Note: Descriptions are shown in the official language in which they were submitted.
CA 02315974 2000-06-20
SMB
Insulin-like Growth Factor Bindin4 Protein Fragments
and the Utilization Thereof
The present invention relates to peptides having cell-proliferative and cell-
protective properties, complexes of the peptides with human insulin-like
growth
factors (IGF) I and II, and the related nucleic acids, antisense nucleotides,
antibodies and inhibitors.
Insulin-like growth factor binding proteins have been described, inter alia,
by
Shimasaki, S., and Ling, N., in Prog. Growth Factor Res. 3 (1991) 243-266, and
Zapf, J., in Eur. J. Endocrinol. 132 (1995) 645-654.
The present invention relates to peptides the amino acid sequence of which
corresponds to parts of the amino acid sequence of insulin-like growth factor
binding proteins, and cyclic, glycosylated, phosphorylated, acetylated,
amidated
and/or sulfated derivatives thereof. These peptides according to the invention
are
designated as IGFBP or IBP.
Preferred peptides are those which are naturally occurring and can be
isolated, for
example, from hemofiltrate. Preferably, the peptides have lengths of from 61
to
115 amino acids. Particularly preferred are peptides having sequences which
correspond to N- or C-terminal sequences of insulin-like growth factor binding
proteins.
Preferred peptides are peptides having an amino acid sequence of formula
Rl-C-X1-PNC-XZ-QC-X3-CWCV-X4-C-R2
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wherein
Rl is NH2, an amino acid or a peptide having an amino acid sequence comprising
up to 41 amino acids, Xl is a peptide having an amino acid sequence comprising
from 24 to 31 amino acids, X2 is a peptide having an amino acid sequence com-
prising 9 amino acids, X3 is a peptide having an amino acid sequence
comprising
amino acids, X4 is a peptide having an amino acid sequence comprising from 18
to 24 amino acids, R2 is COOH, CONH2 or a peptide having up to 12 amino acids,
and cyclic, glycosylated, phosphorylated, acetylated, amidated, sulfated
derivatives
and or fragments thereof having the physiological activity of IGFBP.
The peptide has cell-proliferative and cell-protective properties.
The peptides according to the invention can have disulfide bridges to
correspond to
the general formula:.
---.~ ~ r----z
R,-C-X,-PNC-Xz-QC-X,-C~~hCV-Xa-C-R,
In a preferred embodiment, the peptides have a glycine on one or more of the
following positions of the amino acid sequence. X2 on position 4, X3 on
position 9,
X4 on position 4 or 5, and/or X4 on position 9 or 10.
It is further preferred that X1 is L or V on position 8, and/or X1 is L or I
on position
11, and/or XZ is D or N on position 1, and/or XZ is K or R on position 9,
and/or X3 is
S or A on position 3 and/or R or A on position 8.
In a particularly preferred embodiment, Rl is selected from:
APSEEDHSILWDAISTYDGSKALHVTNIKKWKEP (SEQ ID NO: 1),
GGKHHLGLEEPKKLRPPPARTP (SEQ ID NO: 2),
GKGGKHHLGLEEPKKLRPPPARTP (SEQ ID NO: 3),
GHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGP (SEQ ID NO: 4),
KVNGAPREDARPVPQGS (SEQ ID NO: 5),
LTQSKFVGGAENTAHPRIISAPEMRQESEQGP (SEQ ID NO: 6),
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PQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGP (SEQ ID NO: 7); and/or
X1 is selected from
RIELYRWESLAKAQETSGEEISKFYL (SEQ ID NO: 8),
QQELDQVLERISTMRLPDERGPLEHLYSLHI (SEQ ID NO: 9),
RREMEDTLNHLKFLNVLSPRGVHI (SEQ ID NO: 10),
QSELHRALERLAASQSRTHEDLYIIPI (SEQ ID NO: 11),
RRHMEASLQELKASPRMVPRAVYL (SEQ ID NO: 12),
RRHLDSVLQQLQTEVYRGAQTLYV (SEQ ID NO: 13); and/or
XZ is selected from
NKNGFYHSR (SEQ ID NO: 14),
DKHGLYNLK (SEQ ID NO: 15),
DKKGFYKKK (SEQ ID NO: 16),
DRNGNFHPK (SEQ ID NO: 17),
DRKGFYKRK (SEQ ID NO: 18),
DHRGFYRKR (SEQ ID NO: 19); and/or
X3 is selected from
ETSMDGEAGL (SEQ ID NO: 20),
KMSLNGQRGE (SEQ ID NO: 21),
RPSKGRKRGF (SEQ ID NO: 22),
HPALDGQRGK (SEQ ID NO: 23),
KPSRGRKRGI (SEQ ID NO: 24),
RSSQGQRRGP (SEQ ID NO: 25); and/or
X4 is selected from
YPWNGKRIPGSPEIRGDPN (SEQ ID NO: 26),
NPNTGKLIQGAPTIRGDPE (SEQ ID NO: 27),
DKYGQPLPGYTTKGKEDVH (SEQ ID NO: 28),
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-4-
DRKTGVKLPGGLEPKGELD (SEQ ID NO: 29),
DKYGMKLPGMEYVDGDFQ (SEQ ID NO: 30),
DRMGKSLPGSPDGNGSSS (SEQ ID NO: 31); and/or
R2 is selected from
QIYFNVQN (SEQ ID NO: 32),
HLFYNEQQEARGVHTQRMQ (SEQ ID NO: 33),
HLFYNEQQE (SEQ ID NO: 34),
YSMQSK (SEQ ID NO: 35),
HQLADSFRE (SEQ ID NO: 36),
HTFDSSNVE (SEQ ID NO: 37),
PTGSSG (SEQ ID NO: 38).
Preferred peptides have the following sequences:
IGFBP-1
APSEEDHSILWDAISTYDGSKALHVTNIKKWKEPCRIELYRVVESLAKAQETSGEEISKFYL
PNCNKNGFYHSRQCETSMDGEAGLCWCVYPWNGKRIPGSPEIRGDPNCQIYFNVQN
(SEQ ID NO: 39)
IG FBP-2
GKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDK
HGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQ
RMQ (SEQ ID NO: 40)
GGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKH
GLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQR
MQ (SEQ ID NO: 45)
IGFBP-3
GHAKDSQRYKVDYESQSTDTQN FSSESKRETEYGPCRREM EDTLN H LKFLNVLSPRGVHI
PNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK (SEQ
ID NO: 41)
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KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNCDK
KGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK (SEQ ID NO:
46)
HPLHSKIIIIKKGHAKDSQRY (SEQ ID NO: 47)
IGFBP-4
DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGLRCYP
PRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHDRRCLQKH
FAKIRDRSTSGGKM (SEQ ID NO: 48)
KVNGAPREDARPVPQGSCQSELHRALERLAASQSRTHEDLYIIPIPNCDRNGNFHPKQCHP
ALDGQRGKCWCVDRKTGVKLPGGLEPKGELDCHQLADSFRE (SEQ ID NO: 42)
IGFBP-5
LTQSKFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNC
DRKGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCHTFDSSNVE (SEQ
ID NO: 43)
KFVGGAENTAHPRIISAPEMRQESEQGPCRRHMEASLQELKASPRMVPRAVYLPNCDR
KGFYKRKQCKPSRGRKRGICWCVDKYGMKLPGMEYVDGDFQCHTFDSSNVE (SEQ ID
NO: 49)
HTRISELKAEAVKKDRRKKLTQS (SEQ ID NO: 50)
IGFBP-6
PQAGTARPQDVN RRDQQRN PGTSTTPSQPNSAGVQDTEMGPCRRH LDSVLQQLQTEVY
RGAQTLYVPNCDHRGFYRKRQCRSSQGQRRGPCWCVDRMGKSLPGSPDGNGSSSCPTG
SSG (SEQ ID NO: 44).
The peptides according to the invention can be obtained by purification from
human hemofiltrate or urine, by solid-phase peptide synthesis, or by
expression in
recombinant microorganisms.
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The invention further relates to complexes of the peptides according to the
invention with human insulin-like growth factor I and/or human insulin-like
growth
factor II and its physiologically active fragments and/or derivatives,
especially
amidated, acetylated, sulfated, phosphorylated and/or glycosylated
derivatives.
In addition, the invention relates to nucleic acids coding for the peptides
according
to the invention, antisense nucleotides which, under stringent conditions,
bind to a
nucleic acid coding for the peptide according to the invention, antibodies
which
bind to the peptides according to the invention, inhibitors which inhibit the
biological activity of insulin-like growth factor binding proteins, inhibitors
which
inhibit the expression of insulin-like growth factor binding proteins.
The peptides, complexes, nucleic acids, antisense nucleotides, antibodies and
inhibitors according to the invention are especially suitable for the
preparation of a
medicament for treating the over- or underexpression of insulin-like growth
factor
binding proteins, for treating muscular atrophy, osteoporosis, diabetes,
amyloid
lateral sclerosis, peripheral and central neuropathies, inflammatory
processes,
disordered inflammatory reactions, tumor diseases, inflammatory and neoplastic
diseases, disturbance of growth, muscular affections, affections of the bone
system, and/or for wound and bone healing.
Especially complexes of IGFBP with IGF-I or IGF-II are useful for the
treatment of
bone diseases.
The peptides according to the invention and the complexes of the peptides with
insulin-like growth factor have a cell-proliferative activity.
The peptides according to the invention control the release of IGF-I and IGF-
II
from the complexes at their site of action. The coadministration of the
peptides
according to the invention with IGF-I or IGF-II extends the biological half-
life and
thus the availability of the latter. Hypoglycemia induced by the injection of
free
IGF-I or IGF-II is prevented by the coadministration of the peptides according
to
the invention.
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_ 7 _
In addition, the peptides according to the invention have a growth-promoting
effect on bone cells and lead to an enhancement or modulation of the activity
of
growth hormones.
In a preferred embodiment, the peptides, complexes, nucleic acids, antisense
nucleotides, antibodies and inhibitors according to the invention are
contained in a
pharmaceutically acceptable dosage form in a medicament. They are suitable for
oral, intravenous, intramuscular, intracutaneous, intrathecal administration,
or as
an aerosol for transpulmonary administration. The amount of peptide to be
administered is from 1 Ng to 1 g per dosage unit per day.
The nucleic acids and/or antisense nucleotides according to the invention are
also
suitable for the preparation of a medicament for the treatment of somatic or
non-
somatic genetic diseases.
The diagnostic agent according to the invention contains the peptides,
complexes,
nucleic acids, antisense nucleotides, antibodies and/or inhibitors according
to the
invention.
Preferably, the diagnostic agent contains poly- or monoclonal antibodies
against
the peptide according to the invention. Such antibodies may be fluorescence-
labeled or radioactively labeled to be used in the known ELISA or RIA.
However,
the diagnostic agent may also contain nucleic acids which, in a modified or
labeled
form, are employed in test known to those skilled in the art, such as PCR or
fingerprinting.
The diagnostic agents according to the invention are especially useful for
diagnos-
ing functional disorders in bones, muscles, the nervous system, lymph organs,
the
gastrointestinal tract, the immune system, and of diabetes and inflammatory
and
neoplastic processes, and as a marker in cancer.
Especially the simultaneous occurrence of several fragments of BP-4 or BP-5 in
the
plasma, in particular, of the N- and C-terminal domains, is useful as a marker
for
CA 02315974 2000-06-20
8 _
diseases of bone metabolism. The corresponding peptides can be detected by
mass
spectroscopy, preferably by an immune reaction with corresponding antibodies.
Figure 1 shows an alignment of the consensus sequences of C-terminal fragments
of the insulin-like growth factor proteins.
Figure 2 shows the schematic structure of the insulin-like growth factor
proteins
with the cysteine-rich N- and C-terminal domains.
Figure 3 shows the schematic structure of the insulin-like growth factor
proteins
and the sequence of the biologically active fragments isolated from
hemofiltrate.
Figure 4 shows the isolation of the osteoanabolic factor IGFBP-4-11 from human
hemofiltrate (see Example 3).
Figure 5 shows the sequence and sulfur bridge analysis of the osteoanabolic
factor
IGFBP-4-11. The cysteines 153-183, 194-205 and 207-228 are bridged.
Figure 6 shows the biological effect of the osteoanabolic factor IGFBP-4-11.
After
incubation of primary rat osteoblasts, which had been kept in a serum-free
medium, for (A) 24 hours, (B) 48 hours, and (C) 72 hours with IGFBP-4-11, the
proliferation-promoting effect of IGFBP-4-11 can be seen. An increase of the
DNA
synthetic rate in a dose-dependent way is found, measured as incorporation of
bromodeoxyuridine (BrdU).
Figure 7 shows the specific binding to osteoblasts and the possible receptor
for the
osteoanabolic factor IGFBP-4-11 (designated as IGFBP-413x-z3'). A:
Radioactively
labeled IGFBP-4-11 exhibits specific binding to primary osteoblast cells which
can
be displaced by increasing amounts of non-labeled IGFBP-4-11. B: After having
bound to osteoblasts, radioactively labeled IGFBP-4-11 could be chemically
cross-
linked with its putative receptor molecule and subsequently be detected by gel
electrophoresis followed by autoradiography. The ligand-receptor complex has a
molecular mass of about 120 kDa. The formation of the complex is favored by
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_g_
saponin, a membrane pore generator. Complex formation is prevented by adding
an excess of unlabeled IGFBP-4-11 to the incubation mixture.
The purification of the peptide according to the invention or its complex is
illus-
trated by the following Examples:
Example 1
Purification and peptide-chemical analysis of IGFBP-2-13
The peptides according to the invention can be obtained by a purification
method
starting from human hemofiltrate. This patent method (Forssmann, W.-G. (1988),
German Laid-Open DE 36 33 707 A1), which was developed for the recovery of
proteins from hemofiltrate, was also employed, in a modified form, for the
purification of the peptide complex.
Hemofiltrate batch extraction
Hemofiltrate is obtained in large amounts in the ultrafiltration of the blood
of
kidney disease sufferers. 800 to 1,000 I of hemofiltrate are adjusted to pH
2.7 with
HCI and diluted with water to a conductivity of 5.5 mS/cm, and applied to a
strong
cation-exchanger at a flow rate of 3 I/min.
Conditions of chromatography
column: Vantage VA 250 (Amicon, Witten)
column material: Fractogel TSK SP 650 (M), 25 cm x 20 cm
flow rate: 3 I/min
detection: 280 nm, pH, conductivity
buffer A: hemofiltrate, pH 2.7, conductivity
5.5 mS/cm
buffer B: 0.5 M ammonium acetate
plant: Autopilot Chromatographiesystem (PerSeptive Biosystems,
Wiesbaden)
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After application of a total of 1,000 I of liquid over night, the column is
washed
with several column volumes of 5 mM HCI. The elution of the bound peptides is
performed as a batch elution with 0.5 M ammonium acetate. Complete elution of
the peptides is achieved with an increasing pH value (6.8 to 7.2) and
increased
conductivity (56 mS/cm) in about 5 I of eluate.
First preparative separation
The ammonium acetate eluates from the batch extraction are combined in
amounts of 5,000 to 10,000 I of hemofiltrate peptide. After adjusting the pH
to
2.7, the peptide extract is applied to the preparative cation-exchanger with
admixing completely desalted water having a conductivity of 5.5 mS/cm.
Conditions of chromatography:
column: Vantage 250 VA
column material: Fractogel TSK SP 650 (M), 25 cm x 20 cm
flow rate: up to 3 I/min during application
0.5 to 1 I during elution
detection: 280 nm, pH, conductivity
sample: hemofiltrate, pH 2.7, conductivity 5.5 mS/cm
plant: Autopilot Chromatographiesystem (PerSeptive Biosystems,
Wiesbaden)
After application of the raw extract over a period of 240 min, the column is
washed
with 0.01 M HCI until the conductivity has fallen below 1 mS/cm. Elution is
effected
in several steps with the following buffers:
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buffer pH value buffer substances conductivity (mS/cm)
washing buffer2.0 0.01 M HCI 1
elution buffer3.6 0.1 M citric acid 1-hydrate2.9
1
elution buffer4.5 0.1 M acetic acid + 4.0
2
0.1 M sodium acetate
elution buffer5.0 0.1 M malic acid 6.2
3
elution buffer5.6 0.1 M succinic acid 6.1
4
elution buffer6.6 0.1 M NaHZP04 4.9
elution buffer7.4 0.1 M NaHzP04 6.7
6
elution buffer9.0 0.1 M ammonium carbonate6.7
7
Eluates 1-7 are referred to as pH pool I-VII. They are separately collected
and
finally washed with completely desalted water. Elution is performed until a
new
baseline is reached. For the individual pH pools I to VII, elution volumes of
10 to
25 I are reached.
Second preparative separation
The individual pH pools are separated by reversed-phase chromatography for
fractionating and desalting at the same time.
Conditions of chromatography:
column: FineLine 100 (Pharmacia, Freiburg)
column material: Source RPC, 15 Nm, 10 x 12.5 cm (FineLine 100)
flow rate: 150 ml/min (FineLine 100)
detection: 280 nm, conductivity, pH
buffer A: 10 mM HCI
buffer B: 80% acetonitrile in 10 mM HCI
gradient: 0-60% buffer B in 5 column volumes
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After application of the pH pools, the column is washed with buffer A. During
the
elution, fractions of 200 ml are collected. Aliquots of the fractions are
tested in a
bioassay. The fractions are freeze-dried and stored at -20 °C.
Semipreparative reversed-lahase C18 chromatogra~hy
Fractions 11 and 12 from pH pool V, which had been biologically active in the
assay, were separated through a semipreparative reversed-phase column.
Fractions 21 to 25 contained the substance according to the invention.
Conditions of chromatography
column: 4.7 cm x 30 cm steel column
column material:Vydac RP-C18 15-20 pm, 300 ~
buffer A: 0.1% TFA
buffer B: 0.1% TFA, 80% acetonitrile
gradient: 5-50% B in 45 min, 50-100% B in
10 min
flow rate: 42 ml/min
detection: 214 nm and 280 nm
chromatographic
plant: BioCad
fractions: every 1.5 min from the start of
the gradient
Seminreparative reversed-phase C18 chromatoara~hy
The biologically active fractions 21 to 25 from the preceding chromatography
were
separated through the same semipreparative reversed-phase column. However,
methanol was used as the eluent. Fraction 24 contained the substance according
to the invention.
Conditions of chromatography
column: 4.7 cm x 30 cm steel column
column material: Vydac RP-C18 15-20 Nm, 300 r~
buffer A: 0.1% TFA, 20% methanol
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buffer B: 0.1% TFA, 100% methanol
gradient: 0-20% B in 6.5 min, 20-80% B in 55 min, 80-100% B in
13 min
flow rate: 30 ml/min
detection: 214 nm and 280 nm
chromatographic plant: BioCad
fractions: every 1.5 min from the start of the gradient
Cation exchange chromatographx
The biologically active fractions 19 and 20 from the preceding chromatography
were separated through a cation-exchange column. Fractions 45 to 47 contained
the substance according to the invention.
Conditions of chromatoarap
column: 1 cm x 5 cm steel column
column material: Pepkat, Biotek 5 pm, 300 ~
buffer A: 20 mM sodium phosphate, pH 3.0
buffer B: 20 mM sodium phosphate, pH 3.0, 1.5 M NaCI
gradient: 0-50% B in 50 min, SO-100% B in 10 min
flow rate: 3 ml/min
detection: 280 nm
chromatographic plant: BioCad Sprint
fractions: every 1.5 min from the start of the gradient
Analytical reversed-phase chromatographx
The biologically active fractions 45 to 47 from the preceding chromatography
were
successively separated in several identical runs through a reversed-phase
column.
Fraction 56 contained the substance according to the invention.
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Conditions of chromatography
column: 1 cm x 25 cm steel column
column material:Vydac RP-C18 5 um, 300 ~
buffer A: 0.1% TFA
buffer B: 0.1% TFA, 80% acetonitrile
gradient: 5-50% B in 45 min, 50-100% B in
10 min
flow rate: 2 ml/min
detection: 220 nm
chromatographiclant: Kontron
p
fractions: every 1 min from the start of the
gradient
Second analytical reversed-phase C18 chromatography
The biologically active fraction 56 from the preceding separation step was
further
purified on an analytical reversed-phase column.
Conditions of chromatoara~hv:
column: 0.46 cm x 25 cm steel column
column material:YMC RP-C18, 5 Nm, 300 A
buffer A: 0.1% TFA
buffer B: 0.1% TFA, 80% acetonitrile
gradient: 15-50% B in 75 min
75-100% B in 10 min
,
flow rate: 0.7 ml/min
detection: 214 nm
chromatographiclant: Kontron
p
Third analytical reversed-phase C3 chromatogr~~hX
Part of the biologically active fraction 45 from the preceding separation step
was
directly subjected to mass and sequence analyses. Another part was reduced and
alkylated (as described under Example 2) and then further purified on an
analytical
reversed-phase column.
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Conditions of chromatography
column: 0.1 cm x 15 cm steel column ,
column material: Zorbax RP-C3, 5 Nm, 300 A
Mass determinations
All mass determinations of the unmodified and modified peptides were performed
on a MALDI TOF mass spectrometer. The molecular masses of the peptides were
determined as:
IGF-II: 7,471 Da;
IGFBP-2: 12,681 Da;
IGFBP-2: 12,865 Da.
Determination of cysteines/modification ofa~eptides
Cysteines can be detected after previous chemical derivatization, for example,
after reduction with ~3-mercaptoethanol and carboxamidomethylation with io-
doacetamide, in the peptide sequencing. Derivatization is followed by
desalting,
preferably through analytical reversed-phase chromatography on a Vydac RP-C18
column (4.6 mm x 25 cm). Part of the thus modified peptides are subjected to
sequence analysis, and mass determinations performed on the rest yield a
corresponding molecular mass. From the mass difference to the native peptide,
it
is concluded that the peptides from hemofiltrate contain six cysteines which
are
additionally interconnected by three disulfide bridges.
Sequence determination
Both the purified native and the carboxamidomethylated peptides are analyzed
by
Edman degradation on an ABI 473 A Sequencer using the standard program.
The samples are applied to a Polybrene membrane in amounts of between 100 and
400 pmol. In accordance with the results of mass determinations, the following
N-
terminal sequences were found:
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IGFBP-2-13, MW 12,681
(reduced molecule modified with iodoacetamide, MW 13,045)
Amino acids
GGKHHLGLEEPKKLRPPPARTPCQQELDQV...
IGFBP-2-13, MW 12,865
(reduced molecule modified with iodoacetamide, MW 13,223)
Amino acids
GKGGKHHLGLEEPKKLRPPPARTPCQQELDQV...
IGF-II, MW 7471
Amino acids
AYRPSETLCGGEL....
The C terminus was determined by comparing the measured molecular mass with
the mass calculated from the sequence. The identity of these masses is within
the
measuring accuracy of the MALDI TOF mass spectrometer, i.e., 0.1% of the total
mass.
Data base comparison
A data base comparison was performed on the SwissProt and EMBL nucleic acid
data bases using the HUSAR program package. The peptide sequences have 100%
identity with the amino acids of human IGFBP-2 as derived from the cDNA or
with
the amino acid sequence of IGF-II.
To date, IGFBP-2 has been described as a 34 kD binding protein the sequence of
which was completely analyzed by analyzing the related cDNA (Binkert, C., et
al.,
EMBO Journal Vol. 8 (1989), pages 2497 to 2502). In contrast, IGF-II and also
IGF-I, which also binds to IGFBP-2, have already been extensively described in
terms of their structures on the protein and DNA sequence levels (as a review:
Rechler, M.M., & Nissley, S.P. (1990) "Insulin-like growth factors" in:
Peptide
growth factors and their receptors (Spori, M.B., Roberts, A.B. eds.), pages
263 to
367, Springer-Verlag, Berlin).
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Example 2
Determination of the biological activitx of IGF/IGFBP-2-13
The isolation of IGF/IGFBP-2-13 was based on its biological activity in a
survival
assay of the PC-12 (pheochromocytoma cells) cell line. Thus, aliquots of each
of
the individual chromatographic stages described under Example 1 were freeze-
dried and then subjected to a biological assay. The fractions which gave a
positive
signal were respectively subjected to further purification.
The assay measures the survival of the cells after having been kept in serum-
free
medium by determining the activity of mitochondria) enzymes 24 hours after
serum starvation. As a positive control, nerve growth factor (NGF) or fetal
calf
serum (FCS) are employed in this assay.
In 96-well plates, 10,000 PC-12 cells per well were seeded in a serum-free
medium, following by the addition of aliquots (about 100 ml equivalent of
starting
material) to the wells. The survival rate of the cells is measured 20 hours
later
using a Wst-1 substrate. This substrate is converted by mitochondria) enzymes.
The color intensity obtained is measured at 405 nm in an ELISA reader; the
reference wavelength was over 600 nm.
The IGF/IGFBP-2-13 complex has a dose-dependent survival-promoting effect on
PC-12 cells. These cells correspond to neuronal precursor cells so that it can
be
considered that IGF/IGFBP-2-13 is a neuroprotective factor.
Example 3
Purification of the peptide IGFBP-4-11 according to the invention
The purification of the peptide IGFBP-4-11 according to the invention was per-
formed by complete analogy with the purification of IGFBP-2-13 described under
Example 1 up to the step of the second preparative separation. The further
purification was effected by:
CA 02315974 2000-06-20
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Analytical reversed-phase C18 chromatogr~~hx
Fraction 33 from pH pool IV, which was found biologically active in the assay,
was
separated through an analytical reversed-phase column. Fraction 34 contained
the
substance according to the invention.
Conditions of chromatograph rL:
column: 1 cm x 25 cm steel column
column material:Vydac RP-C4 5 Nm, 300 A
buffer A: 0.1% TFA
buffer B: 0.1% TFA, 80% acetonitrile
gradient: 0-80% B in 80 min, 80-100% B in
10 min
flow rate: 2.5 ml/min
detection: 230 nm
chromatographic
plant: Kontron
fractions: every 1 min from the start of the
gradient
Mass determinations
The mass determinations were performed on an electrospray mass spectrometer.
The molecular mass of the peptide was determined as:
IGFBP-4-11, 11,344 Da.
Sequence determination
The amino acid sequence of the purified native biologically active peptide
IGFBP-4-
11 was determined as described under Example 1.
The following N-terminal sequence was found:
IGFBP-4-11, MW 11,344 Da
KVNGAPREDARPVPQGSXQSELIIRALERL...
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The C terminus was determined by comparing the measured molecular mass with
the mass calculated from the sequence. The identity of these masses is within
the
measuring accuracy of the electrospray mass spectrometer, i.e., 0.1% of the
total
mass.
Data base comparison
A data base comparison was performed on the SwissProt and EMBL nucleic acid
data bases using the HUSAR program package. The peptide sequence has 100%
identity with the amino acids of human IGfBP-4 as derived from the cDNA.
Determination of the sulfur bridge cross-links in IGFBP-4-11
The analysis of the sulfur-bridge cross-linking was performed by cleaving the
native peptide IGFBP-4-11 in two different parallel reactions with the
endoprote-
ases chymotrypsin and Arg-C. The cleaving fragments obtained were then sepa-
rated by analytical reversed-phase chromatography and subjected to molecular
mass and sequence analyses. The following fragments containing two cysteines
and one sulfur bridge each were obtained:
HPKQCHPALDGQRGKCW, MW 1960
CVDRKTGVKLPGGLEPKGELDCHQLADSF, MW 3112
PVPQGSCQSELHR
MW 3236
THEDLYIIPIPNCDR
It can be seen therefrom that the sulfur bridges between cysteines 1 and 2,
between cysteines 3 and 4 and between cysteines 5 and 6 are realized in the
native IGFBP-4-11.
CA 02315974 2000-06-20
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Example 4
Determination of the biological activity of IGFBP-4-11
The isolation of IGFBP-4-11 was based on its biological activity in a
proliferation
assay with primary bone cells (osteoblasts), which were first isolated from
rat
embryonal calvarias.
Thus, aliquots of each of the individual chromatographic stages described
under
Example 3 were freeze-dried and then subjected to a biological assay. The
fractions which gave a positive signal were respectively subjected to further
purification. The assay measures the proliferation of the cells by determining
the
incorporation of radioactive thymidine, i.e., the DNA synthesis rate, 48 or 72
hours
after the addition of the fractions. As a positive control, transforming
growth
factor-beta (TGF-~) or fetal calf serum (FCS) are employed in this assay. In
96-
well plates, 5000 osteoblasts per well were seeded in a serum-containing
medium,
following by the addition of aliquots (about 100 ml equivalent of starting
material)
to the wells. The proliferation rate (DNA synthesis rate) of the cells is
measured 48
or 72 hours later using the addition and incorporation of radioactive
thymidine. The
peptide IGFBP-4-11 has a dose-dependent proliferation-promoting effect on
these
primary osteoblasts. These cells correspond to typical bone cells so that it
can be
considered that IGFBP-4-11 is an osteoanabolic factor.
Example 5
Isolation of the C-terminal domain of IGFBP-3
By a similar method to that used in Examples 1 and 3, a peptide could be
isolated
from hemofiltrate, having a mass of 2,470 Dalton (MALDI: 2481 Dalton) and the
following sequence:
HTRISELKAEAVKKDRRKKLTQS (?)
from which the following sequence results as the C-terminal sequence of IGFBP-
3:
CA 02315974 2000-06-20
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KVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNCDKK
GFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK
Example 6
By a similar method to that used in Examples 1 and 3, the N-terminal domain of
IGFBP-4 could be isolated, having the following sequence:
DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGLRCYP
PRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHDRRCLQKH
FAKIRDRSTSGGKM
Example 7
Determination of the C-terminal sequence of IPB-5
By a method as in Examples 1 and 3, a peptide with a mass of 13.5 kD could be
determined. The sequence determination gave the following sequence:
KFVGGAENTAH PRIISAPEM RQESEQG PCRRH M EASLQELKASPRMVPRAVYLPNCDRKG
FYKRKQCKPSRGRKRGICWCVDKYGM KLPGMEYVDGDFQCHTFDSSNVE
The theoretical mass is 12.5 kD, and therefore, it has to be considered that
the
peptide is glycosylated at serine or threonine.
CA 02315974 2000-06-20
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Prof. Dr. Wolf-Georg Forssmann
(B) STREET: Feodor-Lynen-Str. 31
(C) CITY: Hannover
(E) COUNTRY: Germany
(F) POSTAL CODE: 30625
(ii) TITLE OF INVENTION: Insulin-like Growth Factor Binding Protein
Fragments and the Utilization Thereof
(iii) NUMBER OF SEQUENCES: 50
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER; IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ala Pro Ser Glu Glu Asp His Ser Ile Leu Trp Asp Ala Ile Ser Thr
1 5 10 15
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Tyr Asp Gly Ser Lys Ala Leu His Val Thr Asn Ile Lys Lys Trp Lys
20 25 30
Glu Pro
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gly Gly Lys His His Leu Gly Leu Glu Glu Pro Lys Lys Leu Arg Pro
1 5 10 15
Pro Pro Ala Arg Thr Pro
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3
Gly Lys Gly Gly Lys His His Leu Gly Leu Glu Glu Pro Lys Lys Leu
1 5 10 15
Arg Pro Pro Pro Ala Arg Thr Pro
CA 02315974 2000-06-20
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(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gly His Ala Lys Asp Ser Gln Arg Tyr Lys Val Asp Tyr Glu Ser Gln
1 5 10 1 5
Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu Ser Lys Arg Glu Thr Glu
20 25 30
Tyr Gly Pro
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Lys Val Asn Gly Ala Pro Arg Glu Asp Ala Arg Pro Val Pro Gln Gly
1 5 10 15
Ser
Arg Pro Pro Pr
CA 02315974 2000-06-20
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(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Leu Thr Gln Ser Lys Phe Val Gly Gly Ala Glu Asn Thr Ala His Pro
1 5 10 15
Arg Ile Ile Ser Ala Pro Glu Met Arg Gln Glu Ser Glu Gln Gly Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 7:
Pro Gln Gly Thr Arg Gln Asp Val Asn Arg Arg Asp
Ala Ala Pro Gln
1 5 10 15
Gln Arg Pro Gly Ser Thr Pro Ser Gln Pro Asn Ser
Asn Thr Thr Ala
20 25 30
Gly Val Asp Thr Met Pro
Gln Glu Gly
35 40
CA 02315974 2000-06-20
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(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Arg Ile Glu Leu Tyr Arg Val Val Glu Ser Leu Ala Lys Ala Gln Glu
1 5 10 15
Thr Ser Gly Glu Glu Ile Ser Lys Phe Tyr Leu
20 25
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Gln Gln Glu Leu Asp Gln Val Leu Glu Arg Ile Ser Thr Met Arg Leu
1 5 10 15
Pro Asp Glu Arg Gly Pro Leu Glu His Leu Tyr Ser Leu His Ile
20 25 30
CA 02315974 2000-06-20
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(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Arg Arg Glu Met Glu Asp Thr Leu Asn His Leu Lys Phe Leu Asn Val
1 5 10 15
Leu Ser Pro Arg Gly Val His Ile
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Gln Ser Glu Leu His Arg Ala Leu Glu Arg Leu Ala Ala Ser Gln Ser
1 5 10 . 15
Arg Thr His Glu Asp Leu Tyr Ile Ile Pro Ile
20 25
CA 02315974 2000-06-20
_28_
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Arg Arg His Met Glu Ala Ser Leu Gln Glu Leu Lys Ala Ser Pro Arg
1 5 10 15
Met Val Pro Arg Ala Val Tyr Leu
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Arg Arg His Leu Asp Ser Val Leu Gln Gln Leu Gln Thr Glu Val Tyr
1 5 10 15
Arg Gly Ala Gln Thr Leu Tyr Val
CA 02315974 2000-06-20
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(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Asn Lys Asn Gly Phe Tyr His Ser Arg
1 5
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Asp Lys His Gly Leu Tyr Asn Leu Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
CA 02315974 2000-06-20
-30-
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Asp Lys Lys Gly Phe Tyr Lys Lys Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Asp Arg Asn Gly Asn Phe His Pro Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Asp Arg Lys Gly Phe Tyr Lys Arg Lys
1 5
CA 02315974 2000-06-20
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(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Asp His Arg Gly Phe Tyr Arg Lys Arg
1 5
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Glu Thr Ser Met Asp Gly Glu Ala Gly Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
CA 02315974 2000-06-20
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(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Lys Met Ser Leu Asn Gly Gln Arg Gly Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe
1 5 10
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
His Pro Ala Leu Asp Gly Gln Arg Gly Lys
1 5 10
CA 02315974 2000-06-20
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(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Lys Pro Ser Arg Gly Arg Lys Arg Gly Ile
1 5 10
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Arg Ser Ser Gln Gly Gln Arg Arg Gly Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
CA 02315974 2000-06-20
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(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Tyr Pro Trp Asn Gly Lys Arg Ile Pro Gly Ser Pro Glu Ile Arg Gly
1 5 10 15
Asp Pro Asn
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Asn Pro Asn Thr Gly Lys Leu Ile Gln Gly Ala Pro Thr Ile Arg Gly
1 5 10 15
Asp Pro Glu
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
CA 02315974 2000-06-20
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Asp Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu
1 5 10 15
Asp Val His
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Asp Arg Lys Thr Gly Val Lys Leu Pro Gly Gly Leu Glu Pro Lys Gly
1 5 10 15
Glu Leu Asp
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Asp Lys Tyr Gly Met Lys Leu Pro Gly Met Glu Tyr Val Asp Gly Asp
1 5 10 15
Phe Gln
CA 02315974 2000-06-20
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(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Asp Arg Met Gly Lys Ser Leu Pro Gly Ser Pro Asp Gly Asn Gly Ser
1 5 10 15
Ser Ser
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Gln Ile Tyr Phe Asn Val Gln Asn
1 5
CA 02315974 2000-06-20
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(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
His Leu Phe Tyr Asn Glu Gln Gln Glu Ala Arg Gly Val His Thr Gln
1 5 10 15
Arg Met Gln
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
His Leu Phe Tyr Asn Glu Gln Gln Glu
1 5
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
CA 02315974 2000-06-20
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(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Tyr Ser Met Gln Ser Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
His Gln Leu A1a Asp Ser Phe Arg Glu
1 5
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
CA 02315974 2000-06-20
-39-
His Thr Phe Asp Ser Ser Asn Val Glu
1 5
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
Pro Thr Gly Ser Ser Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE SEQ
DESCRIPTION: ID
NO:
39:
Ala Pro Glu Glu Asp Ser Ile Leu Asp Ala Ile Ser
Ser His Trp Thr
1 5 10 15
Tyr Asp Ser Lys Ala His Val Thr Ile Lys Lys Trp
Gly Leu Asn Lys
20 25 30
Glu Pro Arg Ile Glu Tyr Arg Val Glu Ser Leu Ala
Cys Leu Val Lys
35 40 45
CA 02315974 2000-06-20
-40-
Ala Gln Glu Thr Ser Gly Glu Glu Ile Ser Lys Phe Tyr Leu Pro Asn
50 55 60
Cys Asn Lys Asn Gly Phe Tyr His Ser Arg Gln Cys Glu Thr Ser Met
65 70 75 80
Asp Gly Glu Ala Gly Leu Cys Trp Cys Val Tyr Pro Trp Asn Gly Lys
85 90 95
Arg Ile Pro Gly Ser Pro Glu Ile Arg Gly Asp Pro Asn Cys Gln Ile
100 105 110
Tyr Phe Asn Val Gln Asn
115
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
40:
GlyLys Gly GlyLys His HisLeu Gly Leu GluGlu Pro Lys LysLeu
1 5 10 15
ArgPro Pro ProAla Arg ThrPro Cys Gln GlnGlu Leu Asp GlnVal
20 25 30
LeuGlu Arg IleSer Thr MetArg Leu Pro AspGlu Arg Gly ProLeu
35 40 45
GluHis Leu TyrSer Leu HisIle Pro Asn CysAsp Lys His GlyLeu
50 55 60
TyrAsn Leu LysGln Cys LysMet Ser Leu AsnGly Gln Arg GlyGlu
65 70 75 80
CysTrp Cys ValAsn Pro AsnThr Gly Lys LeuIle Gln Gly AlaPro
85 90 95
ThrIle Arg GlyAsp Pro GluCys His Leu PheTyr Asn Glu GlnGln
100 105 110
CA 02315974 2000-06-20
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Glu Ala Arg Gly Val His Thr Gln Arg Met Gln
115 120
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 114 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
41:
Gly HisAla Lys AspSer GlnArg Tyr Lys Val AspTyr Glu Ser Gln
1 5 10 15
Ser ThrAsp Thr GlnAsn PheSer Ser Glu Ser LysArg Glu Thr Glu
20 25 30
Tyr GlyPro Cys ArgArg GluMet Glu Asp Thr LeuAsn His Leu Lys
35 40 45
Phe LeuAsn Val LeuSer ProArg Gly Val His IlePro Asn Cys Asp
50 55 60
Lys LysGly Phe TyrLys LysLys Gln Cys Arg ProSer Lys Gly Arg
65 70 75 80
Lys ArgGly Phe CysTrp CysVal Asp Lys Tyr GlyGln Pro Leu Pro
85 90 95
Gly TyrThr Thr LysGly LysGlu Asp Val His CysTyr Ser Met Gln
100 105 110
Ser Lys
CA 02315974 2000-06-20
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(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
42:
LysVal Asn Gly AlaPro ArgGlu Asp Ala ArgPro Val Pro GlnGly
1 5 10 15
SerCys Gln Ser GluLeu HisArg Ala Leu GluArg Leu Ala AlaSer
20 25 30
GlnSer Arg Thr HisGlu AspLeu Tyr Ile IlePro Ile Pro AsnCys
35 40 45
AspArg Asn Gly AsnPhe HisPro Lys Gln CysHis Pro Ala LeuAsp
50 55 60
GlyGln Arg Gly LysCys TrpCys Val Asp ArgLys Thr Gly ValLys
65 70 75 80
LeuPro Gly Gly LeuGlu ProLys Gly Glu LeuAsp Cys His GlnLeu
85 90 95
AlaAsp Ser Phe ArgGlu
100
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
CA 02315974 2000-06-20
- 43 -
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID
NO:
43:
Leu ThrGln Ser LysPhe Val GlyGly Ala Glu AsnThr Ala His Pro
1 5 10 15
Arg IleIle Ser AlaPro Glu MetArg Gln Glu SerGlu Gln Gly Pro
20 25 30
Cys ArgArg His MetGlu Ala SerLeu Gln Glu LeuLys Ala Ser Pro
35 40 45
Arg MetVal Pro ArgAla Val TyrLeu Pro Asn CysAsp Arg Lys Gly
50 55 60
Phe TyrLys Arg LysGln Cys LysPro Ser Arg GlyArg Lys Arg Gly
65 70 75 80
Ile CysTrp Cys ValAsp Lys TyrGly Met Lys LeuPro Gly Met Glu
85 90 95
Tyr ValAsp Gly AspPhe Gln CysHis Thr Phe AspSer Ser Asn Val
100 105 110
Glu
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID
NO:
44:
ProGln Ala Gly ThrAla Arg Gln Asp Asn Arg Arg Asp Gln
Pro Val
1 5 10 15
GlnArg Asn Pro GlyThr Ser Thr Pro Gln Pro Asn Ser Ala
Thr Ser
20 25 30
GlyVal Gln Asp ThrGlu Met Pro Cys Arg His Leu Asp Ser
Gly Arg
35 40 45
CA 02315974 2000-06-20
-44-
Val Leu Gln Gln Leu Gln Thr Glu Val Tyr Arg Gly Ala Gln Thr Leu
50 55 60
Tyr Val Pro Asn Cys Asp His Arg Gly Phe Tyr Arg Lys Arg Gln Cys
65 70 75 80
Arg Ser Ser Gln Gly Gln Arg Arg Gly Pro Cys Trp Cys Val Asp Arg
85 90 95
Met Gly Lys Ser Leu Pro Gly Ser Pro Asp Gly Asn Gly Ser Ser Ser
100 105 110
Cys Pro Thr Gly Ser Ser Gly
115
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 121 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
45:
GlyGly Lys His HisLeu GlyLeu Glu Glu ProLys Lys Leu ArgPro
1 5 10 15
ProPro Ala Arg ThrPro CysGln Gln Glu LeuAsp Gln Val LeuGlu
20 25 30
ArgIle Ser Thr MetArg LeuPro Asp Glu ArgGly Pro Leu GluHis
35 40 45
LeuTyr Ser Leu HisIle ProAsn Cys Asp LysHis Gly Leu TyrAsn
50 55 60
LeuLys Gln Cys LysMet SerLeu Asn Gly GlnArg Gly Glu CysTrp
65 70 75 80
CysVal Asn Pro AsnThr GlyLys Leu Ile GlnGly Ala Pro ThrIle
85 90 95
ArgGly Asp Pro GluCys HisLeu Phe Tyr AsnGlu Gln Gln GluAla
100 105 110
CA 02315974 2000-06-20
-45-
Arg Gly Val His Thr Gln Arg Met Gln
115 120
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Lys Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser
1 5 10 15
Glu Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu
20 25 30
Asp Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly
35 40 45
Val His Ile Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln
50 55 60
Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val Asp
65 70 75 80
Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp
85 90 95
Val His Cys Tyr Ser Met Gln Ser Lys
100 105
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
CA 02315974 2000-06-20
-46-
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
His Pro Leu His Ser Lys Ile Ile Ile Ile Lys Lys Gly His Ala Lys
1 5 10 15
Asp Ser Gln Arg Tyr
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 135 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
48:
AspGlu Ala Ile HisCys Pro ProCys Ser GluGlu Lys Leu AlaArg
1 5 10 15
CysArg Pro Pro ValGly Cys GluGlu Leu ValArg Glu Pro GlyCys
20 25 30
GlyCys Cys Ala ThrCys Ala LeuGly Leu GlyMet Pro Cys GlyVal
35 40 45
TyrThr Pro Arg CysGly Ser GlyLeu Arg CysTyr Pro Pro ArgGly
50 55 60
ValGlu Lys Pro LeuHis Thr LeuMet His GlyGln Gly Val CysMet
65 70 75 80
GluLeu Ala Glu IleGlu Ala IleGln Glu SerLeu Gln Pro SerAsp
85 90 95
CA 02315974 2000-06-20
-47-
Lys Asp Glu Gly Asp His Pro Asn Asn Ser Phe Ser Pro Cys Ser Ala
100 105 110
His Asp Arg Arg Cys Leu Gln Lys His Phe Ala Lys Ile Arg Asp Arg
115 120 125
Ser Thr Ser Gly Gly Lys Met
130 135
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQ
SEQUENCE ID
DESCRIPTION: NO:
49:
LysPhe Val Gly GlyAla GluAsn Thr Ala HisPro Arg Ile IleSer
1 5 10 15
AlaPro Glu Met ArgGln GluSer Glu Gln GlyPro Cys Arg ArgHis
20 25 30
MetGlu Ala Ser LeuGln GluLeu Lys Ala SerPro Arg Met ValPro
35 40 45
ArgAla Val Tyr LeuPro AsnCys Asp Arg LysGly Phe Tyr LysArg
50 55 60
LysGln Cys Lys ProSer ArgGly Arg Lys ArgGly Ile Cys TrpCys
65 70 75 80
ValAsp Lys Tyr GlyMet LysLeu Pro Gly MetGlu Tyr Val AspGly
85 90 95
AspPhe Gln Cys HisThr PheAsp Ser Ser AsnVal Glu
100 105
CA 02315974 2000-06-20
-48-
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE type: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
His Thr Arg Ile Ser Glu Leu Lys Ala Glu Ala Val Lys Lys Asp Arg
1 5 10 15
Arg Lys Lys Leu Thr Gln Ser
