGACCA CAGCTCACAA GGATCCCAAT GCCATCTTCC TGAGCTTCCA ACAC~lG~lC
CGAGGAAAGG TGC~~ C~l~ GAl~G~ ~lA GGAGGGTCCA CCCTCTGCGT CAGGGAATTC
120
GGCGGCAACA TGGCGTCTCC GGCGCCGCCT GCTTGTGACC TCCGAGTCCT CAGTAAACTG
180
C~l~ACT CCCATGTCCT TCACAGCAGA CTGAGCCAGT GCCCAGAGGT TCACC~l~llG
240
CCTACACCTG ~ G~l~GCC TGCTGTGGAC TTTAGCTTGG GAGAATGGAA AACCCAGATG
300
GAGGAGACCA AGGCACAGGA CATTCTGGGA GCAGTGACCC TTCTGCTGGA GGGAGTGATG
360
GCAGCACGGG GACAACTGGG ACCCACTTGC CTCTCATCCC TCCTGGGGCA G~ ~GA
420
CAG~lCC~l-- TC~ lGG GGCCCTGCAG AGCCTCCTTG GAACCCAGCT TCCTCCACAG
480
(2) INFORMATION FOR SEQ ID NO 70
(i~ SEQUENCE CHARACTERISTICS
(A) LENGTH 480 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = nDNA (synthetic)"
(xi) ~Q~N~ DESCRIPTION SEQ ID NO 70
GCTCACAAGG ATCCCAATGC CA~ C~l~G AGCTTCCAAC AC~ CC~ AGGAAAGGTG
~ A lG~ll~lAGG AGGGTCCACC CTCTGCGTCA GGGAATTCGG CGGCAACATG
120
G~l~lCCGG CGCCGCCTGC TTGTGACCTC CGAGTCCTCA GTAAACTGCT TCGTGACTCC
180
CA~l~lC~l~lC ACAGCAGACT GAGCCAGTGC CCAGAGGTTC ACC~ l~CC TACACCTGTC
240
CA 022340~9 1998-04-06
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G~lGC~l~ CTGTGGACTT TAGCTTGGGA GAATGGAAAA CCCAGATGGA GGAGACCAAG
300
GCACAGGACA TTCTGGGAGC AGTGACCCTT ~l~G~l~GGAGG GAGTGATGGC AGCACGGGGA
360
CAACTGGGAC CCA~ll~GC~l~ CTCATCCCTC CTGGGGCAGC lll~ GGACA G~l~CC~l~lC
420
~lC~llGGGG CCCTGCAGAG C~lC~ll~GGA ACCCAGCTTC CTCCACAGGG CAGGACCACA
480
(2) INFORMATION FOR SEQ ID NO 71
( i ) ~QU ~:N~ CHARACTERISTICS
(A) LENGTH 480 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = ~DNA (synthetic) n
(xi) ~yUrN~ DESCRIPTION SEQ ID NO 71
GATCCCAATG CCAl-~llC~l GAGCTTCCAA CAC~lG~lCC GAGGAAAGGT GC~l-llC~l~
ATGCTTGTAG GAGGGTCCAC CCTCTGCGTC AGGGAATTCG GCGGCAACAT GGC~l~lCCG
120
GCGCCGC~l~G CTTGTGACCT CCGAGTCCTC AGTAAACTGC TTCGTGACTC CCA~ C~
180
CACAGCAGAC TGAGCCAGTG CCCAGAGGTT CACC~~ lGC CTACACCTGT C~lG~lGC~l
240
G~1~1~GACT TTAGCTTGGG AGAATGGAAA ACCCAGATGG AGGAGACCAA GGCACAGGAC
300
All~lGGGAG CAGTGACCCT ~ GGAG GGAGTGATGG CAGCACGGGG ACAACTGGGA
360
CCCACTTGCC TCTCATCCCT C~l~GGGCAG ~ lGGAC AG~l~CC~ C~l~C~l~ ~GG
420
GCC~l~GCAGA GC~l~C~ GG AACCCAGCTT CCTCCACAGG GCAGGACCAC AGCTCACAAG
480
CA 022340~9 1998-04-06
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124
(2) INFORNATION FOR SEQ ID NO 72
(i) ~Q~N~ CHARACTERISTICS
(A) LENGTH 480 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = nDNA (synthetic)"
(xi) ~U~N~ DESCRIPTION SEQ ID NO 72
GCCATCTTCC TGAGCTTCCA ACAC~l~l~ CGAGGAAAGG ~l~GC~ lC~l GAlG~lu~l~A
GGAGGGTCCA CC~l~lGC~l~ CAGGGAATTC GGCGGCAACA 'l~GC~l~lCC CG~lCCGC~l
120
G~ll~lGACC TCCGAGTCCT CAGTAAACTG ~llC~lGACT CCCATGTCCT TCACAGCAGA
180
CTGAGCCAGT GCCCAGAGGT TCACC~lllG CCTACACCTG TC~lG~l~GCC TGCTGTGGAC
240
TTTAGCTTGG GAGAATGGAA AACCCAGATG GAGGAGACCA AGGCACAGGA CAll~l~GGA
300
GCAGTGACCC ~ ~GA GGGAGTGATG GCAGCACGGG GACAACTGGG ACCCACTTGC
360
CTCTCATCCC lC~l~GGGCA G~-lll~lGGA CAG~l~CC~lC l-~lC~ll~G GGCC~lGCAG
420
AGCCTCCTTG GAACCCAGCT TCCTCCACAG GGCAGGACCA CAGCTCACAA GGATCCCAAT
480
(2) INFORMATION FOR SEQ ID NO 73
( i ) ~:yU~N - '~ CHARACTERISTICS
(A) LENGTH 4 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS unknown
(D) TOPOLOGY unknown
(ii) MOLECULE TYPE peptide
CA 02234059 1998-04-06
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125
(xi) ~uu N~ DESCRIPTION: SEQ ID NO: 73:
Gly Gly Gly Ser
(2) INFORMATION FOR SEQ ID NO: 74:
(i) ~Q~N~ CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nN~S: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
Gly Gly Gly Ser Gly Gly Gly Ser
l 5
(2) INFORMATION FOR SEQ ID NO: 75:
(i) ~uu~ CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
l 5 l0
(2) INFORMATION FOR SEQ ID NO: 76:
~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~nN~cs unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
CA 022340~9 1998-04-06
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
Ser Gly Gly Ser Gly Gly Ser
l 5
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) ~Q~ DESCRIPTION: SEQ ID NO: 77:
Glu Phe Gly Asn Met Ala
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANn~n~F~S: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
Glu Phe Gly Gly Asn Met Ala
l 5
(2)~INFORMATION FOR SEQ ID NO: 79:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: l0 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nN~S: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
CA 02234059 1998-04-06
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127
(xi) ~yU~N~: DESCRIPTION: SEQ ID NO: 79:
Glu Phe Gly Gly Asn Gly Gly Asn Met Ala
(2) INFORMATION FOR SEQ ID NO: 80:
Qu~: CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRA~~ sS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) ~:~U~N~ DESCRIPTION: SEQ ID NO: 80:
Gly Gly Ser Asp Met Ala Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 81:
(i) ~:QU~N~: CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = ~DNA (synthetic)"
(xi) ~ u~ DESCRIPTION: SEQ ID NO: 81:
GGATCCACCA TGAGCCGCCT GCCC~LC~l~G ~l~C~lG~l~CC AA~L~ G~l~ CCGCCCCGC
59
(2) INFORMATION FOR SEQ ID NO: 82:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 459 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA (synthetic)"
CA 022340~9 1998-04-06
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128
(xi) ~QU~N~ DESCRIPTION SEQ ID NO 82
l~lCCCG~l~ CGCCTGCTTG TGACCTCCGA ~l~C~lCAGTA AA~l~G~ lCG TGACTCCCAT
~ ACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC ~ GC~lAC AC~
120
GC~lGLlG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA
180
CAGGACATTC TGGGAGCAGT GACC~ lG CTGGAGGGAG TGATGGCAGC ACGGGGACAA
240
CTGGGACCCA ~lU~C~l~lC A~lCC~lC~l~G GGGCAGCTTT CTGGACAGGT CC~~ C~lC
300
~ GGGCCC TGCAGAGCCT CCTTGGAACC CAG~llC~lC CACAGGGCAG GACCACAGCT
360
CACAAGGATC CCAATGCCAT ~ilC~l~GAGC TTCCAACACC lG~l~CCGAGG AAAGGTGCGT
420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG
459
(2) INFORMATION FOR SEQ ID NO 83
yu~ CHARACTERISTICS
(A) LENGTH 936 base p~irs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE other nucleic acid
(A) DESCRIPTION /desc = ~DNA (synthetic)~
(Xi) ~UU~N-~ DESCRIPTION SEQ ID NO 83
TCCCrAr~CTC CAC~lG~l~l~ TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT
CACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC ~lluGC~l~AC AC~l~lC~lG
120
~ C~l~l~G TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA
180
CA 022340~9 1998-04-06
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CAGGACATTC TGGGAGCAGT GACC~ll~lG CTGGAGGGAG TGATGGCAGC ACGGGGACAA
240
~ CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CC~ C~lC
300
CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAG~llC~l~C CACAGGGCAG GACCACAGCT
360
CACAAGGATC CCAATGCCAT ~ C~lGAGC TTCCAACACC ~l~G~lCCGAGG AAAGGTGCGT
420
TTCCTGATGC TTGTAGGAGG GTCCACCCTC ~lGC~l~AGGG AATTCGGCAA CATGGCGTCT
480
CCCG~lCCGC ~l~ GA CCTCCGAGTC CTCAGTAAAC ~l~G~LlC~lGA CTCCCATGTC
540
CTTCACAGCA GACTGAGCCA GTGCCCAGAG GTTCACCCTT TGCCTACACC TGTCCTGCTG
600
CCTGCTGTGG ACTTTAGCTT GGGAGAATGG AAAACCCAGA TGGAGGAGAC CAAGGCACAG
660
GACATTCTGG GAGCAGTGAC C~ G~l~G GAGGGAGTGA TGGCAGCACG GGGACAACTG
720
GGACCCACTT GCCTCTCATC C~lC~l~GGGG CAG~lll~-L~G GACAGGTCCG l~lC~lC~ll
780
GGGGCC~l~C AGAGC~lC~l TGGAACCCAG ~llC~lCCAC AGGGCAGGAC CACAGCTCAC
840
AAGGATCCCA ATGCCATCTT CCTGAGCTTC CAACACCTGC TCCGAGGAAA GGTGCGTTTC
900
CTGATGCTTG TAGGAGGGTC CACCCTCTGC GTCAGG
936
