U.S. patent application number 12/070643 was filed with the patent office on 2009-01-08 for peptides and related compounds having thrombopoietic activity.
This patent application is currently assigned to Amgen Inc.. Invention is credited to Cynthia Hartley, Hosung Min, Karen C. Sitney.
Application Number | 20090011497 12/070643 |
Document ID | / |
Family ID | 26953908 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090011497 |
Kind Code |
A1 |
Min; Hosung ; et
al. |
January 8, 2009 |
Peptides and related compounds having thrombopoietic activity
Abstract
The present invention relates generally to novel peptides and
related compounds that have thrombopoietic activity. The compounds
of the invention may be used to increase production of platelets or
platelet precursors (e.g. megakaryocytes) in a mammal.
Inventors: |
Min; Hosung; (Newbury Park,
CA) ; Sitney; Karen C.; (Studio City, CA) ;
Hartley; Cynthia; (Ventura, CA) |
Correspondence
Address: |
AMGEN INC.
MAIL STOP 28-2-C, ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Amgen Inc.
|
Family ID: |
26953908 |
Appl. No.: |
12/070643 |
Filed: |
February 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10269806 |
Oct 10, 2002 |
7332474 |
|
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12070643 |
|
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60328666 |
Oct 11, 2001 |
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Current U.S.
Class: |
435/325 ;
435/252.3; 435/252.33; 435/320.1; 530/350; 530/391.1; 536/23.1 |
Current CPC
Class: |
C07K 14/524 20130101;
A61P 7/04 20180101; A61P 7/00 20180101; A61K 38/00 20130101 |
Class at
Publication: |
435/325 ;
536/23.1; 530/350; 530/391.1; 435/320.1; 435/252.33; 435/252.3 |
International
Class: |
C12N 5/06 20060101
C12N005/06; C12N 15/11 20060101 C12N015/11; C07K 14/00 20060101
C07K014/00; C07K 16/18 20060101 C07K016/18; C12N 15/00 20060101
C12N015/00; C12N 1/20 20060101 C12N001/20 |
Claims
1. A compound that binds to an mpl receptor comprising the
sequence: X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18
wherein X1-X4, X9-X10, and X13-X18 are each independently an amino
acid, said compound having a binding affinity for the mpl receptor
greater than that of the sequence:
X19-X20-1-E-G-P-T-L-R-Q-W-L-A-A-R-A-X21-X22, wherein X19-X20 and
X21-X22 are each independently an amino acid, and physiologically
acceptable salts thereof.
2. A compound that binds to an mpl receptor comprising the
sequence: X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18
wherein X1-X4, X9-X10, and X13-X18 are each independently an amino
acid, said compound having a bioactivity greater than that of the
sequence: X19-X20-I-E-G-P-T-L-R-Q-W-L-A-A-R-A-X21-X22, wherein
X19-X20 and X21-X22 are each independently an amino acid, and
physiologically acceptable salts thereof.
3. The compound according to claim 1 wherein: X1 is selected from
the group consisting of A, V, W, M, G, Y, C, Q, E, R and H; X2 is
selected from the group consisting of A, V, L, I, G, S, and C; X3
is selected from the group consisting of L, I, P, W, G, S, D, K and
R; X4 is selected from the group consisting of L, G, Q, D, E and H;
X9 is selected from the group consisting of K and R; X10 is
selected from the group consisting of Q and E; X13 is selected from
the group consisting of A, V, L, S, Q, E and R X14 is selected from
the group consisting of A, W, T, Y, C and Q; X15 is selected from
the group consisting of V, L, G, Y and R; X16 is selected from the
group consisting of A, L, F, G and R; X17 is selected from the
group consisting of A, V, L, M, G, C, Q and N; X18 is selected from
the group consisting of A, V, P, M, F, G, C, Q and K.
4. The compound according to claim 2 wherein: X1 is selected from
the group consisting of A, V, W, M, G, C, E, and R; X2 is selected
from the group consisting of A, V, L, M, F, G, S, C, D and R; X3 is
selected from the group consisting of A, L, I, P, W, Q, K and R; X4
is selected from the group consisting of L G Q D and E; X9 is
selected from the group consisting of K, R, and H; X10 is selected
from the group consisting of Q and E; X13 is selected from the
group consisting of A, L P, F, G, Q, N, E and R; X14 is selected
from the group consisting of L, W, M, C, Q and H; X15 is selected
from the group consisting of V, L, P, G, Y and R; X16 is selected
from the group consisting of A, V, L, F, S, Q, K and R; X17 is
selected from the group consisting of A, V, L, W, M, G, S, C and N;
X18 is selected from the group consisting of A, V, P, M, G, C, Q
and K;
5. A compound that binds to an mpl receptor comprising the
sequence: X1-X2-R-E-G-P-T-L-R-Q-W-L-X13-W-R-R-X17-X18 wherein X1,
X2, X13, X17 and X18 are each independently an amino acid.
6. A compound that binds to an mpl receptor comprising a sequence
which is selected from the group consisting of SEQ ID NO 2 to SEQ
ID NO 30, inclusive. TABLE-US-00017 SEQ ID PEPTIDE SEQUENCE NO:
GAREGPTLRQWLEWVRVG 2 RDLDGPTLRQWLPLPSVQ 3 ALRDGPTLKQWLEYRRQA 4
ARQEGPTLKEWLFWVRMG 5 EALLGPTLREWLAWRRAQ 6 MARDGPTLREWLRTYRMM 7
WMPEGPTLKQWLFHGRGQ 8 HIREGPTLRQWLVALRMV 9 QLGHGPTLRQWLSWYRGM 10
ELRQGPTLHEWLQHLASK 11 VGIEGPTLRQWLAQRLNP 12 WSRDGPTLREWLAWRAVG 13
AVPQGPTLKQWLLWRRCA 14 RIREGPTLKEWLAQRRGF 15 RFAEGPTLREWLEQRKLV 16
DRFQGPTLREWLAAIRSV 17 AGREGPTLREWLNMRVWQ 18 ALQEGPTLRQWLGWGQWG 19
YCDEGPTLKQWLVCLGLQ 20 WCKEGPTLREWLRWGFLC 21 CSSGGPTLREWLQCRRMQ 22
CSWGGPTLKQWLQCVRAK 23 CQLGGPTLREWLACRLGA 24 CWEGGPTLKEWLQCLVER 25
CRGGGPTLHQWLSCFRWQ 26 CRDGGPTLRQWLACLQQK 27 ELRSGPTLKEWLVWRLAQ 28
GCRSGPTLREWLACREVQ 29 TCEQGPTLRQWLLCRQGR 30
7. The compound according to claim 1, 2 or 6 which is cyclic
8. The compound according to claim 1, 2 or 6 wherein at least one
of the amino acid residues has a D configuration.
9. The compound according to claim 1, 2 or 6 wherein all of the
amino acid residues have a D configuration.
10. A dimer or multimer of the compounds according to claims 1, 2
or 6.
11. A composition of matter that binds to an mpl receptor
comprising the formula:
(LN1).sub.1-(TMP1).sub.a-(LN2).sub.m-(TMP2).sub.b-(LN3).sub.n-(-
TMP3).sub.c-(LN4).sub.o-(TMP4).sub.d wherein TMP1, TMP2, TMP3 and
TMP4 are each independently selected from the group consisting of
the compounds of claims 1, 2 and 6; LN1, LN2, LN3 and LN4 are each
independently a linker; a, b, c and d are each independently an
integer from zero to 20; and l, m, n and o are each independently
an integer from zero to twenty.
12. The composition according to claim 11 further comprising a
vehicle and having the formula:
(V1).sub.v-(LN1).sub.1-(TMP1).sub.a-(LN2).sub.m-(TMP2).sub.b-(LN3).sub.n--
(TMP3).sub.c-(LN4).sub.o-(TMP4).sub.d-(V2).sub.w wherein V1 and V2
are each independently a vehicle, and v and w are each
independently an integer from 0 to 1.
13. The compound according to claim 12 wherein LN1, LN2, LN3 and
LN4 comprise peptides.
14. The composition according to claim 12 wherein V1 and/or V2
comprise an Fc domain.
15. The composition according to claim 12 wherein V1 and/or V2
comprise an IgG1 Fc domain.
16. A polynucleotide encoding a composition of matter selected from
the group consisting of the compositions of claim 12.
17. An expression vector comprising the polynucleotide of claim
12.
18. A host cell comprising the expression vector of claim 12.
19. The host cell according to claim 12 wherein the cell is an E.
coli cell.
20. The host cell according to claim 12 wherein the cell is a
prokaryotic cell.
21. The host cell according to claim 12 wherein the cell is a
eukaryotic cell.
22. A pharmaceutical composition comprising an effective amount of
a composition according to claim 12 in admixture with a
pharmaceutically acceptable carrier thereof.
23. A method of treating thrombocytopenia in a mammal comprising
administering a therapeutically effective amount of the composition
according to claim 12.
24. A method of increasing megakaryocytes or platelets in a patient
in need thereof, which comprises administering to said patient an
effective amount of a compound according to claim 12.
25. A compound that binds to an mpl receptor comprising the
formula: (V1).sub.v-(TMP1).sub.a-(V2).sub.w wherein V1 and V2 are
each an IgG1 Fc domain, provided that where v is one, w is zero and
where v is zero, w is one and TMP1 is a peptide of SEQ ID NO 2 to
30; a is an integer from one to twenty.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 10/269,806, filed Oct. 10, 2002, which claims the benefit of
U.S. Provisional Application No. 60/328,666 filed Oct. 11, 2001,
which is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to peptides and
related compounds that have thrombopoietic activity. The compounds
of the invention may be used to increase production of platelets or
platelet precursors (e.g. megakaryocytes) in a mammal.
BACKGROUND OF THE INVENTION
[0003] This invention relates to compounds, especially peptides,
that have the ability to stimulate in vitro and in vivo production
of platelets and their precursor cells, e.g., megakaryocytes. The
following is provided as background regarding two proteins that are
known to have thrombopoietic activity: thrombopoietin (TPO) and
megakaryocyte growth and development factor (MGDF).
[0004] The cloning of endogenous thrombopoietin (TPO) (Lok et al.,
Nature 369:568-571 (1994); Bartley et al., Cell 77:1117-1124
(1994); Kuter et al., Proc. Natl. Acad. Sci. USA 91:11104-11108
(1994); de Sauvage et al., Nature 369:533-538 (1994); Kato et al.,
Journal of Biochemistry 119:229-236 (1995); Chang et al., Journal
of Biological Chemistry 270:511-514 (1995)) has rapidly increased
our understanding of megakaryopoiesis (megakaryocyte production)
and thrombopoiesis (platelet production).
[0005] Endogenous human TPO, a 60 to 70 kDa glycosylated protein
primarily produced in the liver and kidney, consists of 332 amino
acids (Bartley et al., Cell 77:1117-1124 (1994); Chang et al.,
Journal of Biological Chemistry 270:511-514 (1995)). The protein is
highly conserved between different species, and has 23% homology
with human erythropoietin (Gurney et al., Blood 85:981-988 (1995))
in the amino terminus (amino acids 1 to 172) (Bartley et al., Cell
77:1117-1124 (1994)). Endogenous TPO has been shown to possess all
of the characteristics of the key biological regulator of
thrombopoiesis. Its in vitro actions include specific induction of
megakaryocyte colonies from both purified murine hematopoietic stem
cells (Zeigler et al., Blood 84:4045-4052 (1994)) and human
CD34.sup.+ cells (Lok et al., Nature 369:568-571 (1994); Rasko et
al., Stem Cells 15:33-42 (1997)), the generation of megakaryocytes
with increased ploidy (Broudy et al., Blood 85:402-413 (1995)), and
the induction of terminal megakaryocyte maturation and platelet
production (Zeigler et al., Blood 84:4045-4052 (1994); Choi et al.,
Blood 85:402-413 (1995)). Conversely, synthetic antisense
oligodeoxynucleotides to the TPO receptor (c-mpl) significantly
inhibit the colony-forming ability of megakaryocyte progenitors
(Methia et al., Blood 82:1395-1401 (1993)). Moreover, c-mpl
knock-out mice are severely thrombocytopenic and deficient in
megakaryocytes (Alexander et al., Blood 87:2162-2170 (1996)).
[0006] Recombinant human MGDF (rHuMGDF, Amgen Inc., Thousand Oaks,
Calif.) is another thrombopoietic polypeptide related to TPO. It is
produced using E. coli transformed with a plasmid containing cDNA
encoding a truncated protein encompassing the amino-terminal
receptor-binding domain of human TPO (Ulich et al., Blood
86:971-976 (1995)). The polypeptide is extracted, refolded, and
purified, and a poly[ethylene glycol] (PEG) moiety is covalently
attached to the amino terminus. The resulting molecule is referred
to herein as PEG-rHuMGDF or MGDF for short.
[0007] Various studies using animal models (Ulich, T. R. et al.,
Blood 86:971-976 (1995); Hokom, M. M. et al., Blood 86:4486-4492
(1995)) have clearly demonstrated the therapeutic efficacies of TPO
and MGDF in bone marrow transplantation and in the treatment of
thrombocytopenia, a condition that often results from chemotherapy
or radiation therapy. Preliminary data in humans have confirmed the
utility of MGDF in elevating platelet counts in various settings.
(Basser et al., Lancet 348:1279-81 (1996); Kato et al., Journal of
Biochemistry 119:229-236 (1995); Ulich et al., Blood 86:971-976
(1995)). MGDF might be used to enhance the platelet donation
process, since administration of MGDF increases circulating
platelet counts to about three-fold the original value in healthy
platelet donors.
[0008] TPO and MGDF exert their action through binding to the c-mpl
receptor which is expressed primarily on the surface of certain
hematopoietic cells, such as megakaryocytes, platelets, CD34.sup.+
cells and primitive progenitor cells (Debili, N. et al., Blood
85:391-401 (1995); de Sauvage, F. J. et al, Nature 369:533-538
(1994); Bartley, T. D., et al., Cell 77:1117-1124 (1994); Lok, S.
et al., Nature 369: 565-8 (1994)). Like most receptors for
interleukins and protein hormones, c-mpl belongs to the class I
cytokine receptor superfamily (Vigon, I. et al., Proc. Natl. Acad.
Sci. USA 89:5640-5644 (1992)). Activation of this class of
receptors involves ligand-binding induced receptor homodimerization
which in turn triggers the cascade of signal transducing
events.
[0009] In general, the interaction of a protein ligand with its
receptor often takes place at a relatively large interface.
However, as demonstrated in the case of human growth hormone bound
to its receptor, only a few key residues at the interface actually
contribute to most of the binding energy (Clackson, T. et al.,
Science 267:383-386 (1995)). This and the fact that the bulk of the
remaining protein ligand serves only to display the binding
epitopes in the right topology makes it possible to find active
ligands of much smaller size. Accordingly, molecules of only
"peptide" length (e.g., 2 to 80 amino acids) can bind to the
receptor protein of a given large protein ligand. Such peptides may
mimic the bioactitivy of the large protein ligand or, through
competitive binding, inhibit the bioactivity of the large protein
ligand, and are commonly referred to as "peptide mimetics" or
"mimetic peptides."
[0010] Phage display peptide libraries have emerged as a powerful
technique in identifying such peptide mimetics. See, e.g., Scott,
J. K. et al., Science 249:386 (1990); Devlin, J. J. et al., Science
249:404 (1990); U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S.
Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530,
issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11,
1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No.
5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19,
1996; and WO 98/15833, published Apr. 16, 1998 (each of which is
incorporated by reference in its entirety). In such libraries,
random peptide sequences are displayed by fusion with coat proteins
of filamentous phage. Typically, the displayed peptides are
affinity-eluted against an antibody-immobilized extracellular
domain of a receptor. The retained phages may be enriched by
successive rounds of affinity purification and repropagation. The
best binding peptides may be sequenced to identify key residues
within one or more structurally related families of peptides. See,
e.g., Cwirla, et al. (1997), Science 276: 1696-9. The peptide
sequences may also suggest which residues may be safely replaced by
alanine scanning or by mutagenesis at the DNA level. Mutagenesis
libraries may be created and screened to further optimize the
sequence of the best binders. Lowman (1997), Ann. Rev. Biophys.
Biomol. Struct. 26: 401-24.
[0011] Structural analysis of protein-protein interaction may also
be used to suggest peptides that mimic the binding activity of
large protein ligands. In such an analysis, the crystal structure
may suggest the identity and relative orientation of critical
residues of the large protein ligand, from which a peptide may be
designed. See, e.g., Takasaki, et al. (1997), Nature Biotech, 15:
1266-70. These analytical methods may also be used to investigate
the interaction between a receptor protein and peptides selected by
phage display, which may suggest further modification of the
peptides to increase binding affinity.
[0012] Other methods compete with phage display in peptide
research. A peptide library can be fused to the carboxyl terminus
of the lac repressor and expressed in E. coli. Another E.
coli-based method allows display on the cell's outer membrane by
fusion with a peptidoglycan-associated lipoprotein (PAL).
Hereinafter, these and related methods are collectively referred to
as "E. coli display." In another method, translation of random RNA
is halted prior to ribosome release, resulting in a library of
polypeptides with their associated RNA still attached. Hereinafter,
this and related methods are collectively referred to as "ribosome
display." Other methods employ peptides linked to RNA; for example,
PROfusion technology, Phylos, Inc. See, for example, Roberts &
Szostak (1997), Proc. Natl. Acad. Sci. USA, 94: 12297-303.
Hereinafter, this and related methods are collectively referred to
as "RNA-peptide screening." Chemically derived peptide libraries
have been developed in which peptides are immobilized on stable,
non-biological materials, such as polyethylene rods or
solvent-permeable resins. Another chemically derived peptide
library uses photolithography to scan peptides immobilized on glass
slides. Hereinafter, these and related methods are collectively
referred to as "chemical-peptide screening." Chemical-peptide
screening may be advantageous in that it allows use of D-amino
acids and other unnatural analogues, as well as non-peptide
elements. Both biological and chemical methods are reviewed in
Wells & Lowman (1992), Curr. Opin. Biotechnol, 3: 355-62.
Conceptually, one may discover peptide mimetics of any protein
using phage display, RNA-peptide screening, and the other methods
mentioned above.
[0013] By using the phage display peptide library technique, small
peptide molecules that act as agonists of the c-mpl receptor were
discovered (Cwirla, S. E. et al., Science 276:1696-1699 (1997)). In
such a study, random small peptide sequences displayed as fusions
to the coat proteins of filamentous phage were affinity-eluted
against the antibody-immobilized extracellular domain of c-mpl and
the retained phages were enriched for a second round of affinity
purification. This binding selection and repropagation process was
repeated many times to enrich the pool of tighter binders. As a
result, two families of c-mpl-binding peptides, unrelated to each
other in their sequences, were first identified. Mutagenesis
libraries were then created to further optimize the best binders,
which finally led to the isolation of a very active peptide with an
IC.sub.50=2 nM and an EC.sub.50=400 nM (Cwirla, S. E. et al.,
Science 276:1696-1699 (1997)). This 14-residue TPO mimetic peptide
has no apparent sequence homology to TPO or MGDF. The structure of
this particular TPO mimetic peptide (TMP) compound is as
follows:
TABLE-US-00001 (SEQ ID NO: 1) Ile Glu Gly Pro Thr Leu Arg Gln Trp
Leu Ala Ala Arg Ala or, IEGPTLRQWLAARA
using single letter amino acid abbreviations.
[0014] Previously, in a similar study on EPO mimetic peptides, an
EPO mimetic peptide (EMP) was discovered using the same technique
(Wrighton, N. C. et al., Science, 273:458-463 (1996)), and was
found to act as a dimer in binding to the EPO receptor (EPOR). The
(ligand).sub.2/(receptor).sub.2 complex thus formed had a C2
symmetry according to X-ray crystallographic data (Livnah, O. et
al., Science 273:464-471 (1996)). Based on this structural
information, a covalently linked dimer of EMP in which the
C-termini of two EMP monomers were crosslinked with a flexible
spacer was designed and found to have greatly enhanced binding as
well as in vitro/in vivo bioactivity (Wrighton, N. C., et al.,
Nature Biotechnology 15:1261-1265 (1997)).
[0015] A similar C-terminal dimerization strategy was applied to
the TPO mimetic peptide (TMP). (Cwirla, S. E. et al., Science
276:1696-1699 (1997)). It was found that a C-terminally linked
dimer (C--C link) of a particular TPO mimetic peptide had an
improved binding affinity of 0.5 nM and an increased in vitro
activity (EC.sub.50=0.1 nM) in cell proliferation assays (Cwirla,
S. E. et al., Science 276:1696-1699 (1997)).
[0016] The availability of recombinant proteins for therapeutic use
has led to advances in protein modifications in order to enhance or
improve the properties of such proteins as pharmaceutical agents.
Such modifications can provide enhanced protein protection and
decreased degradation by reducing or eliminating proteolysis.
Additional advantages include, under certain circumstances,
increasing the stability, circulation time and biological activity
of the therapeutic protein. A review article describing protein
modifications is Francis, Focus on Growth Factors 3:4-10 (May 1992)
(published by Mediscript, London, UK).
[0017] Useful modifications of protein therapeutic agents include
linkage to polymers such as polyethylene glycol (PEG) and dextran.
Such modifications are discussed in detail in a patent application
entitled "Modified Peptides as Therapeutic Agents," U.S. Ser. No.
09/428,082, PCT appl. no. WO 00/24782, which is hereby incorporated
by reference in its entirety.
[0018] Another such modification is the use of an Fc region of an
immunoglobulin molecule. Antibodies comprise two functionally
independent parts; a variable domain known as "Fab" which binds an
antigen, and a constant domain known as "Fc" which provides the
link to effector functions such as complement or phagocytic cells.
The Fc portion of an immunoglobulin has a long plasma half-life,
whereas the Fab is short-lived. (Capon, et al. Nature 337,
525-531(1989)).
[0019] Therapeutic protein products have been constructed using the
Fc domain to provide longer half life or to incorporate functions
such as Fc receptor binding, protein A binding, complement fixation
and placental transfer which all reside in the Fc proteins of
immunoglobulins. (Capon, et al., Nature 337:525-531 (1989)). For
example, the Fc region of an IgG1 antibody has been fused to
CD30-L, a molecule which binds CD30 receptors expressed on
Hodgkin's Disease tumor cells, anaplastic lymphoma cells, T-cell
leukemia cells and other malignant cell types. See, U.S. Pat. No.
5,480,981. IL-10, an anti-inflammatory and antirejection agent has
been fused to murine Fc 2a in order to increase the cytokine's
short circulating half-life (Zheng, X. et al., The Journal of
Immunology, 154: 5590-5600 (1995)). Studies have also evaluated the
use of tumor necrosis factor receptor linked with the Fc protein of
human IgG1 to treat patients with septic shock (Fisher, C. et al.,
N. Engl. J. Med., 334: 1697-1702 (1996); Van Zee, K. et al., The
Journal of Immunology, 156: 2221-2230 (1996)). Fc has also been
fused with CD4 receptor to produce a therapeutic protein for
treatment of AIDS. See, Capon et al., Nature, 337:525-531 (1989).
In addition, interleukin 2 has been fused to the Fc portion of IgG1
or IgG3 to overcome the short half life of interleukin 2 and its
systemic toxicity. See, Harvill et al., Immunotechnology, 1: 95-105
(1995).
[0020] Published PCT Application No. WO 00/24770 discloses specific
thrombopoietic compounds, generally peptides, having a tandem
(i.e., N- to C-terminus) orientation and tandem peptide dimers
attached at the N-terminus thereof to a carrier molecule, such as a
linear polymer, an oligosaccharide or an Fc group.
[0021] There remains a need to provide additional compounds having
a superior biological activity of stimulating the production of
platelets (thrombopoietic activity) and/or platelet precursor
cells, especially megakaryocytes (megakaryopoietic activity). There
also remains a need to provide compounds that exhibit
thrombopoietic activity and that also possess superior therapeutic
qualities, such as a long half-life. Such compounds will exhibit
advantageous properties relating to production, isolation,
purification, biological activity, stability and circulation time.
The present invention provides new compounds having such
activity(ies), and related aspects.
SUMMARY OF THE INVENTION
[0022] The present invention concerns therapeutic compounds that
bind to the c-mpl receptor (hereinafter "the mpl receptor"). More
particularly, the present invention provides a group of compounds
that demonstrate an improved ability to bind to and/or trigger a
transmembrane signal through, i.e., activating, the mpl receptor,
which is the same receptor that mediates the activity of endogenous
thrombopoietin (TPO). Thus, the inventive compounds have superior
thrombopoietic activity, i.e., the ability to stimulate, in vivo
and in vitro, the production of platelets and/or
megakaryocytopoietic activity, i.e., the ability to stimulate, in
vivo and in vitro, the production of platelet precursors. Further,
certain of the inventive compounds also exhibit superior
therapeutic properties, such as improved plasma half-life,
biological activity and in vivo circulation time.
[0023] In one aspect, the present invention provides a compound
that binds to an mpl receptor comprising the sequence:
X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18
wherein X1-X4, X9-X10, and X13-X18 are each independently an amino
acid as defined herein, and wherein the compound has a binding
affinity for the mpl receptor and/or a bioactivity greater than
that of the sequence:
I-E-G-P-T-L-R-Q-W-L-A-A-R-A.
[0024] In yet a further aspect, the present invention provides a
compound that binds to an mpl receptor having the sequence:
X1-X2-R-E-G-P-T-L-R-Q-W-L-X13-W-R-R-X17-X18
wherein X1, X2, X13, X17 and X18 are each independently an amino
acid.
[0025] In yet another aspect, the present invention provides a
compound that binds to an mpl receptor comprising a sequence which
is selected from the group consisting of SEQ ID NO 2 to SEQ ID NO
30, inclusive.
[0026] In another aspect, the present invention is a dimer or
multimer of a compound comprising a sequence which is selected from
the group consisting of SEQ ID NO 2 to SEQ ID NO 30.
[0027] In another aspect, the present invention provides a
composition of matter that binds to an mpl receptor having the
formula:
(LN1).sub.1-(TMP1).sub.a-(LN2).sub.m-(TMP2).sub.b-(LN3).sub.n-(TMP3).sub-
.c-(LN4).sub.o-(TMP4).sub.d
wherein TMP1, TMP2, TMP3 and TMP4 are each independently selected
from the group consisting of the TMPs disclosed herein; LN1, LN2,
LN3 and LN4 are each independently a linker; a, b, c and d are each
independently an integer from zero to ten; and l, m, n and o are
each independently an integer from zero to twenty.
[0028] In yet another aspect, the present invention provides a
composition of matter that binds to an mpl receptor having the
formula:
(V1).sub.v-(LN1).sub.1-(TMP1).sub.a-(LN2).sub.m-(TMP2).sub.b-(LN3).sub.n-
-(TMP3).sub.c-(LN4).sub.o-(TMP4).sub.d-(V2).sub.w
wherein V1 and V2 are each independently a vehicle, and v and w are
each independently an integer from 0 to 1.
[0029] The compounds of this invention may be prepared by standard
synthetic methods, recombinant DNA techniques, or any other methods
of preparing peptides and fusion proteins. Compounds of this
invention that encompass non-peptide portions may be synthesized by
standard organic chemistry reactions, in addition to standard
peptide chemistry reactions when applicable.
[0030] The compounds of this invention may be used for therapeutic
or prophylactic purposes by formulating them with appropriate
pharmaceutical carrier materials and administering an effective
amount to a patient, such as a human (or other mammal) in need
thereof. The vehicle-linked peptide may have activity comparable
to--or even greater than--the natural ligand mimicked by the
peptide, here, thrombopoietin.
[0031] In another aspect, the present invention provides methods of
treating thrombocytopenic disorders. In other aspects, the present
invention provides methods of increasing megakaryocytes or
platelets and methods of producing compounds described herein.
[0032] In yet another aspect, the present invention also provides
for related pharmaceutical compositions.
[0033] In other aspects, the present invention provides for
polynucleotides encoding the compositions of matter disclosed
herein, expression vectors comprising the polynucleotides and host
cells comprising the expression vectors.
BRIEF DESCRIPTION OF THE FIGURES
[0034] Numerous other aspects and advantages of the present
invention will therefore be apparent upon consideration of the
following detailed description thereof, reference being made to the
drawings wherein:
[0035] FIG. 1 shows exemplary structures of peptide and
peptide-linker compounds of the present invention.
[0036] FIG. 2 shows exemplary structures of peptide-vehicle and
peptide-linker-vehicle compounds of the present invention.
[0037] FIG. 3 shows the nucleic acid and amino acid sequences (SEQ
ID NOS: 31 and 32, respectively) of human IgG1 Fc that may be used
as a preferred vehicle in this invention.
[0038] FIG. 4 shows exemplary Fc monomer and dimers compounds of
the present invention that may be derived from an IgG1 antibody.
"Fc" in the figure represents any of the Fc variants within the
meaning of Fc domain herein. "Peptide" represent any of the
peptides, linker-peptides, peptide-peptide combinations, or any
combination thereof, as disclosed herein. The specific dimers are
as follows:
[0039] FIGS. 4A and 4D show single disulfide-bonded dimers. IgG1
antibodies typically have two disulfide bonds at the hinge region
of the antibody. The Fc domain in FIGS. 4A and 4 D may be formed by
truncation between the two disulfide bond sites or by substitution
of a cysteinyl residue with an unreactive residue (e.g., alanyl).
In FIG. 4A, the Fc domain is linked to the amino terminus of the
peptide; in 4D, at the carboxyl terminus of the peptide.
[0040] FIGS. 4B and 4E show doubly disulfide-bonded dimers. This Fc
domain may be formed by truncation of the parent antibody to retain
both cysteinyl residues in the Fc domain chains or by expression
from a construct including a sequence encoding such an Fc domain.
In FIG. 4B, the Fc domain is linked to the amino terminus of the
peptide; in 4E, at the carboxyl terminus of the peptide.
[0041] FIGS. 4C and 4F show noncovalent dimers. This Fc domain may
be formed by elimination of the cysteinyl residues by either
truncation or substitution. One may desire to eliminate the
cysteinyl residues to avoid impurities formed by reaction of the
cysteinyl residue with cysteinyl residues of other proteins present
in the host cell. The noncovalent bonding of the Fc domains is
sufficient to hold together the dimer. Other dimers may be formed
by using Fc domains derived from different types of antibodies
(e.g., IgG2, IgM).
[0042] FIGS. 4G and 4H show single chain Fc domains attached at the
N-terminus of a peptide (FIG. 4G) and at the C-terminus of a
peptide (FIG. 4H).
[0043] FIG. 5 shows exemplary structures of preferred compounds of
the invention that feature tandem repeats of the pharmacologically
active peptide attached to an Fc domain. FIG. 5A shows a single
chain (or Fc monomer) molecule having attached thereto a tandem
peptide dimer, and may also represent the DNA construct for the
molecule. FIG. 5B shows an Fc dimer in which the linker-peptide
portion is present on only one chain of the Fc dimer. FIG. 5C shows
an Fc dimer having the peptide portion (in this case, a tandem
peptide dimer) on both chains. The dimer of FIG. 5C will form
spontaneously in certain host cells upon expression of a DNA
construct encoding the single chain shown in FIG. 5A. In other host
cells, the cells could be placed in conditions favoring formation
of dimers or the dimers can be formed in vitro. FIGS. 5D through 5I
represent additional exemplary single chain (Fc monomer) and double
chain (Fc dimer) preferred embodiments.
[0044] FIG. 6 shows the nucleic acid sequence (SEQ ID NO 33) and
amino acid sequence (SEQ ID NO 34) for a preferred vector
(20003180) for use in constructing TMP-Fc fusions compounds as
shown in Example 3 herein.
[0045] FIG. 7 shows fragments of exemplary pairs of
oligonucleotides used to create preferred peptides of the present
invention as shown in Example 3. Nucleic acid and amino acid
sequences are provided for each. (SEQ ID NOS 35-150)
[0046] FIG. 8 shows the nucleic acid sequence (SEQ ID NO 151) and
the amino acid sequence (SEQ ID NO 152) of an exemplary vector
(20003182) for use in constructing C-terminal Fc fusion compounds
(i.e., peptide attached at its N-terminus to the C-terminus of the
Fc).
[0047] FIG. 9 shows ELISA dose-response of selected phage
clones.
[0048] FIGS. 10, 11 and 12 show the bioactivity of select compounds
of the present invention.
[0049] FIGS. 13 and 14 show in vivo platelet counts after a single
injection of select compounds of the present invention into
mice.
DETAILED DESCRIPTION OF THE INVENTION
I. Definition of Terms
[0050] The terms used throughout this specification are defined as
follows, unless otherwise limited in specific instances.
[0051] The term "peptide" refers to molecules of approximately 2 to
80 amino acids, with molecules of 3 to 40 amino acids preferred.
Exemplary peptides may be randomly generated by any of the methods
set forth herein such as carried in peptide library (e.g. phage
display library), generated by chemical synthesis, derived by
digestion of proteins and the like.
[0052] The term "randomized" used in connection with peptide
sequences refers to fully random sequences (e.g., selected by phage
display methods or RNA-peptide screening) and sequences in which
one or more residues of a naturally occurring molecule is replaced
by an amino acid residue not present in that position in the
naturally occurring molecule. Exemplary methods for creating and
identifying randomized peptide sequences include phage display, E.
coli display, ribosome display, RNA-peptide screening, chemical
screening, and the like.
[0053] The term "dimer" as applied to peptides refers to molecules
having two peptide chains associated covalently or non-covalently,
with or without linkers. Peptide dimers wherein the peptides are
linked C-terminus to N-terminus may also be referred to as "tandem
repeats" or "tandem dimers." Peptide dimers wherein the peptides
are linked C- to C-terminus, or N- to N-terminus may also be
referred to as "parallel repeats" or "parallel dimers."
[0054] The term "multimer" as applied to peptides refers to
molecules having three or more peptide chains associated
covalently, noncovalently, or by both covalent and non-covalent
interactions, with or without linkers.
[0055] The terms "derivatizing" and "derivative" or "derivatized"
involve processes and resulting compounds in which (1) the compound
has a cyclic portion; for example, cross-linking between cysteinyl
residues within the compound; (2) the compound is cross-linked or
has a cross-linking site; for example, the compound has a cysteinyl
residue and thus forms cross-linked dimers in culture or in vivo;
(3) one or more peptidyl linkage is replaced by a non-peptidyl
linkage; (4) the N-terminus is replaced by --NRR.sup.1,
NRC(O)R.sup.1, --NRC(O)OR.sup.1, --NRS(O).sub.2R.sup.1,
--NHC(O)NHR, a succinimide group, or substituted or unsubstituted
benzyloxycarbonyl-NH-- wherein R and R.sup.1 and the ring
substituents are as defined hereinafter; (5) the C-terminus is
replaced by --C(O)R.sup.2 or --NR.sup.3R.sup.4 wherein R.sup.2,
R.sup.3 and R.sup.4 are as defined hereinafter; and (6) compounds
in which individual amino acid moieties are modified through
treatment with agents capable of reacting with selected side chains
or terminal residues. Derivatives are further described
hereinafter.
[0056] The term "thrombopoietin mimetic peptide," "TPO mimetic
peptide" or "TMP" refers to a peptide that binds to the mpl
receptor and/or has thrombopoietic activity, i.e., the ability to
stimulate, in vivo or in vitro, the production of platelets or
platelet precursors, including but not limited to
megakaryocytes.
[0057] The term "mpl-binding domain" refers to any amino acid
sequence that binds the mpl receptor and comprises naturally
occurring sequences or randomized sequences. Exemplary mpl-binding
domains can be identified or derived by phage display or other
methods mentioned herein.
[0058] The term "mpl receptor agonist" refers to a molecule that
binds to the mpl receptor and increases or decreases one or more
assay parameters as does endogenous thrombopoietin (eTPO), the
native mpl receptor ligand.
[0059] The term "comprising" means that a compound may include
additional amino acids on either or both of the N- or C-termini of
the given sequence. Of course, these additional amino acids should
not significantly interfere with the activity of the compound.
[0060] Additionally, physiologically acceptable salts of the
compounds of this invention are also encompassed herein. The term
"physiologically acceptable salts" refers to any salts that are
known or later discovered to be pharmaceutically acceptable. Some
specific examples are: acetate; trifluoroacetate; hydrohalides,
such as hydrochloride and hydrobromide; sulfate; citrate; tartrate;
glycolate; and oxalate.
[0061] The term "vehicle" refers to a molecule that prevents
degradation and/or increases half-life, reduces toxicity, reduces
immunogenicity and/or increases biological activity of a
therapeutic protein. Exemplary vehicles include an Fc domain (which
is preferred) as well as a linear polymer (e.g., polyethylene
glycol (PEG), polylysine, dextran, etc.); a branched-chain polymer
(see, for example, U.S. Pat. Nos. 4,289,872 to Denkenwalter et al.,
issued Sep. 15, 1981; 5,229,490 to Tam, issued Jul. 20, 1993; WO
93/21259 by Frechet et al., published 28 Oct. 1993); a lipid; a
cholesterol group (such as a steroid); a carbohydrate or
oligosaccharide (e.g., dextran); any natural synthetic protein,
polypeptide or peptide that binds to a salvage receptor; albumin,
including human serum albumin (HSA), leucine zipper domain, and
other such proteins and protein fragments.
[0062] The term "native Fc" refers to molecule or sequence
comprising the sequence of a non-antigen-binding fragment resulting
from digestion of whole antibody, whether in monomeric or
multimeric form. The original immunoglobulin source of the native
Fc is preferably of human origin and may be any of the
immunoglobulins, although IgG1 and IgG2 are preferred. Native Fcs
are made up of monomeric polypeptides that may be linked into
dimeric or multimeric forms by covalent (i.e., disulfide bonds) and
non-covalent association. The number of intermolecular disulfide
bonds between monomeric subunits of native Fc molecules ranges from
1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g.,
IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a
disulfide-bonded dimer resulting from papain digestion of an IgG
(see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The
term "native Fc" as used herein is generic to the monomeric,
dimeric, and multimeric forms.
[0063] The term "Fc variant" refers to a molecule or sequence that
is modified from a native Fc but still comprises a binding site for
the salvage receptor, FcRn. International applications WO 97/34631
(published 25 Sep. 1997) and WO 96/32478 describe exemplary Fc
variants, as well as interaction with the salvage receptor, and are
hereby incorporated by reference in their entirety. Thus, the term
"Fc variant" comprises a molecule or sequence that is humanized
from a non-human native Fc. Furthermore, a native Fc comprises
sites that may be removed because they provide structural features
or biological activity that are not required for the fusion
molecules of the present invention. Thus, the term "Fc variant"
comprises a molecule or sequence that lacks one or more native Fc
sites or residues that affect or are involved in (1) disulfide bond
formation, (2) incompatibility with a selected host cell (3)
N-terminal heterogeneity upon expression in a selected host cell,
(4) glycosylation, (5) interaction with complement, (6) binding to
an Fc receptor other than a salvage receptor, or (7)
antibody-dependent cellular cytotoxicity (ADCC).
[0064] The term "Fc domain" encompasses native Fc and Fc variant
molecules and sequences as defined above. As with Fc variants and
native Fcs, the term "Fc domain" includes molecules in monomeric or
multimeric form, whether digested from whole antibody or produced
by other means.
[0065] The term "dimer" as applied to Fc domains or molecules
comprising Fc domains refers to molecules having two polypeptide
chains associated covalently or non-covalently.
[0066] The term "multimer" as applied to Fc domains or molecules
comprising Fc domains refers to molecules having two or more
polypeptide chains associated covalently, noncovalently, or by both
covalent and non-covalent interactions. IgG molecules typically
form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers,
dimers, trimers, or tetramers. Multimers may be formed by
exploiting the sequence and resulting activity of the native Ig
source of the Fc or by derivatizing (as defined herein) such a
native Fc.
[0067] The terms "peptibody" and "peptibodies" refer to molecules
comprising an antibody Fc domain attached to at least one peptide.
Such peptibodies may be multimers or dimers or fragments thereof,
and they may be derivatized.
II. Structure of Compounds
[0068] In General. The present invention provides compounds capable
of binding to and/or modulating the biological activity of the mpl
receptor. More particularly, the present invention provides a group
of compounds that are capable of binding to and/or triggering a
transmembrane signal through, i.e., activating, the mpl receptor,
which is the same receptor that mediates the activity of endogenous
thrombopoietin (TPO). Thus, the inventive compounds have
thrombopoietic activity, i.e., the ability to stimulate, in vivo
and in vitro, the production of platelets and/or have
megakaryocytopoietic activity, i.e., the ability to stimulate, in
vivo and in vitro, the production of platelet precursors, including
megakaryocytes.
[0069] Briefly, the compounds of the present invention comprise one
or more peptides having the sequence of formula I:
X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18; I
wherein X1-X4, X9-X10, and X13-X18 are each independently an amino
acid.
[0070] In other compositions of matter prepared in accordance with
this invention, the compounds may comprise one or more peptides
having the sequence of formula I attached or otherwise linked to
each other, for example, as dimers or multimers.
[0071] In other compositions of matter prepared in accordance with
this invention, the compounds may comprise one or more peptides of
formula I which are attached or otherwise linked to a vehicle at
the peptide's N-terminus or C-terminus. Any of these peptides may
be linked in tandem (i.e., sequentially, N to C), or in parallel
(i.e., N- to N-terminus, or C- to C-terminus) with or without
linkers.
[0072] Peptides. Compounds of the present invention comprise TPO
mimetic peptides, either alone or in combination with another TMP
as, for example, dimers or multimers. TMPs of the present invention
comprise the following sequence:
X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18; I
wherein X1-X4, X9-X10, and X13-X18 are each independently an amino
acid. Preferred amino acid residues of the above sequence are
further defined below in Table 1.
TABLE-US-00002 TABLE 1 Preferred Amino Acid Residues Position Amino
Acid Residue X1 A, V, W, M, G, Y, C, Q, E, R, H X2 A, V, L, I, G,
S, C X3 L, I, P, W, G, S, D, K, R X4 L, G, Q, D, E, H X9 K, R X10
Q, E X13 A, V, L, S, Q, E, R X14 A, W, T, Y, C, Q X15 V, L, G, Y, R
X16 A, L, F, G, R X17 A, V, L, M, G, C, Q, N X18 A, V, P, M, F, G,
C, Q, K
[0073] Even more preferred TMP sequences of the present invention
are those having the sequence:
X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18; I
wherein X1-X4, X9-X10, and X13-X18 are each independently an amino
acid and wherein the peptide has a binding affinity for the mpl
receptor and/or a bioactivity equal to or greater than that of the
sequence:
TABLE-US-00003 I-E-G-P-T-L-R-Q-W-L-A-A-R-A. [SEQ ID NO 1]
[0074] Binding affinity can be measured by any assay known or
available to those skilled in the art, including but not limited to
BIAcore measurements, ELISA assays, competition assays, etc.
[0075] Bioactivity can be measured in vivo or in vitro by any assay
known or available to those skilled in the art. Exemplary assays
include, but are not limited to, cell-based assays, i.e.,
megakaryocyte proliferation assays, 32D cell assays (an IL-3
dependent clone of murine 32D cells that have been transfected with
human mpl receptor, described in greater detail in WO 95/26746),
CD34+ assays, CD61 cell assays, etc. Bioactivity can also be
measured by various in vivo animal assays.
[0076] Further preferred TMP sequences of the present invention are
identified in Table 2 below.
TABLE-US-00004 TABLE 2 Preferred TMP sequences SEQ ID TMP No.
PEPTIDE SEQUENCE NO: TMP2 GAREGPTLRQWLEWVRVG 2 TMP3
RDLDGPTLRQWLPLPSVQ 3 TMP4 ALRDGPTLKQWLEYRRQA 4 TMP5
ARQEGPTLKEWLFWVRMG 5 TMP6 EALLGPTLREWLAWRRAQ 6 TMP7
MARDGPTLREWLRTYRMM 7 TMP8 WMPEGPTLKQWLFHGRGQ 8 TMP9
HIREGPTLRQWLVALRMV 9 TMP10 QLGHGPTLRQWLSWYRGM 10 TMP11
ELRQGPTLHEWLQHLASK 11 TMP12 VGIEGPTLRQWLAQRLNP 12 TMP13
WSRDGPTLREWLAWRAVG 13 TMP14 AVPQGPTLKQWLLWRRCA 14 TMP15
RIREGPTLKEWLAQRRGF 15 TMP16 RFAEGPTLREWLEQRKLV 16 TMP17
DRFQGPTLREWLAAIRSV 17 TMP18 AGREGPTLREWLNMRVWQ 18 TMP19
ALQEGPTLRQWLGWGQWG 19 TMP20 YCDEGPTLKQWLVCLGLQ 20 TMP21
WCKEGPTLREWLRWGFLC 21 TMP22 CSSGGPTLREWLQCRRMQ 22 TMP23
CSWGGPTLKQWLQCVRAK 23 TMP24 CQLGGPTLREWLACRLGA 24 TMP25
CWEGGPTLKEWLQCLVER 25 TMP26 CRGGGPTLHQWLSCFRWQ 26 TMP27
CRDGGPTLRQWLACLQQK 27 TMP28 ELRSGPTLKEWLVWRLAQ 28 TMP29
GCRSGPTLREWLACREVQ 29 TMP30 TCEQGPTLRQWLLCRQGR 30
[0077] Binding affinity and bioactivity data for the peptides
TMP2-TMP30 are described further in the Examples. To better mimic
the phage environment from which the peptides were selected, and to
shield the charged amino- and carboxy-terminus ends of the
preferred 18 amino acid peptides, two amino acid "caps" were added
to each end of each peptide. In particular, glutamine (Q) and
cysteine (C) were added to the amino terminus of each of
TMP2-TMP30. Similarly, two amino acid "caps" were added to the
carboxy terminus of each peptide-histadine (H) and serine (S). It
will be appreciated by those skilled in the art that the caps
merely shield the charged ends and are not intended to contribute
to or detract from to the binding affinity and/or bioactivity of
the preferred peptides.
[0078] Since peptide affinity is known to increase with peptide
length, the benchmark bioactive peptide (SEQ ID NO 1) was increased
from 14 amino acids to 22 amino acids to be the same length as the
test peptides, TMP2-TMP30. See Examples 6-11. It will be understood
by those skilled in the art that the bioactive region of the
comparator peptide is the core 14 amino acid sequence identified as
SEQ ID NO 1, and also referred to as TMP1.
[0079] Any peptide containing a cysteinyl residue may be
cross-linked with another Cys-containing peptide, either or both of
which may be linked to a vehicle. Any peptide having more than one
Cys residue may form an intrapeptide disulfide bond, as well. Any
of these peptides may be derivatized as described hereinafter.
[0080] Additional useful peptide sequences may result from
conservative and/or non-conservative modifications of the amino
acid sequences of the TMPs disclosed herein. Conservative
modifications will produce peptides having functional and chemical
characteristics similar to those of the peptide from which such
modifications are made. In contrast, substantial modifications in
the functional and/or chemical characteristics of the peptides may
be accomplished by selecting substitutions in the amino acid
sequence that differ significantly in their effect on maintaining
(a) the structure of the molecular backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the size of the molecule.
[0081] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
normative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described for
"alanine scanning mutagenesis" (see, for example, MacLennan et al.,
1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998,
Adv. Biophys. 35:1-24, which discuss alanine scanning
mutagenesis).
[0082] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. For example, amino acid
substitutions can be used to identify important residues of the
peptide sequence, or to increase or decrease the affinity of the
peptide or vehicle-peptide molecules (see preceding formulae)
described herein. Exemplary amino acid substitutions are set forth
in Table 3.
TABLE-US-00005 TABLE 3 Amino Acid Substitutions Original Exemplary
Preferred Residues Substitutions Substitutions Ala (A) Val, Leu,
Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu
Cys (C) Ser, Ala Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro,
Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Leu
Ala, Phe, Norleucine Leu (L) Norleucine, Ile Ile, Val, Met, Ala,
Phe Lys (K) Arg, 1,4 Arg Diamino- butyric Acid, Gln, Asn Met (M)
Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Leu Ala, Tyr Pro (P) Ala
Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr
Tyr (Y) Trp, Phe, Thr, Phe Ser Val (V) Ile, Met, Leu, Leu Phe, Ala,
Norleucine
[0083] In certain embodiments, conservative amino acid
substitutions also encompass non-naturally occurring amino acid
residues which are typically incorporated by chemical peptide
synthesis rather than by synthesis in biological systems.
[0084] Naturally occurring residues may be divided into classes
based on common sidechain properties that may be useful for
modifications of sequence. For example, non-conservative
substitutions may involve the exchange of a member of one of these
classes for a member from another class. Such substituted residues
may be introduced into regions of the peptide that are homologous
with non-human orthologs, or into the non-homologous regions of the
molecule. In addition, one may also make modifications using P or G
for the purpose of influencing chain orientation.
[0085] In making such modifications, the hydropathic index of amino
acids may be considered. Each amino acid has been assigned a
hydropathic index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0086] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art. Kyte et al., J. Mol. Biol., 157: 105-131
(1982). It is known that certain amino acids may be substituted for
other amino acids having a similar hydropathic index or score and
still retain a similar biological activity. In making changes based
upon the hydropathic index, the substitution of amino acids whose
hydropathic indices are within .+-.2 is preferred, those which are
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred.
[0087] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. The greatest local average hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.,
with a biological property of the protein.
[0088] The following hydrophilicity values have been assigned to
amino acid residues: arginine (+3.0); lysine (+3.0); aspartate
(+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine
(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline
(-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine
(-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
In making changes based upon similar hydrophilicity values, the
substitution of amino acids whose hydrophilicity values are within
.+-.2 is preferred, those which are within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred. One may also identify epitopes from primary amino acid
sequences on the basis of hydrophilicity. These regions are also
referred to as "epitopic core regions."
[0089] A skilled artisan will be able to determine suitable
variants using well known techniques. For identifying suitable
areas of the molecule that may be changed without destroying
activity, one skilled in the art may target areas not believed to
be important for activity. For example, when similar polypeptides
with similar activities from the same species or from other species
are known, one skilled in the art may compare the amino acid
sequence of a peptide to similar peptides. With such a comparison,
one can identify residues and portions of the molecules that are
conserved among similar polypeptides. It will be appreciated that
changes in areas of a peptide that are not conserved relative to
such similar peptides would be less likely to adversely affect the
biological activity and/or structure of the peptide. One skilled in
the art would also know that, even in relatively conserved regions,
one may substitute chemically similar amino acids for the naturally
occurring residues while retaining activity (conservative amino
acid residue substitutions). Therefore, even areas that may be
important for biological activity or for structure may be subject
to conservative amino acid substitutions without destroying the
biological activity or without adversely affecting the peptide
structure.
[0090] The amino acids may have either L or D stereochemistry
(except for Gly, which is neither L nor D) and the TMPs of the
present invention may comprise a combination of stereochemistries.
However, the L stereochemistry is preferred for all of the amino
acids in the TMP chain. The invention also provides reverse TMP
molecules wherein the amino terminal to carboxy terminal sequence
of the amino acids is reversed. For example, the reverse of a
molecule having the normal sequence X.sub.1-X.sub.2-X.sub.3 would
be X.sub.3-X.sub.2-X.sub.1. The invention also provides
retro-reverse TMP molecules wherein, like a reverse TMP, the amino
terminal to carboxy terminal sequence of amino acids is reversed
and residues that are normally "L" enantiomers in TMP are altered
to the "D" stereoisomer form.
[0091] It is also contemplated that "derivatives" of the TMPs may
be substituted for the above-described TMPs. Such derivative TMPs
include moieties wherein one or more of the following modifications
have been made: [0092] one or more of the peptidyl [--C(O)NR--]
linkages (bonds) have been replaced by a non-peptidyl linkage such
as a --CH.sub.2-carbamate linkage [--CH.sub.2--OC(O)NR--]; a
phosphonate linkage; a --CH.sub.2-sulfonamide
[--CH.sub.2--S(O).sub.2NR--] linkage; a urea [--NHC(O)NH--]
linkage; a --CH.sub.2-secondary amine linkage; or an alkylated
peptidyl linkage [--C(O)NR.sup.6-- where R.sup.6 is lower alkyl];
[0093] peptides wherein the N-terminus is derivatized to a
--NRR.sup.1 group; to a --NRC(O)R group; to a --NRC(O)OR group; to
a --NRS(O).sub.2R group; to a --NHC(O)NHR group, where R and
R.sup.1 are hydrogen or lower alkyl, with the proviso that R and
R.sup.1 are not both hydrogen; to a succinimide group; to a
benzyloxycarbonyl-NH-- (CBZ-NH--) group; or to a
benzyloxycarbonyl-NH-- group having from 1 to 3 substituents on the
phenyl ring selected from the group consisting of lower alkyl,
lower alkoxy, chloro, and bromo; and [0094] peptides wherein the
free C terminus is derivatized to --C(O)R.sup.2 where R.sup.2 is
selected from the group consisting of lower alkoxy and
--NR.sup.3R.sup.4 where R.sup.3 and R.sup.4 are independently
selected from the group consisting of hydrogen and lower alkyl. By
"lower" is meant a group having from 1 to 6 carbon atoms.
[0095] Additionally, modifications of individual amino acids may be
introduced into the TMP molecule by reacting targeted amino acid
residues of the peptide with an organic derivatizing agent that is
capable of reacting with selected side chains or terminal residues.
The following are exemplary:
[0096] Lysinyl and amino terminal residues may be reacted with
succinic or other carboxylic acid anhydrides. Derivatization with
these agents has the effect of reversing the charge of the lysinyl
residues. Other suitable reagents for derivatizing
alpha-amino-containing residues include imidoesters such as methyl
picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione;
and transaminase-catalyzed reaction with glyoxylate.
[0097] Arginyl residues may be modified by reaction with one or
several conventional reagents, among them phenylglyoxal,
2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
Derivatization of arginine residues requires that the reaction be
performed in alkaline conditions because of the high pKa of the
guanidine functional group. Furthermore, these reagents may react
with the groups of lysine as well as the arginine guanidino
group.
[0098] The specific modification of tyrosyl residues per se has
been studied extensively, with particular interest in introducing
spectral labels into tyrosyl residues by reaction with aromatic
diazonium compounds or tetranitromethane. Most commonly,
N-acetylimidizole and tetranitromethane may be used to form
O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
[0099] Carboxyl side groups (aspartyl or glutamyl) may be
selectively modified by reaction with carbodiimides
(R'--N.dbd.C.dbd.N--R') such as
1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,
aspartyl and glutamyl residues may be converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0100] Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl residues.
Alternatively, these residues may be deamidated under mildly acidic
conditions. Either form of these residues falls within the scope of
this invention.
[0101] Derivatization with bifunctional agents is useful for
cross-linking the peptides or their functional derivatives to a
water-insoluble support matrix or to other macromolecular carriers.
Commonly used cross-linking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as 3,3'-dithiobis
(succinimidylpropionate), and bifunctional maleimides such as
bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and
4,330,440 may be employed for protein immobilization.
[0102] Other possible modifications include hydroxylation of
proline and lysine, phosphorylation of hydroxyl groups of seryl or
threonyl residues, oxidation of the sulfur atom in Cys, methylation
of the alpha-amino groups of lysine, arginine, and histidine side
chains (Creighton, T. E., Proteins: Structure and Molecule
Properties, W. H. Freeman & Co., San Francisco, pp. 79-86
(1983)), acetylation of the N-terminal amine, and, in some
instances, amidation of the C-terminal carboxyl groups.
[0103] Such derivatized moieties preferably improve one or more
characteristics including thrombopoietic activity, solubility,
absorption, biological half life, and the like of the inventive
compounds. Alternatively, derivatized moieties may result in
compounds that have the same, or essentially the same,
characteristics and/or properties of the compound that is not
derivatized. The moieties may alternatively eliminate or attenuate
any undesirable side effect of the compounds and the like.
[0104] Compounds of the present invention may be changed at the DNA
level, as well. The DNA sequence of any portion of the compound may
be changed to codons more compatible with the chosen host cell. For
E. coli, which is the preferred host cell, optimized codons are
known in the art. Codons may be substituted to eliminate
restriction sites or to include silent restriction sites, which may
aid in processing of the DNA in the selected host cell. The
vehicle, linker and peptide DNA sequences may be modified to
include any of the foregoing sequence changes. Thus, all
modifications, substitution, derivatizations, etc. discussed herein
apply equally to all aspects of the present invention, including
but not limited to peptides, peptide dimers and multimers, linkers,
and vehicles.
[0105] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar peptides
that are important for activity or structure. In view of such a
comparison, one can predict the importance of amino acid residues
in a peptide that correspond to amino acid residues that are
important for activity or structure in similar peptides. One
skilled in the art may opt for chemically similar amino acid
substitutions for such predicted important amino acid residues of
the peptides.
[0106] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of that
information, one skilled in the art may predict the alignment of
amino acid residues of a peptide with respect to its three
dimensional structure. One skilled in the art may choose not to
make radical changes to amino acid residues predicted to be on the
surface of the protein, since such residues may be involved in
important interactions with other molecules. Moreover, one skilled
in the art may generate test variants containing a single amino
acid substitution at each desired amino acid residue. The variants
can then be screened using activity assays know to those skilled in
the art. Such data could be used to gather information about
suitable variants. For example, if one discovered that a change to
a particular amino acid residue resulted in destroyed, undesirably
reduced, or unsuitable activity, variants with such a change would
be avoided. In other words, based on information gathered from such
routine experiments, one skilled in the art can readily determine
the amino acids where further substitutions should be avoided
either alone or in combination with other mutations.
[0107] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in
Biotech., 7(4): 422-427 (1996), Chou et al., Biochemistry, 13(2):
222-245 (1974); Chou et al., Biochemistry, 113(2): 211-222 (1974);
Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45-148
(1978); Chou et al., Ann. Rev. Biochem., 47: 251-276 and Chou et
al., Biophys. J., 26: 367-384 (1979). Moreover, computer programs
are currently available to assist with predicting secondary
structure. One method of predicting secondary structure is based
upon homology modeling. For example, two polypeptides or proteins
which have a sequence identity of greater than 30%, or similarity
greater than 40% often have similar structural topologies. The
recent growth of the protein structural data base (PDB) has
provided enhanced predictability of secondary structure, including
the potential number of folds within a polypeptide's or protein's
structure. See Holm et al., Nucl. Acid. Res., 27(1): 244-247
(1999). It has been suggested (Brenner et al., Curr. Op. Struct.
Biol., 7(3): 369-376 (1997)) that there are a limited number of
folds in a given polypeptide or protein and that once a critical
number of structures have been resolved, structural prediction will
gain dramatically in accuracy.
[0108] Additional methods of predicting secondary structure include
"threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3): 377-87
(1997); Sippl et al., Structure, 4(1): 15-9 (1996)), "profile
analysis" (Bowie et al., Science, 253: 164-170 (1991); Gribskov et
al., Meth. Enzym., 183: 146-159 (1990); Gribskov et al., Proc. Nat.
Acad. Sci., 84(13): 4355-8 (1987)), and "evolutionary linkage" (See
Home, supra, and Brenner, supra).
[0109] Formulae for preferred peptide and peptide-linker molecules
of the present invention are shown in FIG. 1. Additionally,
physiologically acceptable salts of the TMPs are also
encompassed.
Peptide Compounds
[0110] In addition to novel peptides, the present invention
provides novel peptide compounds wherein one or more peptides of
the present invention are attached or otherwise linked to each
other, to a linker (LN) and/or to a vehicle (V). TMPs may be linked
in tandem (i.e., sequentially, N-terminus to C-terminus) or in
parallel (i.e., N- to N-terminus or C- to C-terminus). TMPs may be
attached to other TMPs or the same TMPs, with or without linkers.
TMPs may also be attached to other TMPs or the same TMPs with or
without linkers and with or without vehicles.
Peptide-linker-vehicle compounds of the present invention may be
described by the following formula:
(V1).sub.v-(LN1).sub.1-(TMP1).sub.a-(LN2).sub.m-(TMP2).sub.b-(LN3).sub.n-
-(TMP3).sub.c-(LN4).sub.o-(TMP4).sub.d-(V2).sub.w
wherein: V1 and V2 are vehicles; LN1, LN2, LN3 and LN4 are each
independently linkers; TMP1, TMP2, TMP3 and TMP4 are each
independently peptide sequences of the formula I; a, b, c and d and
l, m, n and o are each independently an integer from zero to
twenty, and v and w are each independently an integer from zero to
one.
[0111] Exemplary compounds of the present invention are shown by
the following formulae:
TABLE-US-00006 TMP1-V1 TMP1-LN1-V1 TMP1-TMP2-V1
TMP1-LN1-TMP2-LN2-V1
and additional multimers thereof wherein V1 is a vehicle
(preferably an Fc domain) and is attached at the C-terminus of a
TMP, either with or without a linker;
TABLE-US-00007 V1-TMP1 V1-LN1-TMP1 V1-TMP1-TMP2
V1-LN1-TMP1-LN2-TMP2
and multimers thereof wherein V1 is a vehicle (preferably an Fc
domain) and is attached at the N-terminus of a TMP, either with or
without a linker. Formulae for preferred peptide-vehicle and
peptide-linker-vehicle molecules of the present invention are shown
in FIG. 2.
[0112] Many of the preferred compounds of the invention are dimers
or multimers in that they possess two TMP moieties or multimers in
that they possess multiple TMP moieties. Each of TMP1 through TMP4
etc. can have the same or different structures. Preferably the
compounds of the present invention will have from 2-5 TMP moieties,
particularly preferably 2-3 and most preferably 2.
[0113] These compounds are preferably dimers which are either
attached directly or are linked by a linker group (see below). The
monomeric TMP moieties are shown in the conventional orientation
from N- to C-terminus reading left to right. Accordingly, it can be
seen that the inventive compounds can be oriented so that the
C-terminus of TMP1 is attached either directly or through a linker
to the N-terminus of TMP2 (a tandem dimer). Alternately, the
inventive compounds can be oriented so that the C-terminus of TMP1
is attached either directly or through a linker to the C-terminus
of TMP2, or the N-terminus of TMP1 is attached either directly or
through a linker to the N-terminus of TMP2 (a parallel dimer).
These compounds are referred to as dimers even if TMP 1 and TMP2
are structurally distinct. That is, both homodimers and
heterodimers are envisioned.
Linkers.
[0114] In another embodiment, the present invention provides one or
more TMPs covalently bonded or otherwise linked or attached to
another TMP peptide of via a "linker" group (LN1, LN2, etc.). Any
linker group is optional. When it is present, it is not critical
what its chemical structure, since it serves primarily as a spacer.
The linker should be chosen so as not to interfere with the
biological activity of the final compound and also so that
immunogenicity of the final compound is not significantly
increased. The linker is preferably made up of amino acids linked
together by peptide bonds. Thus, in preferred embodiments, the
linker is made up of from 1 to 30 amino acids linked by peptide
bonds, wherein the amino acids are selected from the 20 naturally
occurring amino acids. Some of these amino acids may be
glycosylated, as is well understood by those in the art. In a more
preferred embodiment, the 1 to 20 amino acids are selected from
glycine, alanine, proline, asparagine, glutamine, and lysine. Even
more preferably, a linker is made up of a majority of amino acids
that are sterically unhindered, such as glycine and alanine. Thus,
preferred linkers are polyglycines (particularly (Gly).sub.4,
(Gly).sub.5), poly(Gly-Ala), and polyalanines. Other specific
examples of linkers are:
TABLE-US-00008 (Gly).sub.3Lys(Gly).sub.4; (SEQ ID NO: 96)
(Gly).sub.3AsnGlySer(Gly).sub.2; (SEQ ID NO: 97)
(Gly).sub.3Cys(Gly).sub.4; (SEQ ID NO: 98) and GlyProAsnGlyGly.
(SEQ ID NO: 99)
TO explain the above nomenclature, for example,
(Gly).sub.3Lys(Gly).sub.4 means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly.
Combinations of Gly and Ala are also preferred. The linkers shown
here are exemplary; linkers within the scope of this invention may
be much longer and may include other residues.
[0115] Non-peptide linkers are also possible. For example, alkyl
linkers such as --NH--(CH.sub.2).sub.n--C(O)--, wherein s=2-20
could be used. These alkyl linkers may further be substituted by
any non-sterically hindering group such as lower alkyl (e.g.,
C.sub.1-C.sub.6) lower acyl, halogen (e.g., Cl, Br), CN, NH.sub.2,
phenyl, etc. An exemplary non-peptide linker is a PEG linker,
##STR00001##
wherein n is such that the linker has a molecular weight of 100 to
5000 kD, preferably 100 to 500 kD. The peptide linkers may be
altered to form derivatives in the same manner as described
above.
[0116] In general, it has been discovered that a linker of a length
of about 0-14 sub-units (e.g., amino acids) is preferred for the
thrombopoietic compounds of the present invention. The peptide
linkers may be altered to form derivatives in the same manner as
described above for the TMPs. In addition, the compounds of this
embodiment may further be linear or cyclic. By "cyclic" is meant
that at least two separated, i.e., non-contiguous, portions of the
molecule are linked to each other. For example, the amino and
carboxy terminus of the ends of the molecule could be covalently
linked to form a cyclic molecule. Alternatively, the molecule could
contain two or more Cys residues (e.g., in the linker), which could
cyclize via disulfide bond formation. It is further contemplated
that more than one tandem peptide dimer can link to form a dimer of
dimers. Thus, for example, a tandem dimer containing a Cys residue
can form an intermolecular disulfide bond with a Cys of another
such dimer. Exemplary peptide-linker compounds of the invention are
shown below:
TABLE-US-00009 (SEQ ID NO 100)
CSSGGPTLREWLQCRRMQ--GGGGG--CSSGGPTLREWLQCRRMQ; (SEQ ID NO 101)
QLGHGPTLRQWLSWYRGN--(Gly).sub.3Lys(Gly).sub.4-- ALRDGPTLKQWLEYRRQA;
(SEQ ID NO 102)
RFAEGPTLREWLEQRKLV-GGG(PEG)GGG-RFAEGPTLREWLEQRKLV.
[0117] Thus, in preferred embodiments, the linker comprises
(LN1).sub.n, wherein LN1 is a naturally occurring amino acid or a
stereoisomer thereof and "n" is any one of 1 through 20. Formulae
for preferred peptide-linker molecules are shown in FIG. 1. Further
preferred peptide-linker molecules include:
i) TMP1-LN1-TMP2-LN2
ii) LN1-TMP1-LN2-TMP2
[0118] iii) LN1-TMP1-LN2-TMP1
iv) TMP1-LN1-TMP1-LN1-TMP1-LN1
v) LN1-TMP1-LN2-TMP2-LN3-TMP3-LN4-TMP4
[0119] wherein LN1-LN4 are each independent linkers.
Vehicles
[0120] In yet another embodiment, peptides or peptide compounds of
the present invention may be linked or attached to a vehicle (V). A
vehicle generally refers to a molecule that prevents degradation
and/or increases half-life, reduces toxicity, reduces
immunogenicity, or increases biological activity of a therapeutic
protein. The vehicle (V) may be attached to a peptide through the
N-terminus, C terminus, peptide backbone or a sidechain.
[0121] The vehicle (V) may be a carrier molecule, such as a linear
polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a
branched-chain polymer (see, for example, U.S. Pat. Nos. 4,289,872
to Denkenwalter et al., issued Sep. 15, 1981; 5,229,490 to Tam,
issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published 28
Oct. 1993); a lipid; a cholesterol group (such as a steroid); or a
carbohydrate or oligosaccharide. Other possible carriers include
one or more water soluble polymer attachments such as
polyoxyethylene glycol, or polypropylene glycol as described U.S.
Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and
4,179,337. Still other useful polymers known in the art include
monomethoxy-polyethylene glycol, dextran, cellulose, or other
carbohydrate based polymers, poly-(N-vinyl
pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures
of these polymers. Exemplary vehicles also include: [0122] an Fc
domain; [0123] other proteins, polypeptides, or peptides capable of
binding to a salvage receptor; [0124] human serum albumin (HSA);
[0125] a leucine zipper (LZ) domain; [0126] polyethylene glycol
(PEG), including 5 kD, 20 kD, and 30 kD PEG, as well as other
polymers; [0127] dextran; and other molecules known in the art to
provide extended half-life and/or protection from proteolytic
degradation or clearance.
[0128] An exemplary carrier is polyethylene glycol (PEG). The PEG
group may be of any convenient molecular weight and may be straight
chain or branched. The average molecular weight of the PEG will
preferably range from about 2 kDa to about 100 kDa, more preferably
from about 5 kDa to about 50 kDa, most preferably from about 5 kDa
to about 10 kDa.
[0129] The PEG groups will generally be attached to the compounds
of the invention via acylation, reductive alkylation, Michael
addition, thiol alkylation or other chemoselective
conjugation/ligation methods through a reactive group on the PEG
moiety (e.g., an aldehyde, amino, ester, thiol, -haloacetyl,
maleimido or hydrazino group) to a reactive group on the target
compound (e.g., an aldehyde, amino, ester, thiol, -haloacetyl,
maleimido or hydrazino group).
[0130] Carbohydrate (oligosaccharide) groups may conveniently be
attached to sites that are known to be glycosylation sites in
proteins. Generally, O-linked oligosaccharides are attached to
serine (Ser) or threonine (Thr) residues while N-linked
oligosaccharides are attached to asparagine (Asn) residues when
they are part of the sequence Asn-X-Ser/Thr, where X can be any
amino acid except proline. X is preferably one of the 19 naturally
occurring amino acids not including proline. The structures of
N-linked and O-linked oligosaccharides and the sugar residues found
in each type are different. One type of sugar that is commonly
found on both is N-acetylneuraminic acid (referred to as sialic
acid). Sialic acid is usually the terminal residue of both N-linked
and O-linked oligosaccharides and, by virtue of its negative
charge, may confer acidic properties to the glycosylated compound.
Such site(s) may be incorporated in the linker of the compounds of
this invention and are preferably glycosylated by a cell during
recombinant production of the polypeptide compounds (e.g., in
mammalian cells such as CHO, BHK, COS). However, such sites may
further be glycosylated by synthetic or semi-synthetic procedures
known in the art.
[0131] In a more preferred embodiment, the vehicle (V) may comprise
one or more antibody Fc domains. Thus, the peptide compounds
described above may further be fused to one or more Fc domains,
either directly or through linkers. The Fc vehicle may be selected
from the human immunoglobulin IgG-1 heavy chain, see Ellison, J. W.
et al., Nucleic Acids Res. 10:4071-4079 (1982), or any other Fc
sequence known in the art (e.g. other IgG classes including but not
limited to IgG-2, IgG-3 and IgG-4, or other immunoglobulins).
[0132] It is well known that Fc regions of antibodies are made up
of monomeric polypeptide segments that may be linked into dimeric
or multimeric forms by disulfide bonds or by non-covalent
association. The number of intermolecular disulfide bonds between
monomeric subunits of native Fc molecules ranges from 1 to 4
depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g.,
IgG1, IgG2, IgG3, IgA1, IgGA2) of antibody involved. The term "Fc"
as used herein is generic to the monomeric, dimeric, and multimeric
forms of Fc molecules. It should be noted that Fc monomers will
spontaneously dimerize when the appropriate Cys residues are
present unless particular conditions are present that prevent
dimerization through disulfide bond formation. Even if the Cys
residues that normally form disulfide bonds in the Fc dimer are
removed or replaced by other residues, the monomeric chains will
generally dimerize through non-covalent interactions. The term "Fc"
herein is used to mean any of these forms: the native monomer, the
native dimer (disulfide bond linked), modified dimers (disulfide
and/or non-covalently linked), and modified monomers (i.e.,
derivatives).
[0133] Variants, analogs or derivatives of the Fc portion may be
constructed by, for example, making various substitutions of
residues or sequences.
[0134] Variant (or analog) polypeptides include insertion variants,
wherein one or more amino acid residues supplement an Fc amino acid
sequence. Insertions may be located at either or both termini of
the protein, or may be positioned within internal regions of the Fc
amino acid sequence. Insertional variants with additional residues
at either or both termini can include for example, fusion proteins
and proteins including amino acid tags or labels. For example, the
Fc molecule may optionally contain an N-terminal Met, especially
when the molecule is expressed recombinantly in a bacterial cell
such as E. coli.
[0135] In Fc deletion variants, one or more amino acid residues in
an Fc polypeptide are removed. Deletions can be effected at one or
both termini of the Fc polypeptide, or with removal of one or more
residues within the Fc amino acid sequence. Deletion variants,
therefore, include all fragments of an Fc polypeptide sequence.
[0136] In Fc substitution variants, one or more amino acid residues
of an Fc polypeptide are removed and replaced with alternative
residues. In one aspect, the substitutions are conservative in
nature, however, the invention embraces substitutions that are also
non-conservative.
[0137] For example, cysteine residues can be deleted or replaced
with other amino acids to prevent formation of some or all
disulfide crosslinks of the Fc sequences. One may remove each of
these cysteine residues or substitute one or more such cysteine
residues with other amino acids, such as Ala or Ser. As another
example, modifications may also be made to introduce amino acid
substitutions to (1) ablate the Fc receptor binding site; (2)
ablate the complement (C1q) binding site; and/or to (3) ablate the
antibody dependent cell-mediated cytotoxicity (ADCC) site. Such
sites are known in the art, and any known substitutions are within
the scope of Fc as used herein. For example, see Molecular
Immunology, Vol. 29, No. 5, 633-639 (1992) with regards to ADCC
sites in IgG1.
[0138] Likewise, one or more tyrosine residues can be replaced by
phenylalanine residues as well. In addition, other variant amino
acid insertions, deletions (e.g., from 1-25 amino acids) and/or
substitutions are also contemplated and are within the scope of the
present invention. Conservative amino acid substitutions will
generally be preferred. Furthermore, alterations may be in the form
of altered amino acids, such as peptidomimetics or D-amino
acids.
[0139] Fc sequences of the present invention may also be
derivatized, i.e., bearing modifications other than insertion,
deletion, or substitution of amino acid residues. Preferably, the
modifications are covalent in nature, and include for example,
chemical bonding with polymers, lipids, other organic, and
inorganic moieties. Derivatives of the invention may be prepared to
increase circulating half-life, or may be designed to improve
targeting capacity for the polypeptide to desired cells, tissues,
or organs.
[0140] It is also possible to use the salvage receptor binding
domain of the intact Fc molecule as the Fc part of the inventive
compounds, such as described in WO 96/32478, entitled "Altered
Polypeptides with Increased Half-Life". Additional members of the
class of molecules designated as Fc herein are those that are
described in WO 97/34631, entitled "Immunoglobulin-Like Domains
with Increased Half-Lives". Both of the published PCT applications
cited in this paragraph are hereby incorporated by reference.
[0141] The Fc fusions may be at the N- or C-terminus of TMP.sub.1
or TMP.sub.2 or at both the N- and C-termini of TMP.sub.1 or
TMP.sub.2. Similarly, the Fc fusions may be at the N- or C-terminus
of the Fc domain.
[0142] Preferred compounds of the present invention include IgG1 Fc
fusion dimers linked or otherwise attached to dimers or multimers
of the TMPs disclosed herein. In such cases, each Fc domain will be
linked to a dimer or multimer of TMP peptides, either with or
without linkers. Schematic examples of such compounds are shown in
FIG. 2.
[0143] Multiple vehicles may also be used; e.g., Fc's at each
terminus or an Fc at a terminus and a PEG group at the other
terminus or a sidechain.
[0144] Exemplary peptide-vehicle compounds are provided in Table 4
below.
TABLE-US-00010 TABLE 4 Exemplary Peptide-Vehicle Compounds SEQ ID
AMINO ACID SEQUENCE NO: HIREGPTLRQWLVALRMV-GGG(PEG)GGG- 153
HIREGPTLRQWLVALRMV Fc-TCEQGPTLRQWLLCRQGR-GGGKGGG- 154
TCEQGPTLRQWLLCRqGR-Fc Fc-QLGHGPTLRQWLSWYRGM-GPNG-ELRSGPTLKEWLVWRLAq
155 CSWGGPTLKQWLQCVRAK-Fc 156 SWGGPTLKQWLQCVRAK
Fc-GGGKGGG-AVPQGPTLKQWLLWRRCA 157 PEG-CSSGGPTLREWLQCRRMQ 158 |
CSSGGPTLREWLQCRRMQ Fc-GGGGG-YCDEGPTLKQWLVCLGLQ-GGGGG- 159
YCDEGPTLKQWLVCLGLQ CSWGGPTLKQWLQCVRAK-GGGAGGG-CSWGGPTLKQWLQCVRAK-
160 GGGAGGG-CSWGGPTLKQWLQCVPAK-GGGAGGG-Fc
VGIEGPTLRQWLAQRLNP-GGGCGGG-VGIEGPTLRQWLAQRLNP- 161 PEG
Fc-ELRSGPTLKEWLVWRLAq-GGGG-ELRSGPTLKEWLVWRLAQ 162
Fc-ALRDGPTLKQWLEYRRQA-GGGKGGG- 163 ALRDGPTLKQWLEYRRQA-Fc
[0145] Further, preferred embodiments of the present invention are
listed in Table 5.
TABLE-US-00011 TABLE 5 Specific Preferred Embodiments AMINO ACID
SEQUENCE SEQ ID NO: ALRDGPTLKQWLEYRRQA-ALRDGPTLKQWLEYRRQA 164
EALLGPTLREWLAWRRAQ-EALLGPTLREWLAWRRAQ 165
AVPQGPTLKQWLLWRRCA-AVPQGPTLKQWLLWRRCA 166
YCDEGPTLKQWLVCLGLQ-YCDEGPTLKQWLVCLGLQ 167
CSSGGPTLREWLQCRRMQ-CSSGGPTLREWLQCRRMQ 168
CSWGGPTLKQWLQCVRAK-CSWGGPTLKQWLQCVRAK 169
ALRDGPTLKQWLEYRRQA-GGGGG-ALRDGPTLKQWLEYRRQA 170
EALLGPTLREWLAWRRAQ-GGGGG-EALLGPTLREWLAWRRAQ 171
AVPQGPTLKQWLLWRRCA-GGGGG-AVPQGPTLKQWLLWRRCA 172
YCDEGPTLKQWLVCLGLQ-GGGGG-YCDEGPTLKQWLVCLGLQ 173
CSSGGPTLREWLQCRRMQ-GGGGG-CSSGGPTLREWLQCRRMQ 174
CSWGGPTLKQWLQCVRAK-GGGGG-CSWGGPTLKQWLQCVRAK 175
Fc-GGGGG-ALRDGPTLKQWLEYRRQA 176 Fc-GGGGG-EALLGPTLREWLAWRRAQ 177
Fc-GGGGG-AVPQGPTLKQWLLWRRCA 178 Fc-GGGGG-YCDEGPTLKQWLVCLGLQ 179
Fc-GGGGG-CSSGGPTLREWLQCRRMQ 180 Fc-GGGGG-CSWGGPTLKQWLQCVRAK 181
Fc-GGGGG-ALRDGPTLKQWLEYRRQA-GGGGG-ALRDGPTLKQWLEYRRQA 182
Fc-GGGGG-EALLGPTLREWLAWRRAQ-GGGGG-EALLGPTLREWLAWRRAQ 183
Fc-GGGGG-AVPQGPTLKQWLLWRRCA-GGGGG-AVPQGPTLKQWLLWRRCA 184
Fc-GGGGG-YCDEGPTLKQWLVCLGLQ-GGGGG-YCDEGPTLKQWLVCLGLQ 185
Fc-GGGGG-CSSGGPTLREWLQCRRMQ-GGGGG-CSSGGPTLREWLQCRRMQ 186
Fc-GGGGG-CSWGGPTLKQWLQCVRAK-GGGGG-CSWGGPTLKQWLQCVRAK 187
ALRDGPTLKQWLEYRRQA-GGGGG-ALRDGPTLKQWLEYRRQA-GGGGG-Fc 188
EALLGPTLREWLAWRRAQ-GGGGG-EALLGPTLREWLAWRRAQ-GGGGG-Fc 189
AVPQGPTLKQWLLWRRCA-GGGGG-AVPQGPTLKQWLLWRRCA-GGGGG-Fc 190
YCDEGPTLKQWLVCLGLQ-GGGGG-YCDEGPTLKQWLVCLGLQ-GGGGG-Fc 191
CSSGGPTLREWLQCRRMQ-GGGGG-CSSGGPTLREWLQCRRMQ-GGGGG-Fc 192
CSWGGPTLKQWLQCVRAK-GGGGG-CSWGGPTLKQWLQCVRAK-GGGGG-Fc 193
ALRDGPTLKQWLEYRRQA-GGGGG-Fc 194 EALLGPTLREWLAWRRAQ-GGGGG-Fc 195
AVPQGPTLKQWLLWRRCA-GGGGG-Fc 196 YCDEGPTLKQWLVCLGLQ-GGGGG-Fc 197
CSSGGPTLREWLQCRRMQ-GGGGG-Fc 198
III. Methods of Making
[0146] The compounds of this invention may be made in a variety of
ways. Since many of the compounds are peptides, or include a
peptide, methods for synthesizing peptides are of particular
relevance here. Solid phase synthesis techniques may be used.
Suitable techniques are well known in the art, and include those
described in Merrifield, in Chem. Polypeptides, pp. 335-61
(Katsoyannis and Panayotis eds. 1973); Merrifield, J. Am. Chem.
Soc. 85:2149 (1963); Davis et al., Biochem. Intl. 10:394-414
(1985); Stewart and Young, Solid Phase Peptide Synthesis (1969);
U.S. Pat. No. 3,941,763; Finn et al., The Proteins, 3rd ed., vol.
2, pp. 105-253 (1976); and Erickson et al., The Proteins, 3rd ed.,
vol. 2, pp. 257-527 (1976). Solid phase synthesis is the preferred
technique of making individual peptides since it is the most
cost-effective method of making small peptides.
[0147] The peptides may also be made in transformed host cells
using recombinant DNA techniques. To do so, a recombinant DNA
molecule coding for the peptide is prepared. Methods of preparing
such DNA and/or RNA molecules are well known in the art. For
instance, sequences coding for the peptides could be excised from
DNA using suitable restriction enzymes. The relevant sequences can
be created using the polymerase chain reaction (PCR) with the
inclusion of useful restriction sites for subsequent cloning.
Alternatively, the DNA/RNA molecule could be synthesized using
chemical synthesis techniques, such as the phosphoramidite method.
Also, a combination of these or other techniques could be used.
[0148] The invention also includes a vector encoding the peptides
in an appropriate host. The vector comprises the DNA molecule that
encodes the peptides operatively linked to appropriate expression
control sequences. Methods of effecting this operative linking,
either before or after the peptide-encoding DNA molecule is
inserted into the vector, are well known. Expression control
sequences include promoters, activators, enhancers, operators,
ribosomal binding sites, start signals, stop signals, cap signals,
polyadenylation signals, and other signals involved with the
control of transcription or translation.
[0149] The resulting vector comprising the peptide-encoding DNA
molecule is used to transform an appropriate host. This
transformation may be performed using methods well known in the
art.
[0150] Any of a large number of available and well-known host cells
may be used in the practice of this invention. The selection of a
particular host is dependent upon a number of factors recognized by
the art. These factors include, for example, compatibility with the
chosen expression vector, toxicity to the host cell of the peptides
encoded by the DNA molecule, rate of transformation, ease of
recovery of the peptides, expression characteristics, bio-safety
and costs. A balance of these factors must be struck with the
understanding that not all hosts may be equally effective for the
expression of a particular DNA sequence.
[0151] Within these general guidelines, useful microbial hosts
include bacteria (such as E. coli), yeast (such as Saccharomyces
sp. and Pichia pastoris) and other fungi, insects, plants,
mammalian (including human) cells in culture, or other hosts known
in the art. The transformed host is cultured under conventional
fermentation conditions so that the desired peptides are expressed.
Such fermentation conditions are well known in the art. The
peptides are then purified from the fermentation culture or from
the host cells in which they are expressed. These purification
methods are also well known in the art.
[0152] Compounds that contain derivatized peptides or which contain
non-peptide groups may be synthesized by well-known organic
chemistry techniques. For example, solid phase synthesis techniques
may be used. Suitable techniques are well known in the art, and
include those described in Merrifield (1973), Chem. Polypeptides,
pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J.
Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10:
394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis;
U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.)
2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2:
257-527. Solid phase synthesis is the preferred technique of making
individual peptides since it is the most cost-effective method of
making small peptides.
IV. Uses of the Compounds
[0153] The compounds of this invention have the ability to bind to
and/or activate the mpl receptor, and/or have the ability to
stimulate the production (both in vivo and in vitro) of platelets
("thrombopoietic activity") and platelet precursors
("megakaryocytopoietic activity"). To measure the activity (-ies)
of these compounds, one can utilize standard assays, such as those
described in WO95/26746 entitled "Compositions and Methods for
Stimulating Megakaryocyte Growth and Differentiation". In vivo
assays are further described in the Examples section herein.
[0154] The conditions to be treated by the methods and compositions
of the present invention are generally those which involve an
existing megakaryocyte/platelet deficiency or an expected or
anticipated megakaryocyte/platelet deficiency in the future (e.g.,
because of planned surgery or platelet donation). Such conditions
may be the result of a deficiency (temporary or permanent) of
active mpl ligand in vivo. The generic term for platelet deficiency
is thrombocytopenia, and hence the methods and compositions of the
present invention are generally available for prophylactically or
therapeutically treating thrombocytopenia in patients in need
thereof.
[0155] The World Health Organization has classified the degree of
thrombocytopenia on the number of circulating platelets in the
individual (Miller, et al., Cancer 47:210-211 (1981)). For example,
an individual showing no signs of thrombocytopenia (Grade 0) will
generally have at least 100,000 platelets/mm.sup.3. Mild
thrombocytopenia (Grade 1) indicates a circulating level of
platelets between 79,000 and 99,000/mm.sup.3. Moderate
thrombocytopenia (Grade 2) shows between 50,000 and 74,000
platelets/mm.sup.3 and severe thrombocytopenia is characterized by
between 25,000 and 49,000 platelets/mm.sup.3. Life-threatening or
debilitating thrombocytopenia is characterized by a circulating
concentration of platelets of less than 25,000/mm.sup.3.
[0156] Thrombocytopenia (platelet deficiencies) may be present for
various reasons, including chemotherapy and other therapy with a
variety of drugs, radiation therapy, surgery, accidental blood
loss, and other specific disease conditions. Exemplary specific
disease conditions that involve thrombocytopenia and may be treated
in accordance with this invention are: aplastic anemia; idiopathic
or immune thrombocytopenia (ITP), including idiopathic
thrombocytopenic purpura associated with breast cancer; HIV
associated ITP and HIV-related thrombotic thrombocytopenic purpura;
metastatic tumors which result in thrombocytopenia; systemic lupus
erythematosus; including neonatal lupus syndrome splenomegaly;
Fanconi's syndrome; vitamin B12 deficiency; folic acid deficiency;
May-Hegglin anomaly; Wiskott-Aldrich syndrome; chronic liver
disease; myelodysplastic syndrome associated with thrombocytopenia;
paroxysmal nocturnal hemoglobinuria; acute profound
thrombocytopenia following C7E3 Fab (Abciximab) therapy; alloimmune
thrombocytopenia, including maternal alloimmune thrombocytopenia;
thrombocytopenia associated with antiphospholipid antibodies and
thrombosis; autoimmune thrombocytopenia; drug-induced immune
thrombocytopenia, including carboplatin-induced thrombocytopenia,
heparin-induced thrombocytopenia; fetal thrombocytopenia;
gestational thrombocytopenia; Hughes' syndrome; lupoid
thrombocytopenia; accidental and/or massive blood loss;
myeloproliferative disorders; thrombocytopenia in patients with
malignancies; thrombotic thrombocytopenia purpura, including
thrombotic microangiopathy manifesting as thrombotic
thrombocytopenic purpura/hemolytic uremic syndrome in cancer
patients; autoimmune hemolytic anemia; occult jejunal diverticulum
perforation; pure red cell aplasia; autoimmune thrombocytopenia;
nephropathia epidemica; rifampicin-associated acute renal failure;
Paris-Trousseau thrombocytopenia; neonatal alloimmune
thrombocytopenia; paroxysmal nocturnal hemoglobinuria; hematologic
changes in stomach cancer; hemolytic uremic syndromes in childhood;
hematologic manifestations related to viral infection including
hepatitis A virus and CMV-associated thrombocytopenia. Also,
certain treatments for AIDS result in thrombocytopenia (e.g., AZT).
Certain wound healing disorders might also benefit from an increase
in platelet numbers.
[0157] With regard to anticipated platelet deficiencies, e.g., due
to future surgery, a compound of the present invention could be
administered several days to several hours prior to the need for
platelets. With regard to acute situations, e.g., accidental and
massive blood loss, a compound of this invention could be
administered along with blood or purified platelets.
[0158] The compounds of this invention may also be useful in
stimulating certain cell types other than megakaryocytes if such
cells are found to express mpl receptor. Conditions associated with
such cells that express the mpl receptor, which are responsive to
stimulation by the mpl ligand, are also within the scope of this
invention.
[0159] The compounds of this invention may be used in any situation
in which production of platelets or platelet precursor cells is
desired, or in which stimulation of the mpl receptor is desired.
Thus, for example, the compounds of this invention may be used to
treat any condition in a mammal wherein there is a need of
platelets, megakaryocytes, and the like. Such conditions are
described in detail in the following exemplary sources: WO95/26746;
WO95/21919; WO95/18858; WO95/21920 and are incorporated herein.
[0160] The compounds of this invention may also be useful in
maintaining the viability or storage life of platelets and/or
megakaryocytes and related cells. Accordingly, it could be useful
to include an effective amount of one or more such compounds in a
composition containing such cells.
[0161] By "mammal" is meant any mammal, including humans, domestic
animals including dogs and cats; exotic and/or zoo animals
including monkeys; laboratory animals including mice, rats, and
guinea pigs; farm animals including horses, cattle, sheep, goats,
and pigs; and the like. The preferred mammal is human.
V. Pharmaceutical Compositions
[0162] The present invention also provides pharmaceutical
compositions and methods of using pharmaceutical compositions of
the inventive compounds. Such pharmaceutical compositions may be
for administration for injection, or for oral, nasal, transdermal
or other forms of administration, including, e.g., by intravenous,
intradermal, intramuscular, intramammary, intraperitoneal,
intrathecal, intraocular, retrobulbar, intrapulmonary (e.g.,
aerosolized drugs) or subcutaneous injection (including depot
administration for long term release); by sublingual, anal,
vaginal, or by surgical implantation, e.g., embedded under the
splenic capsule, brain, or in the cornea. The treatment may consist
of a single dose or a plurality of doses over a period of time. In
general, comprehended by the invention are pharmaceutical
compositions comprising effective amounts of a compound of the
invention together with pharmaceutically acceptable diluents,
preservatives, solubilizers, emulsifiers, adjuvants and/or
carriers. Such compositions include diluents of various buffer
content (e.g., Tris-HCl, acetate, phosphate), pH and ionic
strength; additives such as detergents and solubilizing agents
(e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic
acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl
alcohol) and bulking substances (e.g., lactose, mannitol);
incorporation of the material into particulate preparations of
polymeric compounds such as polylactic acid, polyglycolic acid,
etc. or into liposomes. Hyaluronic acid may also be used, and this
may have the effect of promoting sustained duration in the
circulation. The pharmaceutical compositions optionally may include
still other pharmaceutically acceptable liquid, semisolid, or solid
diluents that serve as pharmaceutical vehicles, excipients, or
media, including but are not limited to, polyoxyethylene sorbitan
monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate,
starches, sucrose, dextrose, gum acacia, calcium phosphate, mineral
oil, cocoa butter, and oil of theobroma. Such compositions may
influence the physical state, stability, rate of in vivo release,
and rate of in vivo clearance of the present proteins and
derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th
Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712
which are herein incorporated by reference. The compositions may be
prepared in liquid form, or may be in dried powder, such as
lyophilized form. Implantable sustained release formulations are
also contemplated, as are transdermal formulations.
[0163] Contemplated for use herein are oral solid dosage forms,
which are described generally in Remington's Pharmaceutical
Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at
Chapter 89, which is herein incorporated by reference. Solid dosage
forms include tablets, capsules, pills, troches or lozenges,
cachets or pellets. Also, liposomal or proteinoid encapsulation may
be used to formulate the present compositions (as, for example,
proteinoid microspheres reported in U.S. Pat. No. 4,925,673).
Liposomal encapsulation may be used and the liposomes may be
derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556).
A description of possible solid dosage forms for the therapeutic is
given by Marshall, K., Modern Pharmaceutics, Edited by G. S. Banker
and C. T. Rhodes Chapter 10, 1979, herein incorporated by
reference. In general, the formulation will include the inventive
compound, and inert ingredients which allow for protection against
the stomach environment, and release of the biologically active
material in the intestine.
[0164] Also specifically contemplated are oral dosage forms of the
above inventive compounds. If necessary, the compounds may be
chemically modified so that oral delivery is efficacious.
Generally, the chemical modification contemplated is the attachment
of at least one moiety to the compound molecule itself, where said
moiety permits (a) inhibition of proteolysis; and (b) uptake into
the blood stream from the stomach or intestine. Also desired is the
increase in overall stability of the compound and increase in
circulation time in the body. Examples of such moieties include:
Polyethylene glycol, copolymers of ethylene glycol and propylene
glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,
polyvinyl pyrrolidone and polyproline (Abuchowski and Davis,
Soluble Polymer-Enzyme Adducts, Enzymes as Drugs, Hocenberg and
Roberts, eds., Wiley-Interscience, New York, N.Y., (1981), pp
367-383; Newmark, et al., J. Appl. Biochem. 4:185-189 (1982)).
Other polymers that could be used are poly-1,3-dioxolane and
poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as
indicated above, are polyethylene glycol moieties.
[0165] For the oral delivery dosage forms, it is also possible to
use a salt of a modified aliphatic amino acid, such as sodium
N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to
enhance absorption of the therapeutic compounds of this invention.
The clinical efficacy of a heparin formulation using SNAC has been
demonstrated in a Phase II trial conducted by Emisphere
Technologies. See U.S. Pat. No. 5,792,451, "Oral drug delivery
composition and methods".
[0166] The therapeutic can be included in the formulation as fine
multiparticulates in the form of granules or pellets of particle
size about 1 mm. The formulation of the material for capsule
administration could also be as a powder, lightly compressed plugs
or even as tablets. The therapeutic could be prepared by
compression.
[0167] Colorants and flavoring agents may all be included. For
example, the protein (or derivative) may be formulated (such as by
liposome or microsphere encapsulation) and then further contained
within an edible product, such as a refrigerated beverage
containing colorants and flavoring agents.
[0168] One may dilute or increase the volume of the therapeutic
with an inert material. These diluents could include carbohydrates,
especially mannitol, -lactose, anhydrous lactose, cellulose,
sucrose, modified dextrans and starch. Certain inorganic salts may
also be used as fillers including calcium triphosphate, magnesium
carbonate and sodium chloride. Some commercially available diluents
are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
[0169] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrants include but are not limited to starch including the
commercial disintegrant based on starch, Explotab. Sodium starch
glycolate, Amberlite, sodium carboxymethylcellulose,
ultramylopectin, sodium alginate, gelatin, orange peel, acid
carboxymethyl cellulose, natural sponge and bentonite may all be
used. Another form of the disintegrants are the insoluble cationic
exchange resins. Powdered gums may be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants.
[0170] Binders may be used to hold the therapeutic agent together
to form a hard tablet and include materials from natural products
such as acacia, tragacanth, starch and gelatin. Others include
methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl
cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic.
[0171] An antifrictional agent may be included in the formulation
of the therapeutic to prevent sticking during the formulation
process. Lubricants may be used as a layer between the therapeutic
and the die wall, and these can include but are not limited to;
stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and
waxes. Soluble lubricants may also be used such as sodium lauryl
sulfate, magnesium lauryl sulfate, polyethylene glycol of various
molecular weights, Carbowax 4000 and 6000.
[0172] Glidants that might improve the flow properties of the drug
during formulation and to aid rearrangement during compression
might be added. The glidants may include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0173] To aid dissolution of the therapeutic into the aqueous
environment, a surfactant might be added as a wetting agent.
Surfactants may include anionic detergents such as sodium lauryl
sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethonium chloride. The list of
potential nonionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the protein or
derivative either alone or as a mixture in different ratios.
[0174] Additives which potentially enhance uptake of the compound
are for instance the fatty acids oleic acid, linoleic acid and
linolenic acid.
[0175] Controlled release formulation may be desirable. The drug
could be incorporated into an inert matrix which permits release by
either diffusion or leaching mechanisms e.g., gums. Slowly
degenerating matrices may also be incorporated into the
formulation, e.g., alginates, polysaccharides. Another form of a
controlled release of this therapeutic is by a method based on the
Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in
a semipermeable membrane which allows water to enter and push drug
out through a single small opening due to osmotic effects. Some
enteric coatings also have a delayed release effect.
[0176] Other coatings may be used for the formulation. These
include a variety of sugars which could be applied in a coating
pan. The therapeutic agent could also be given in a film coated
tablet and the materials used in this instance are divided into 2
groups. The first are the nonenteric materials and include methyl
cellulose, ethyl cellulose, hydroxyethyl cellulose,
methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,
providone and the polyethylene glycols. The second group consists
of the enteric materials that are commonly esters of phthalic
acid.
[0177] A mix of materials might be used to provide the optimum film
coating. Film coating may be carried out in a pan coater or in a
fluidized bed or by compression coating.
[0178] Also contemplated herein is pulmonary delivery of the
present protein (or derivatives thereof). The protein (or
derivative) is delivered to the lungs of a mammal while inhaling
and traverses across the lung epithelial lining to the blood
stream. (Other reports of this include Adjei et al., Pharmaceutical
Research 7:565-569 (1990); Adjei et al., International Journal of
Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et
al., Journal of Cardiovascular Pharmacology 13 (suppl.5): s.
143-146 (1989) (endothelin-1); Hubbard et al., Annals of Internal
Medicine 3:206-212 (1989) (1-antitrypsin); Smith et al., J. Clin.
Invest. 84:1145-1146 (1989) (1-proteinase); Oswein et al.,
"Aerosolization of Proteins", Proceedings of Symposium on
Respiratory Drug Delivery II, Keystone, Colo., March, 1990
(recombinant human growth hormone); Debs et al., The Journal of
Immunology 140:3482-3488 (1988) (interferon- and tumor necrosis
factor) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte
colony stimulating factor).
[0179] Contemplated for use in the practice of this invention are a
wide range of mechanical devices designed for pulmonary delivery of
therapeutic products, including but not limited to nebulizers,
metered dose inhalers, and powder inhalers, all of which are
familiar to those skilled in the art.
[0180] Some specific examples of commercially available devices
suitable for the practice of this invention are the Ultravent
nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the
Acorn II nebulizer, manufactured by Marquest Medical Products,
Englewood, Colo.; the Ventolin metered dose inhaler, manufactured
by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler
powder inhaler, manufactured by Fisons Corp., Bedford, Mass.
[0181] All such devices require the use of formulations suitable
for the dispensing of the inventive compound. Typically, each
formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to diluents, adjuvants and/or carriers useful in therapy.
[0182] The inventive compound should most advantageously be
prepared in particulate form with an average particle size of less
than 10 .mu.m (or microns), most preferably 0.5 to 5 .mu.m, for
most effective delivery to the distal lung.
[0183] Carriers include carbohydrates such as trehalose, mannitol,
xylitol, sucrose, lactose, and sorbitol. Other ingredients for use
in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or
synthetic surfactants may be used. Polyethylene glycol may be used
(even apart from its use in derivatizing the protein or analog).
Dextrans, such as cyclodextran, may be used. Bile salts and other
related enhancers may be used. Cellulose and cellulose derivatives
may be used. Amino acids may be used, such as use in a buffer
formulation.
[0184] Also, the use of liposomes, microcapsules or microspheres,
inclusion complexes, or other types of carriers is
contemplated.
[0185] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise the inventive compound
dissolved in water at a concentration of about 0.1 to 25 mg of
biologically active protein per mL of solution. The formulation may
also include a buffer and a simple sugar (e.g., for protein
stabilization and regulation of osmotic pressure). The nebulizer
formulation may also contain a surfactant, to reduce or prevent
surface induced aggregation of the protein caused by atomization of
the solution in forming the aerosol.
[0186] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the inventive
compound suspended in a propellant with the aid of a surfactant.
The propellant may be any conventional material employed for this
purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydrofluorocarbon, or a hydrocarbon, including
trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or
combinations thereof. Suitable surfactants include sorbitan
trioleate and soya lecithin. Oleic acid may also be useful as a
surfactant.
[0187] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing the inventive
compound and may also include a bulking agent, such as lactose,
sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which
facilitate dispersal of the powder from the device, e.g., 50 to 90%
by weight of the formulation.
[0188] Nasal delivery of the inventive compound is also
contemplated. Nasal delivery allows the passage of the protein to
the blood stream directly after administering the therapeutic
product to the nose, without the necessity for deposition of the
product in the lung. Formulations for nasal delivery include those
with dextran or cyclodextran. Delivery via transport across other
mucous membranes is also contemplated.
Dosages
[0189] The dosage regimen involved in a method for treating the
above-described conditions will be determined by the attending
physician, considering various factors which modify the action of
drugs, e.g. the age, condition, body weight, sex and diet of the
patient, the severity of any infection, time of administration and
other clinical factors.
[0190] The inventive compounds may be administered by an initial
bolus followed by a continuous infusion to maintain therapeutic
circulating levels of drug product. As another example, the
inventive compound may be administered as a one-time dose. Those of
ordinary skill in the art will readily optimize effective dosages
and administration regimens as determined by good medical practice
and the clinical condition of the individual patient. The frequency
of dosing will depend on the pharmacokinetic parameters of the
agents and the route of administration. The optimal pharmaceutical
formulation will be determined by one skilled in the art depending
upon the route of administration and desired dosage. See for
example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack
Publishing Co., Easton, Pa. 18042) pages 1435-1712, the disclosure
of which is hereby incorporated by reference. Such formulations may
influence the physical state, stability, rate of in vivo release,
and rate of in vivo clearance of the administered agents. Depending
on the route of administration, a suitable dose may be calculated
according to body weight, body surface area or organ size. Further
refinement of the calculations necessary to determine the
appropriate dosage for treatment involving each of the above
mentioned formulations is routinely made by those of ordinary skill
in the art without undue experimentation, especially in light of
the dosage information and assays disclosed herein, as well as the
pharmacokinetic data observed in the human clinical trials
discussed above. Appropriate dosages may be ascertained through use
of established assays for determining blood levels dosages in
conjunction with appropriate dose-response data. The final dosage
regimen will be determined by the attending physician, considering
various factors which modify the action of drugs, e.g. the drug's
specific activity, the severity of the damage and the
responsiveness of the patient, the age, condition, body weight, sex
and diet of the patient, the severity of any infection, time of
administration and other clinical factors. As studies are
conducted, further information will emerge regarding the
appropriate dosage levels and duration of treatment for various
diseases and conditions.
[0191] The therapeutic methods, compositions and compounds of the
present invention may also be employed, alone or in combination
with other cytokines, soluble mpl receptor, hematopoietic factors,
interleukins, growth factors or antibodies in the treatment of
disease states characterized by other symptoms as well as platelet
deficiencies. It is anticipated that the inventive compound will
prove useful in treating some forms of thrombocytopenia in
combination with general stimulators of hematopoiesis, such as IL-3
or GM-CSF. Other megakaryocytic stimulatory factors, i.e., meg-CSF,
stem cell factor (SCF), leukemia inhibitory factor (LIF),
oncostatin M (OSM), or other molecules with megakaryocyte
stimulating activity may also be employed with mpl ligand.
Additional exemplary cytokines or hematopoietic factors for such
co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), M-CSF, SCF,
GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO,
interferon-alpha (IFN-alpha), consensus interferon, IFN-beta,
IFN-gamma, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15,
IL-16, IL-17, IL-18, thrombopoietin (TPO), angiopoietins, for
example Ang-1, Ang-2, Ang-4, Ang-Y, the human angiopoietin-like
polypeptide, vascular endothelial growth factor (VEGF), angiogenin,
bone morphogenic protein-1, bone morphogenic protein-2, bone
morphogenic protein-3, bone morphogenic protein-4, bone morphogenic
protein-5, bone morphogenic protein-6, bone morphogenic protein-7,
bone morphogenic protein-8, bone morphogenic protein-9, bone
morphogenic protein-10, bone morphogenic protein-11, bone
morphogenic protein-12, bone morphogenic protein-13, bone
morphogenic protein-14, bone morphogenic protein-15, bone
morphogenic protein receptor IA, bone morphogenic protein receptor
IB, brain derived neurotrophic factor, ciliary neutrophic factor,
ciliary neutrophic factor receptor, cytokine-induced neutrophil
chemotactic factor 1, cytokine-induced neutrophil, chemotactic
factor 2, cytokine-induced neutrophil chemotactic factor 2,
endothelial cell growth factor, endothelin 1, epidermal growth
factor, epithelial-derived neutrophil attractant, fibroblast growth
factor 4, fibroblast growth factor 5, fibroblast growth factor 6,
fibroblast growth factor 7, fibroblast growth factor 8, fibroblast
growth factor 8b, fibroblast growth factor 8c, fibroblast growth
factor 9, fibroblast growth factor 10, fibroblast growth factor
acidic, fibroblast growth factor basic, glial cell line-derived
neutrophic factor receptor 1, glial cell line-derived neutrophic
factor receptor 2, growth related protein, growth related protein,
growth related protein, growth related protein, heparin binding
epidermal growth factor, hepatocyte growth factor, hepatocyte
growth factor receptor, insulin-like growth factor I, insulin-like
growth factor receptor, insulin-like growth factor II, insulin-like
growth factor binding protein, keratinocyte growth factor, leukemia
inhibitory factor, leukemia inhibitory factor receptor, nerve
growth factor nerve growth factor receptor, neurotrophin-3,
neurotrophin-4, placenta growth factor, placenta growth factor 2,
platelet-derived endothelial cell growth factor, platelet derived
growth factor, platelet derived growth factor A chain, platelet
derived growth factor AA, platelet derived growth factor AB,
platelet derived growth factor B chain, platelet derived growth
factor BB, platelet derived growth factor receptor, platelet
derived growth factor receptor, pre-B cell growth stimulating
factor, stem cell factor receptor, TNF, including TNF0, TNF1, TNF2,
transforming growth factor, transforming growth factor,
transforming growth factor 1, transforming growth factor 1.2,
transforming growth factor 2, transforming growth factor 3,
transforming growth factor 5, latent transforming growth factor 1,
transforming growth factor binding protein I, transforming growth
factor binding protein II, transforming growth factor binding
protein III, tumor necrosis factor receptor type I, tumor necrosis
factor receptor type II, urokinase-type plasminogen activator
receptor, vascular endothelial growth factor, and chimeric proteins
and biologically or immunologically active fragments thereof. It
may further be useful to administer, either simultaneously or
sequentially, an effective amount of a soluble mammalian mpl
receptor, which appears to have an effect of causing megakaryocytes
to fragment into platelets once the megakaryocytes have reached
mature form. Thus, administration of an inventive compound (to
enhance the number of mature megakaryocytes) followed by
administration of the soluble mpl receptor (to inactivate the
ligand and allow the mature megakaryocytes to produce platelets) is
expected to be a particularly effective means of stimulating
platelet production. 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
conventional methods.
[0192] In cases where the inventive compounds are added to
compositions of platelets and/or megakaryocytes and related cells,
the amount to be included will generally be ascertained
experimentally by techniques and assays known in the art. An
exemplary range of amounts is 0.1 .mu.g-1 mg inventive compound per
10.sup.6 cells.
[0193] It is understood that the application of the teachings of
the present invention to a specific problem or situation will be
within the capabilities of one having ordinary skill in the art in
light of the teachings contained herein. Examples of the products
of the present invention and representative processes for their
isolation, use, and manufacture appear below.
EXAMPLES
[0194] The following sets forth exemplary methods for making and
characterizing some of the compounds disclosed herein.
Example 1
1. Construction of Secondary Peptide Libraries
[0195] A. Preparation of Electrocompetent E. coli Cells: Overnight
E. coli (TG1 strain; Amersham Pharmacia Biotech, Piscataway, N.J.)
culture was prepared in 10 ml of 2xYT medium (1.6% Bacto Tryptone,
1% Yeast Extract, 85.5 mM NaCl) at 37.degree. C. One milliliter of
this overnight culture was used to inoculate 1 liter of 2xYT medium
containing 0.4% glucose and 10 mM MgCl.sub.2, and this one liter
culture was grown in a shaker at 37.degree. C. until
OD.sub.600=0.8. The culture was chilled on ice for 15 min and
centrifuged at 4000 rpm (Beckman JA-10 rotor) for 20 min at
4.degree. C. The bacteria pellets were resuspended in 500 ml of
ice-chilled 10% glycerol solution, and the resulting mixture was
centrifuged at 4000 rpm for 20 min at 4.degree. C. The bacteria
pellets were resuspended again in 500 ml of ice-chilled 10%
glycerol solution, and the resulting mixture again was centrifuged
at 4000 rpm for 20 min at 4.degree. C. The cell pellets were then
resuspended in 25 ml of ice-chilled 10% glycerol solution. This
concentrated bacteria sample was transferred to ice-chilled 50 ml
conical tube and centrifuged at 3500 rpm in a tabletop centrifuge
(Beckman CS-6R) for 15 min at 4.degree. C. The cell pellets were
resuspended in a small volume of ice-chilled glycerol solution, and
100 or 300 .mu.l bacteria stocks were immediately frozen in an
ethanol/dry-ice bath and stored in -80.degree. C. freezer.
[0196] B. Modification of pCES1 Vector
[0197] PCR reaction was performed using Extend Long Template PCR
Systems (Roche Diagnostics Corp., Indianapolis, Ind.) with 1 .mu.g
of pCES1 vector (TargetQuest Inc.) as a template. The volume of PCR
mixture was 100 .mu.l which contains 1.times.PCR buffer, 200 nM of
each of the two primer 5'-CAAACGAATGGATCCTCATTAAAGCCAGA-3' and
5'-GGTGGTGCGGCCGCACTCGAGACTGTTGAAAGTTGTTTAGCA-3', 200 nM DNTP, 3 U
of Tag DNA polymerase. The TRIO-Thermoblock (Biometra) PCR system
was used to run the following program: 94.degree. C. for 5 min; 30
cycles of [94.degree. C. for 30 second, 50.degree. C. for 30
second, 72.degree. C. for 45 second]; 72.degree. C. for 10 min;
cool to 4.degree. C. The PCR products were run on a 1% agarose gel
and purified with QIAGEN Spin Column (QIAGEN Inc., Valencia,
Calif.) according to the manufacturer's protocols. A second PCR
reaction was performed with 5 .mu.l of PCR products and 200 nM of
each of the two primer 5'-CAAACGAATGGATCCTCATTAAAGCCAGA-3' and
5'-AACACAAAAGTGCACAGGGTGGAGGTGGTGGTGCGGCCGCACT-3' under the same
PCR conditions as described above.
[0198] The PCR products and original pCES1 vector were digested
separately in a 100 .mu.l reaction containing 1.times.NEB2 buffer,
60 U of ApaLI (New England Biolabs, Beverly, Mass.), 60 U of BamHI
(New England Biolabs) at 37.degree. C. for 1 hr. Both digested DNA
were purified with QIAGEN Spin Column and ligated together in a 40
.mu.l reaction containing 1.times. ligation buffer and 40 U of T4
DNA ligase (New England Biolabs) at room temperature overnight.
[0199] The vectors were transfected into E. coli and incubated at
37.degree. C. overnight. Isolated single colonies were selected and
plasmid was purified with QIAGEN Spin Column. The correct insert
was confirmed by DNA sequencing.
[0200] C. Preparation of Vector DNA
[0201] One microgram of the modified pCES1 vector DNA (section 1B)
was transformed into 100 .mu.l of electrocompetent TG1 E. coli
(section 1A) using the Gene Pulser II (BIO-RAD, Hercules, Calif.)
with the setting of 2500 V, 25.degree. F., and 200 ohms. The
transformed bacteria sample was then transferred immediately into a
tube containing 900 .mu.l of SOC (2% tryptone, 0.5% yeast extract,
10 mM NaCl, 2.5 mM KCl, 20 mM glucose, 10 mM MgSO.sub.4, 10 mM
MgCl.sub.2), and this culture was allowed to grow at 37.degree. C.
with shaking for 1 hour. The cells were then spread onto the 2xYTAG
(2xYT with 10 ug/ml ampicillin and 2% glucose) agar plate and
incubated at 37.degree. C. overnight. A single colony was used to
inoculate 1 liter of 2xYTAG media at 37.degree. C. with shaking
overnight. The plasmid vector DNA was purified with QIAGEN Plasmid
Maxi Kit according to the manufacturer's protocols.
[0202] D. Digestion of Vector DNA
[0203] Fifty microgram of vector DNA (section 1C) was digested in a
400 .mu.l reaction containing 1.times.NEB buffer2, 200 U of ApaLI,
and 200 U of XhoI at 37.degree. C. overnight. This restriction
digest reaction was incubated overnight at 37.degree. C. and
analyzed in a pre-made 1% agarose gel (Embi Tec, San Diego,
Calif.). The linearized vector DNA was excised from the gel and
extracted with QIAquick Gel Extraction Kit (QIAGEN Inc.) according
to the manufacturer's directions.
[0204] E. Preparation of Library Oligonucleotides
[0205] Two library oligonucleotides (fixed and doped) were
designed. The fixed library oligonucleotide
5'-CACAGTGCACAGGGTNNKNNKNNKNNKGGTCCTACTCTGMRKSARTGGCTGNNKNNKNNK
NNKNNKNNKCATTCTCTCGAGATCG-3' and the doped library oligonucleo-tide
5'-CACAGTGCAC-AGGGTNNKNNKNNKNNKggKcc-KacKctKNNKNNKtgKNNKNNKNNKNNKNNKNNKNN-
KCATTCTCTCGAGATCG-3' (lower case letters represent a mixture of 70%
of the indicated base and 10% of each of the other three
nucleotides) were synthesized. Each of these oligonucleotides was
used as templates in Polymerase Chain Reactions.
[0206] Expand High Fidelity PCR System kit (Roche Diagnostics
Corp.) was used for the PCR reactions. Each PCR reaction was 100
.mu.l in volume and contained 10 nM of a library oligonucleotide,
1.times.PCR buffer, 300 nM of each of the primers
5'-CACAGTGCACAGGGT-3' and 5'-TGATCTCGAGAGAATG-3', 200 nM DNTP, 2 mM
CaCl.sub.2, and 5 U of the Expand polymerase. The thermocycler
(GeneAmp PCR System 9700, Applied Biosystem) was used to run the
following program: 94.degree. C. for 5 min; 30 cycles of
[94.degree. C. for 30 second, 55.degree. C. for 30 second,
72.degree. C. for 45 second]; 72.degree. C. for 7 min; cool to
4.degree. C. The free nucleotides were removed using the QIAquick
Nucleotide Removal Kit (QIAGEN Inc.) according to the
manufacturer's protocols.
[0207] F. Digestion of Library Oligonucleotides
[0208] Five microgram of the each of the PCR products (section 1E)
was digested in a 400 .mu.l reaction that contained 1.times.NEB
buffer2, 200 U of ApaLI, and 200 U of XhoI at 37.degree. C.
overnight. The digested DNA was separated on a 3% agarose gel (Embi
Tec). The DNA band of interest from each reaction was cut from the
gel and extracted with QIAquick Gel Extraction Kit.
[0209] G. Ligation of Vector with Library Oligonucleotides
[0210] The linearized vector (section 1D, 25 .mu.g) and each
digested PCR product (section 1F, 5 ug) were ligated in a 400 .mu.l
reaction containing 1.times.NEB ligation buffer and 80 U of the T4
DNA ligase at 16.degree. C. overnight. The ligated products were
incubated at 65.degree. C. for 20 minutes to inactivate the DNA
ligase and further incubated with 8 U NotI at 37.degree. C. for 2
hr to minimize vector self-ligation. The ligated products were then
purified by a standard phenol/chloroform extraction (Molecular
Cloning, Maniatis et al 3.sup.rd Edition) and resuspended in 30
.mu.l of H.sub.2O.
[0211] H. Electroporation Transformation
[0212] For each library, ten electroporation reactions were
performed. For each transformation, 3 .mu.l of the ligated vector
DNA (section 1G) and 300 .mu.l of TG1 cells (section 1A) were mixed
in a 0.2-cm cuvette (BIO-RAD). The resulting mixture was pulsed by
the Gene Pulser II with the setting of 2500 V, 25 uF, and 200 ohms.
The transformed bacteria samples from the ten electroporation
reactions were combined and transferred into a flask containing 27
ml of SOC for incubation at 37.degree. C. for 1 hr. The cells were
then added to 170 ml 2xYTAG and grew at 37.degree. C. with shaking
for 3 hrs. The cells were centrifuged at 5000 rpm for 10 min at
4.degree. C. The cell pellets were then resuspended in 10 ml of 15%
glycerol/2xYT and stored at -80.degree. C. This is the primary
stock of the libraries. Titers showed library sizes of
1.0.times.10.sup.9 independent transformants and 2.4.times.10.sup.9
independent transformants for the fixed and doped library,
respectively.
2. Amplification of the Libraries
[0213] A. Making Secondary Stock of the Libraries
[0214] The primary library cell stock (section 1H) was used to
inoculate 1300 ml (for fixed library) and 2600 ml (for doped
library) of 2xYTAG media so that the starting OD.sub.600=0.1. The
cultures were allowed to grow at 37.degree. C. with shaking for
several hours until OD.sub.600=0.5. A 120 ml aliquot for the fixed
library and a 240 ml aliquot for the doped library were taken out
and grown up in separate flasks for another two hours at 37.degree.
C. These sub-cultures were centrifuged at 5000 rpm (Beckman JA-14
rotor) for 10 min at 4.degree. C., and the bacteria pellets were
resuspended in 10 ml (for each library) of 15% glycerol/2xYT for
storage at -80.degree. C.
[0215] B. Phage Induction
[0216] M13KO7 helper phage aliquots (Amersham Pharmacia Biotech)
were added to the remaining bacteria cultures at OD.sub.600=0.5
(section 2A) to the final concentration of 3.times.10.sup.9 pfu/ml.
The helper phages were allowed to infect bacteria at 37.degree. C.
for 30 min without shaking and 30 min with slow shaking. The
infected cells were centrifuged with 5000 rpm for 10 min at
4.degree. C. The cell pellets were resuspended with 1300 ml (fixed
library) and 2600 ml (doped library) of 2xYTAK (2YT with 100 ug/ml
ampicillin and 40 ug/ml kanamycin). The phagemid production was
allowed to occur at 37.degree. C. overnight while shaking.
[0217] C. Harvest of Phage
[0218] The bacteria cultures (section 2B) were centrifuged at 5000
rpm for 10 min at 4.degree. C. The supernatants were transferred
into new bottles, and 0.2 volume of 20% PEG/2.5M NaCl were added
and incubated on ice for 1 hr to precipitate the phagemids.
Precipitated phagemids were centrifuged at 8000 rpm for 20 min at
4.degree. C. and carefully resuspended with 100 ml of cold PBS. The
phagemid solution was further purified by centrifuging away the
remaining cells with 8000 rpm for 10 min at 4.degree. C. and
precipitating the phagemids by adding 0.2 volume of 20% PEG/2.5M
NaCl. The phagemids were centrifuged at 8000 rpm for 20 min at
4.degree. C., and the phagemid pellets were resuspended with 12 ml
of cold PBS. Four milliliter of 60% glycerol solution was added to
the phagemid solution for storage at -80.degree. C. The phagemid
titers were determined by a standard procedure (Molecular Cloning,
Maniatis et al 3.sup.rd Edition).
3. Selection of Human MPL Binding Phages
[0219] A. Biotinylation of Human MPL
[0220] One milligram of recombinant human MPL was biotinylated
using the EZ-Link Sulfo-NHS-LC-Biotinylation Kit (PIERCE, Rockford,
Ill.) according to the manufacturer's directions.
[0221] B. Immobilization of MPL on Magnetic Beads
[0222] The biotinylated MPL (section 3A) was immobilized on the
Dynabead M-280 Streptavidin (DYNAL, Lake Success, N.Y.) at a
concentration of 1 .mu.g MPL per 100 .mu.l of the bead stock from
the manufacturer. After drawing the beads to one side of a tube
using a magnet and pipetting away the liquid, the beads were washed
twice with the phosphate buffer saline (PBS) and resuspended in
PBS. The biotinylated MPL protein was added to the washed beads at
the above concentration and incubated with rotation for 1 hour at
room temperature. The MPL coated beads were then blocked by adding
BSA to 2% final concentration and incubating overnight at 4.degree.
C. with rotation. The resulting MPL coated beads were then washed
twice with PBST (PBS with 0.05% Tween-20) before being subjected to
the selection procedures.
[0223] C. Selection Using the MPL Coated Beads
[0224] About 100 fold library equivalent phagemids (section 2C,
1.times.10.sup.11 cfu for fixed library, 2.4.times.10.sup.11 cfu
for doped library) were blocked for one hour with 1 ml of PBS
containing 2% BSA. The blocked phagemid sample was subjected to a
negative selection step by adding it to blank beads (same beads as
section 3B but no MPL coated), and this mixture was incubated at
room temperature for 1 hr with rotation. The phagemid containing
supernatant was drawn out using magnet and transferred to a new
tube containing MPL coated beads (section 3B), and this mixture was
incubated at room temperature for 1 hr with rotation. After the
supernatant was discarded, the phagemid-bound-beads were washed 10
times with PBST and 10 times with PBS. The phagemids were then
allowed to elute in 1 ml of 100 mM triethylamine solution (Sigma,
St. Louis, Mo.) for 10 minutes on a rotator. The pH of the phagemid
containing solution was neutralized by adding 0.5 ml of 1 M
Tris-HCl (pH 7.5). The resulting phagemids were used to infect 5 ml
of freshly grown TG1 bacteria (OD.sub.600 about 0.5) at 37.degree.
C. for 30 minutes without shaking and 30 minutes with slow shaking.
All the infected TG1 cells were plated on a large 2xYTAG plate and
incubated at 30.degree. C. overnight.
[0225] D. Induction and Harvesting of Phage
[0226] A 10 ml aliquot of 2xYTAG media was added to the plate
(section 3C) to resuspend TG1 cells. All TG1 cells were collected
in a tube, and a 250 .mu.l aliquot of these cells was added to 25
ml of 2xYTAG and grown at 37.degree. C. until OD.sub.600=0.5. The
M13KO7 helper phages were added to a final concentration of
3.times.10.sup.9 cfu/ml and incubated at 37.degree. C. for 30
minutes without shaking and 30 minutes with slow shaking. The cells
were centrifuged with 5000 rpm for 10 minute at 4.degree. C. and
resuspended with 25 ml of 2xYTAK. These bacteria were allowed to
grow at 30.degree. C. overnight with shaking. The induced phagemids
were harvest and purified as in section 2C.
[0227] E. Second Round Selection
[0228] The second round selection was performed as outlined in
section 3B to 3C except the following. About 0.5 ml aliquot of
phagemid solution resulting from section 3D was used as the input
phagemid. Only 0.1 .mu.g of biotinylated MPL (section 3A) was used
to coat onto the Dynabead M-280 Streptavidin. The phage-bound-beads
were washed 16 times with PBST, where the final wash involved 30
minutes incubation at room temperature in PBST. The beads were
washed 10 more times with PBS.
4. Clonal Analysis
[0229] A. Preparation of Master Plate
[0230] Single colonies from the second round selection were picked
and inoculated into 96 well plates containing 120 .mu.l of 2xYTAG
per well. The 96 well plates were incubated in 30.degree. C. shaker
for overnight. Forty microliters of 60% glycerol were added per
well for storage at -80.degree. C.
[0231] B. Phagemid ELISA
[0232] About 3 .mu.l aliquots of cells from the master plate
(section 4A) were inoculated into a fresh 96 well plate with
containing 120 .mu.l of 2xYTAG per well, and this new plate of
cells were grown at 37.degree. C. until approximate OD.sub.600=0.5.
Forty microliters of 2xYTAG containing M13KO7 helper phage
(1.2.times.10.sup.10 cfu/ml) were added to each well, and the 96
well plate was incubated at 37.degree. C. for 30 minutes without
shaking and another 30 min with slow shaking. The plate was
centrifuged at 2000 rpm (Beckman CS-6R tabletop centrifuge) for 10
min at 4.degree. C. The supernatants were removed from the wells,
and each cell pellet was resuspended using 160 .mu.l of 2xYTAK per
well. The plate was incubated at 30.degree. C. overnight for
phagemid expression.
[0233] Recombinant human MPL was coated onto the 96 well Maxisorp
plate (NUNC) at 5 .mu.g/ml in 0.1 M carbonate buffer pH9.6 at
4.degree. C. overnight. As a control, BSA (Sigma) was coated onto a
separate Maxisorp plate at 5 ug/ml.
[0234] On the following day, the overnight cell cultures were
centrifuged at 2000 rpm for 10 min at 4.degree. C. Twenty
microliters of supernatant from each well were transferred to a new
96 well plate containing 180 .mu.l of 2% BSA/PBS solution per well.
The resulting mixtures were incubated for 1 hour at room
temperature with shaking to block the phagemids. Meanwhile, the MPL
coated plate was blocked with 200 .mu.l of 2% BSA/PBS solution per
well for 1 hour at room temperature while shaking. The BSA solution
was discarded, and each well was washed three times with PBST
solution. After the last washing step, 50 .mu.l of blocked phagemid
solutions was added to each well of the MPL coated plate as well as
the control plate and incubated for 1 hour at room temperature with
shaking. The liquid was discarded, and each well was washed three
times with PBST solution. Fifty microliters of the HRP-conjugated
anti-M13 mAb (Amersham Pharmacia Biotech) at 1:15,000 dilution were
added to each well of the MPL coated and control plates, and these
plates were incubated for 1 hour at room temperature with shaking.
The liquids were discarded again, and each well was washed three
times with PBST solution. Fifty microliters of LumiGLO
chemiluminescent substrates (Kirkegaard & Perry Laboratories,
Gaithersburg, Md.) were added to the wells, and each well was read
by Luminoskan Ascent DLRearly machine (Labsystems, Franklin,
Mass.).
[0235] C. Sequencing of the Phage Clones
[0236] PCR reaction was performed using 1 .mu.l of bacteria from
each well of the master plate (section 4A) as a template. The
volume of each PCR mixture was 20 .mu.l which contains 1.times.PCR
buffer, 300 nM of each of the two primers
5'-GTTAGCTCACTCATTAGGCAC-3' and 5'-GTACCGTAACACTGAGTTTCG-3', 200 nM
DNTP, 2 mM CaCl.sub.2, and 5 U taq DNA polymerase (Roche Molecular
Biochemicals). The GeneAmp PCR System 9700 (Applied Biosystem) was
used to run the following program: 94.degree. C. for 5 min; 40
cycles of [94.degree. C. for 45 second, 55.degree. C. for 45
second, 72.degree. C. for 90 second]; 72.degree. C. for 10 min;
cool to 4.degree. C. The PCR products were purified with QIAquick
96 PCR Purification Kit (QIAGEN Inc.) according to the
manufacturer's directions. All purified PCR products were sequenced
with primer 5'-CGGATAACAATTTCACACAGG-3' using the ABI 3770
Sequencer (Perkin Elmer) according to the manufacturer's
directions.
5. Sequence Ranking
[0237] The peptide sequences that were translated from nucleotide
sequences above were correlated to ELISA data. The clones that
showed high OD reading in the MPL coated wells and low OD reading
in the BSA coated wells were considered as candidates for further
study. The sequences that occur multiple times were also considered
as candidates for further study. The phage clones selected based on
these criteria were further characterized in ELISA titration
experiments. See FIG. 9 (ELISA dose-response of selected phage
clones).
Example 2
Preparation of Peptides
[0238] All peptides were prepared by the well-established stepwise
solid phase synthesis method. Merrifield (1963), J. Am. Chem. Soc.
85:2149. Steward and Young (1969), Solid Phase Peptide Synthesis.
Fmoc-protected amino acids were used as the building blocks and the
peptide-chain was built-up using an ABI or Symphony peptide
synthesizer. Typically, peptide synthesis began with a preloaded
Wang resin to generate a peptide with a free carboxylic acid at the
C-terminus (alternatively, Rink resin can be used to generate a
peptide with a C-terminal amide functionality). Fmoc removal was
carried out with the standard piperidine protocol. The coupling was
effected using uronium (such as HBTU) or carbodiimide (such as
DCC/HOBt) chemistry. Side-chain protecting groups were:
Glu(O-t-Bu), Asp(O-t-Bu), Ser(t-Bu), Thr(t-Bu), Arg(Pbf), Asn(Trt),
Gln(Trt), His(Trt), Lys(t-Boc), Trp(t-Boc) and Cys(Trt). The final
deprotection and cleavage of all peptidyl-resins was effected at RT
for 4 hr, using trifluoroacetic acid (TFA) containing 2.5%
H.sub.2O, 5% phenol, 2.5% triisopropylsilane and 2.5% thioanisole
or mercaptoethanol. After removal of TFA, the cleaved peptide was
precipitated with cold anhydrous ether. For those peptides that
contain disulfide bonds, formation of the cyclic products was
performed directly on the crude material by using 15% DMSO in
H.sub.2O (pH 7.5). All crude peptides were purified by reverse
phase HPLC and the structures of purified peptides were confirmed
by ESI-MS and amino acid analysis.
Example 3
Preparation of TMP-Fc Peptibody Compounds
[0239] Several peptides were chosen for expression as peptide-Fc
fusions (i.e., Fc attached to the C-terminus of the peptide)
(C-terminal fusions). A DNA sequence coding for the Fc region of
human IgG1 fused in-frame to each TPO-mimetic peptide was placed
under control of the luxPR promoter in the plasmid expression
vector pAMG21 as follows.
[0240] The plasmid encoding TMP1-Fc (Amgen strain #3788) was
altered to contain an ApaLI site and a XhoI site to allow for easy
cloning of short peptides from annealed oligonucleotides. The
primer 2396-69 was used to add the ApaLI and XhoI restriction
enzyme sites. PCR was performed with Expand Long polymerase using
2396-69 and the universal 3' primer 191-24 on the 3788 DNA
template. The primer sequences are as follows:
TABLE-US-00012 2396- ACAAACAAACATATGGGTGCACAGAAAGCGGCCGCAAAAAAACT
69 CGAGGGTGGAGGCGGTGGGGACA 191-24 GGTCATTACTGGACCGGATC
[0241] The resulting PCR fragment was digested with NdeI and BsrGI,
gel purified, and used as the insert. The plasmid from strain #3788
was also digested NdeI and BsrGI, gel purified, and used as the
vector. Vector and insert were ligated together, and the resulting
ligation mixture was electroporated into GM221 cells (see below).
Single colonies were picked and plasmid DNA was prepared and DNA
sequenced. One resulting plasmid, 200003180, was shown to have the
correct DNA sequence and was used as the vector for constructing
TMP-Fc fusions. This vector is shown in FIG. 6.
[0242] Plasmid 200003180 was digested with ApaLI and XhoI and
served as the vector. Each pair of oligonucleotides (see FIG. 7)
was annealed to form a duplex with ApaLI and XhoI sticky ends.
These molecules were ligated into the vector to produce the fusion
proteins of interest. The ApaLI to XhoI fragment for each
corresponding pair of oligonucleotides is provided in FIG. 7.
[0243] TMPs 1-23, 25, 26 and 28 were expressed as C-terminal
fusions.
Example 4
Preparation of Fc-TMP Peptibody Compounds
[0244] Some of the peptides were expressed as Fc-peptide fusions
(i.e., Fc attached to the N-terminus of peptide) (N-terminal
fusions). The plasmid encoding Fc-TMP1 (Amgen strain #3728) was
altered to contain an ApaLI site and an XhoI site to allow for easy
cloning of short peptides from annealed oligonucleotides. A primer,
2396-70, was designed to add the ApaLI and XhoI restriction enzyme
sites. PCR was performed with Expand Long polymerase using 2396-70
and the universal 5' primer 1209-85 on the 3728 DNA template. The
primer sequences are as follows:
TABLE-US-00013 1209-85 CGTACAGGTTTACGCAAGAAAATGG 2396-70
TTTGTTGGATCCATTACTCGAGTTTTTTTGCGGCC
GCTTTCTGTGCACCACCACCTCCACCTTTAC
The resulting PCR fragment was digested with BsrGI and BamHI, gel
purified, and used as the insert. The plasmid from strain #3728 was
also digested with BsrGI and BamHI, gel purified, and used as the
vector. Vector and insert were ligated together, and the resulting
ligation mixture was electroporated into GM221 cells. Single
colonies were picked and plasmid DNA was prepared and DNA
sequenced. One resulting plasmid, 200003182 (FIG. 8), was shown to
have the correct DNA sequence and was used as the vector for
constructing Fc-TMP fusions.
[0245] The 200003182 plasmid was digested with ApaLI and XhoI and
served as the vector. Annealed oligos with ApaLI and XhoI sticky
ends were ligated into the vector to produce the fusions of
interest.
[0246] TMP20, TMP24, TMP27, TMP29 and TMP30 were produced as
N-terminal fusions in this manner.
Transformation
[0247] Each of the above ligations were transformed by
electroporation into the host strain GM221 described below. Clones
were screened for the ability to produce the recombinant protein
product and to possess the gene fusion having the correct
nucleotide sequence.
pAMG21
[0248] The expression plasmid pAMG21 is available from the ATCC
under accession number 98113, which was deposited on Jul. 24,
1996.
GM221 (Amgen Host Strain #2596)
[0249] The Amgen host strain #2596 is an E. coli K-12 strain that
has been modified to contain both the temperature sensitive lambda
repressor cI857s7 in the early ebg region and the lacIQ repressor
in the late ebg region (68 minutes). The presence of these two
repressor genes allows the use of this host with a variety of
expression systems, however both of these repressors are irrelevant
to the expression from luxPR. The untransformed host has no
antibiotic resistances.
[0250] The ribosome binding site of the cI857s7 gene has been
modified to include an enhanced RBS. It has been inserted into the
ebg operon between nucleotide position 1170 and 1411 as numbered in
Genbank accession number M64441 Gb_Ba with deletion of the
intervening ebg sequence.
[0251] The construct was delivered to the chromosome using a
recombinant phage called MMebg-cI857s7 enhanced RBS #4 into
F'tet/393. After recombination and resolution only the chromosomal
insert described above remains in the cell. It was renamed
F'tet/GM101.
[0252] F'tet/GM101 was then modified by the delivery of a lacIQ
construct into the ebg operon between nucleotide position 2493 and
2937 as numbered in the Genbank accession number M64441 Gb_Ba with
the deletion of the intervening ebg sequence. The construct was
delivered to the chromosome using a recombinant phage called
AGebg-LacIQ#5 into F'tet/GM101. After recombination and resolution
only the chromosomal insert described above remains in the cell. It
was renamed F'tet/GM221. The F'tet episome was cured from the
strain using acridine orange at a concentration of 25 ug/ml in LB.
The cured strain was identified as tetracycline sensitive and was
stored as GM221.
Expression.
[0253] Cultures of GM221 expressing each of the fusion proteins
were grown at 37.degree. C. in Luria Broth medium. Induction of
gene product expression from the luxPR promoter was achieved
following the addition of the synthetic autoinducer
N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a
final concentration of 20 ng/ml and incubation at 37.degree. C. for
a further 3 hours. After 3 hours, the bacterial cultures were
examined by microscopy for the presence of inclusion bodies and
were then collected by centrifugation. Refractile inclusion bodies
were observed in induced cultures indicating that the fusion
protein was most likely produced in the insoluble fraction in E.
coli. Cell pellets were lysed directly by resuspension in Laemmli
sample buffer containing 10% .beta.-mercaptoethanol and were
analyzed by SDS-PAGE. An intense Coomassie stained band of the
appropriate size (approximately 30 kDa) was observed for each
protein.
Example 5
Purification of Peptibody Compounds
[0254] Cells were broken in water (1/10) by high pressure
homogenization (2 passes at 14,000 PSI) and inclusion bodies were
harvested by centrifugation (4200 RPM in J-6B for 1 hour).
Inclusion bodies were solubilized in 6 M guanidine, 50 mM Tris, 8
mM DTT, pH 8.7 for 1 hour at a 1/10 ratio. The solubilized mixture
was diluted 20 times into 2 M urea, 50 mM Tris, 160 mM arginine, 3
mM cysteine, pH 8.5. The mixture was stirred overnight in the cold.
The mixture was then concentrated about 10 fold by ultrafiltration.
It was then diluted 3 fold with 10 mM Tris, 1.5 M urea, pH 9. The
pH of this mixture was then adjusted to pH 5 with acetic acid. The
precipitate was removed by centrifugation and the supernatant was
loaded onto a SP-Sepharose Fast Flow column equilibrated in 20 mM
NaAc, 100 mM NaCl, pH 5 (10 mg/ml protein load, room temperature).
The protein was eluted using a 20 column volume gradient in the
same buffer ranging from 100 mM NaCl to 500 mM NaCl. The pool from
the column was diluted 3 fold and loaded onto a SP-Sepharose HP
column in 20 mM NaAc, 150 mM NaCl, pH 5 (10 mg/ml protein load,
room temperature). The protein was eluted using a 20 column volume
gradient in the same buffer ranging from 150 mM NaCl to 400 mM
NaCl. The peak was pooled and filtered.
Example 6
Peptide Affinity Binding Studies
[0255] Experiment were carried out using BIACORE 3000 at room
temperature to determine the binding affinity for several TMP
peptides (TMP1-TMP23). Hu-mpl was immobilized on the sensor chip
(CM5) surface using amine coupling procedure (activation by NHS/EDC
and blocking by ethanolamine). 0.78 nM to 100 nM of TMP peptides
were injected over the hu-mpl surface. BIACORE running buffer was
PBS with 0.005% Surfactant P20. Samples were also injected over a
blank surface for a control. The experimental data were analyzed
using BIAEVALUATION 3.1 software package.
[0256] As previously discussed, to better mimic the phage
environment from which the peptides were selected and to conceal
from the receptor the charged amino- and carboxy-terminus ends of
the 18 amino acid preferred peptides (TMP2-TMP30), two amino acid
"caps" were added to each of the carboxy terminus and the amino
terminus of each peptide: glutamine-cysteine (QC) to the amino
terminus and histadine-serine (HS) to the carboxy terminus,
bringing the length of each peptide to 22 amino acids. Since
peptide affinity is known to increase with peptide length, the
benchmark bioactive 14 amino acid peptide sequence (SEQ ID NO 1)
was also increased to a total of 22 amino acids. The bioactive
region of each peptide, however, remains the same and is indicated
in bold below.
TABLE-US-00014 Affinity relative TMP No. Peptide Sequence K.sub.D
(nM) to TMP1 TMP1 SAQGIEGPTLRQWLAARALETV 5.40 -- TMP2
QGGAREGPTLRQWLEWVRVGHS 1.60 3.38 TMP3 QGRDLDGPTLRQWLPLPSVQHS 45.00
0.12 TMP4 QGALRDGPTLKQWLEYRRQAHS 0.86 6.28 TMP5
QGARQEGPTLKEWLFWVRMGHS 6.66 0.81 TMP6 QGEALLGPTLREWLAWRRAQHS 0.37
14.59 TMP7 QGMARDGPTLREWLRTYRMMHS 1.20 4.50 TMP8
QGWMPEGPTLKQWLFHGRGQHS 23.20 0.23 TMP9 QGHIREGPTLRQWLVALRMVHS 1.67
3.23 TMP10 QGQLGHGPTLRQWLSWYRGMHS 1.22 4.43 TMP11
QGELRQGPTLHEWLQHLASKHS 35.90 0.15 TMP12 QGVGIEGPTLRQWLAQRLNPHS 5.20
1.04 TMP13 QGWSRDGPTLREWLAWRAVGHS 4.44 1.22 TMP14
QGAVPQGPTLKQWLLWRRCAHS 0.88 6.14 TMP15 QGRIREGPTLKEWLAQRRGFHS 1.03
5.24 TMP16 QGRFAEGPTLREWLEQRKLVHS 6.58 0.82 TMP17
QGDRFQGPTLREWLAAIRSVHS 12.90 0.42 TMP18 QGAGREGPTLREWLNMRVWQHS
12.80 0.42 TMP19 QGALQEGPTLRQWLGWGQWGHS 78.50 0.07 TMP20
QGYCDEGPTLKQWLVCLGLQHS 0.56 9.64 TMP21 QGWCKEGPTLREWLRWGFLCHS 1.53
3.53 TMP22 QGCSSGGPTLREWLQCRRMQHS <0.1 >54 TMP23
QGCSWGGPTLKQWLQCVPAKHS <0.1 >54
Example 7
Peptide Bioactivity Studies
[0257] Cell-based assays were used to determine the bioactivity of
the peptides TMP1-TMP23.
[0258] The murine 32D cell proliferation assay involves the use of
murine 32D cells that have been transfected with a human mpl
receptor. The results below are reported relative to TMP1.
[0259] The CD61 cell assay involves the use of primary human CD34+
cells, which were cultured for several days in the presence of
peptides TMP1-TMP23. These cells were then sorted to determine the
percentage of cells expressing a megakaryocyte specific marker
(CD61) on the cell surface. While active compounds stimulated the
appearance of these platelet precursors cells in a dose-dependent
fashion, markers for erythroid precursors (CD36+) and neutrophil
precursors (CD15+) remained at baseline. Qualitative results of the
CD61 cell assay, which represent the average of three different
concentrations, are shown below.
TABLE-US-00015 Murine 32D Cell Proliferation Assay CD61 Cell Assay
Peptide (relative to TMP1) (relative to TMP1 TMP01 100% -/+ TMP02
290% + TMP03 39% ++ TMP04 42% - TMP05 85% ++ TMP06 569% ++ TMP07
289% ++ TMP08 39% + TMP09 2% - TMP10 12% - TMP11 21% - TMP12 10% -
TMP13 328% ++ TMP14 635% +++ TMP15 35% - TMP16 32% + TMP17 21% -
TMP18 337% ++ TMP19 27% + TMP20 Not Detectable -/+ TMP21 312% -/+
TMP22 Not Detectable - TMP23 Not Detectable +++
Example 8
Peptibody Binding Studies
[0260] Several TMP peptibodies were tested for their binding
activities to hu-MPL in a direct binding analysis on BIAcore. The
experiments were carried out using BIAcore 2000 (BIACORE Inc.) at
25 C. The running buffer was PBS with 0.005% Surfactant P20.
Recombinant Protein G (Pierce 21193ZZ) was immobilized onto a CM5
chip following a standard amine coupling procedure (activation by
NHS/EDC and blocking by ethanolamine) to capture the TMP
peptibodies to approximate 400 RU. Recombinant hu-MPL (Lot
27315-53) was serially diluted from 1 uM to 0.15 nM in sample
buffer (PBS with 0.005% Surfactant P20 and 100 ug/ml BSA) before
injection over the captured peptibody surfaces at 50 ul/min for 3
minutes. rhu-MPL samples were also injected over a blank protein G
surface to subtract any non-specific binding background. The
protein G surface was regenerated with sequential injection of 100
ul of ImmunoPure IgG elution buffer (Pierce 21009ZZ, pH 2) and 100
ul of 8 mM Glycine pH 1.5, 1 M NaCl at 50 ul/min between two
cycles. Binding affinities (K.sub.D) of the peptibodies to rhu-MPL
were determined by nonlinear regression analysis of the data using
BIAevaluation 3.1 (BIACORE Inc.). The results are summarized as
follows:
TABLE-US-00016 Peptibody (TMP- Fc) k.sub.a (1/Ms) k.sub.d (1/s)
K.sub.D (M) TMP20-Fc 5.06 .times. 10.sup.4 7.34 .times. 10.sup.-3
1.45 .times. 10.sup.-7 Fc-TMP24 4.01 .times. 10.sup.4 8.75 .times.
10.sup.-3 2.18 .times. 10.sup.-7 TMP25-Fc 2.35 .times. 10.sup.4
1.40 .times. 10.sup.-3 5.97 .times. 10.sup.-8 TMP26-Fc 1.3 .times.
10.sup.5 8.42 .times. 10.sup.-3 6.49 .times. 10.sup.-8 TMP28-Fc
6.78 .times. 10.sup.4 2.52 .times. 10.sup.-2 3.71 .times.
10.sup.-7
Example 9
Peptibody Activity Assays
[0261] Primary human CD34+ cells were cultured for several days in
the presence of several TMP-Fc fusion proteins. These cells were
then sorted to determine the percentage of cells expressing a
megakaryocyte specific marker (CD61) on the cell surface. While
active compounds stimulated the appearance of these platelet
precursor cells in a dose-dependent fashion, markers for erythroid
precursors (CD36+) (not shown) and neutrophil precursors (CD15+)
(not shown) remained at baseline. See FIGS. 10, 11 and 12(CD61 cell
assay).
Example 10
In Vivo Activity
[0262] Normal female BDF1 mice, approximately 10-12 weeks of age,
were used for in vivo activity studies.
[0263] Mice were injected subcutaneously for a bolus treatment.
Subcutaneous injections were delivered in a volume of 0.2 ml.
Compounds were diluted in PBS with 0.1% BSA. All experiments
included one control group, labeled "carrier" that were treated
with this diluent only.
[0264] Ten mice per group treated on day 0, two groups started 4
days apart for a total of 20 mice per group. Five mice bled at each
time point, mice were bled a minimum of three times a week. Mice
were anesthetized with isoflurane and a total volume of 140-160 ul
of blood was obtained by puncture of the orbital sinus. Blood was
counted on a Technicon H1E blood analyzer running software for
murine blood. Parameters measured were white blood cells, red blood
cells, hematocrit, hemoglobin, platelets, neutrophils. See FIGS. 13
and 14.
[0265] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto, without departing from the
spirit and scope of the invention as set forth herein.
Sequence CWU 1
1
199118PRTArtificial SequenceSynthesized Peptide Sequence 1Gln Gly
Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala1 5 10 15Leu
Glu218PRTArtificial SequenceSynthesized Peptide Sequence 2Gly Ala
Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu Glu Trp Val Arg1 5 10 15Val
Gly318PRTArtificial SequenceSynthesized Peptide Sequence 3Arg Asp
Leu Asp Gly Pro Thr Leu Arg Gln Trp Leu Pro Leu Pro Ser1 5 10 15Val
Gln418PRTArtificial SequenceSynthesized Peptide Sequence 4Ala Leu
Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr Arg Arg1 5 10 15Gln
Ala518PRTArtificial SequenceSynthesized Peptide Sequence 5Ala Arg
Gln Glu Gly Pro Thr Leu Lys Glu Trp Leu Phe Trp Val Arg1 5 10 15Met
Gly618PRTArtificial SequenceSynthesized Peptide Sequence 6Glu Ala
Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg1 5 10 15Ala
Gln718PRTArtificial SequenceSynthesized Peptide Sequence 7Met Ala
Arg Asp Gly Pro Thr Leu Arg Glu Trp Leu Arg Thr Tyr Arg1 5 10 15Met
Met818PRTArtificial SequenceSynthesized Peptide Sequence 8Trp Met
Pro Glu Gly Pro Thr Leu Lys Gln Trp Leu Phe His Gly Arg1 5 10 15Gly
Gln918PRTArtificial SequenceSynthesized Peptide Sequence 9His Ile
Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu Val Ala Leu Arg1 5 10 15Met
Val1018PRTArtificial SequenceSynthesized Peptide Sequence 10Gln Leu
Gly His Gly Pro Thr Leu Arg Gln Trp Leu Ser Trp Tyr Arg1 5 10 15Gly
Met1118PRTArtificial SequenceSynthesized Peptide Sequence 11Glu Leu
Arg Gln Gly Pro Thr Leu His Glu Trp Leu Gln His Leu Ala1 5 10 15Ser
Lys1218PRTArtificial SequenceSynthesized Peptide Sequence 12Val Gly
Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Gln Arg Leu1 5 10 15Asn
Pro1318PRTArtificial SequenceSynthesized Peptide Sequence 13Trp Ser
Arg Asp Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Ala1 5 10 15Val
Gly1418PRTArtificial SequenceSynthesized Peptide Sequence 14Ala Val
Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu Leu Trp Arg Arg1 5 10 15Cys
Ala1518PRTArtificial SequenceSynthesized Peptide Sequence 15Arg Ile
Arg Glu Gly Pro Thr Leu Lys Glu Trp Leu Ala Gln Arg Arg1 5 10 15Gly
Phe1618PRTArtificial SequenceSynthesized Peptide Sequence 16Arg Phe
Ala Glu Gly Pro Thr Leu Arg Glu Trp Leu Glu Gln Arg Lys1 5 10 15Leu
Val1718PRTArtificial SequenceSynthesized Peptide Sequence 17Asp Arg
Phe Gln Gly Pro Thr Leu Arg Glu Trp Leu Ala Ala Ile Arg1 5 10 15Ser
Val1818PRTArtificial SequenceSynthesized Peptide Sequence 18Ala Gly
Arg Glu Gly Pro Thr Leu Arg Glu Trp Leu Asn Met Arg Val1 5 10 15Trp
Gln1918PRTArtificial SequenceSynthesized Peptide Sequence 19Ala Leu
Gln Glu Gly Pro Thr Leu Arg Gln Trp Leu Gly Trp Gly Gln1 5 10 15Trp
Gly2018PRTArtificial SequenceSynthesized Peptide Sequence 20Tyr Cys
Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu Val Cys Leu Gly1 5 10 15Leu
Gln2118PRTArtificial SequenceSynthesized Peptide Sequence 21Trp Cys
Lys Glu Gly Pro Thr Leu Arg Glu Trp Leu Arg Trp Gly Phe1 5 10 15Leu
Cys2218PRTArtificial SequenceSynthesized Peptide Sequence 22Cys Ser
Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg1 5 10 15Met
Gln2318PRTArtificial SequenceSynthesized Peptide Sequence 23Cys Ser
Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg1 5 10 15Ala
Lys2418PRTArtificial SequenceSynthesized Peptide Sequence 24Cys Gln
Leu Gly Gly Pro Thr Leu Arg Glu Trp Leu Ala Cys Arg Leu1 5 10 15Gly
Ala2518PRTArtificial SequenceSynthesized Peptide Sequence 25Cys Trp
Glu Gly Gly Pro Thr Leu Lys Glu Trp Leu Gln Cys Leu Val1 5 10 15Glu
Arg2618PRTArtificial SequenceSynthesized Peptide Sequence 26Cys Arg
Gly Gly Gly Pro Thr Leu His Gln Trp Leu Ser Cys Phe Arg1 5 10 15Trp
Gln2718PRTArtificial SequenceSynthesized Peptide Sequence 27Cys Arg
Asp Gly Gly Pro Thr Leu Arg Gln Trp Leu Ala Cys Leu Gln1 5 10 15Gln
Lys2818PRTArtificial SequenceSynthesized Peptide Sequence 28Glu Leu
Arg Ser Gly Pro Thr Leu Lys Glu Trp Leu Val Trp Arg Leu1 5 10 15Ala
Gln2918PRTArtificial SequenceSynthesized Peptide Sequence 29Gly Cys
Arg Ser Gly Pro Thr Leu Arg Glu Trp Leu Ala Cys Arg Glu1 5 10 15Val
Gln3018PRTArtificial SequenceSynthesized Peptide Sequence 30Thr Cys
Glu Gln Gly Pro Thr Leu Arg Gln Trp Leu Leu Cys Arg Gln1 5 10 15Gly
Arg31684DNAArtificial SequenceSynthesized Peptide Sequence 31atg
gac aaa act cac aca tgt cca cct tgt cca gct ccg gaa ctc ctg 48Met
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10
15ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc
96Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg
agc 144Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 35 40 45cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc
gtg gag 192His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 50 55 60gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac
aac agc acg 240Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr65 70 75 80tac cgt gtg gtc agc gtc ctc acc gtc ctg cac
cag gac tgg ctg aat 288Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 85 90 95ggc aag gag tac aag tgc aag gtc tcc aac
aaa gcc ctc cca gcc ccc 336Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro 100 105 110atc gag aaa acc atc tcc aaa gcc
aaa ggg cag ccc cga gaa cca cag 384Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125gtg tac acc ctg ccc cca
tcc cgg gat gag ctg acc aag aac cag gtc 432Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140agc ctg acc tgc
ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 480Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160gag
tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 528Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170
175ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc
576Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
180 185 190gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc
tcc gtg 624Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val 195 200 205atg cat gag gct ctg cac aac cac tac acg cag aag
agc ctc tcc ctg 672Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 210 215 220tct ccg ggt aaa 684Ser Pro Gly
Lys22532228PRTArtificial SequenceSynthesized Peptide Sequence 32Met
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10
15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 35 40 45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro 100 105 110Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170
175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val 195 200 205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 210 215 220Ser Pro Gly Lys22533835DNAArtificial
SequenceSynthesized Peptide Sequence 33tagtcgatta atcgatttga
ttctagattt gttttaacta attaaaggag gaataacat 59atg ggt gca cag aaa
gcg gcc gca aaa aaa ctc gag ggt gga ggc ggt 107Met Gly Ala Gln Lys
Ala Ala Ala Lys Lys Leu Glu Gly Gly Gly Gly1 5 10 15ggg gac aaa act
cac aca tgt cca cct tgc cca gca cct gaa ctc ctg 155Gly Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 20 25 30ggg gga ccg
tca gtt ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 203Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 35 40 45atg atc
tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 251Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 50 55 60cac
gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 299His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu65 70 75
80gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg
347Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
85 90 95tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg
aat 395Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn 100 105 110ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc
cca gcc ccc 443Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro 115 120 125atc gag aaa acc atc tcc aaa gcc aaa ggg cag
ccc cga gaa cca cag 491Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln 130 135 140gtg tac acc ctg ccc cca tcc cgg gat
gag ctg acc aag aac cag gtc 539Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val145 150 155 160agc ctg acc tgc ctg gtc
aaa ggc ttc tat ccc agc gac atc gcc gtg 587Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 165 170 175gag tgg gag agc
aat ggg cag ccg gag aac aac tac aag acc acg cct 635Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 180 185 190ccc gtg
ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 683Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 195 200
205gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg
731Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
210 215 220atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc
tcc ctg 779Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu225 230 235 240tct ccg ggt aaa taatggatcc gcggaaagaa
gaagaagaag aagaaagccc gaaa 835Ser Pro Gly Lys34244PRTArtificial
SequenceSynthesized Peptide Sequence 34Met Gly Ala Gln Lys Ala Ala
Ala Lys Lys Leu Glu Gly Gly Gly Gly1 5 10 15Gly Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 20 25 30Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 35 40 45Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 50 55 60His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu65 70 75 80Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 85 90
95Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
100 105 110Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro 115 120 125Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 130 135 140Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val145 150 155 160Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val 165 170 175Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 180 185 190Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 195 200 205Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 210 215
220Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu225 230 235 240Ser Pro Gly Lys3566DNAArtificial
SequenceSynthesized Peptide Sequence 35cat atg ggt gca cag ggt atc
gaa ggt ccg act ctg cgt cag tgg ctg 48 Met Gly Ala Gln Gly Ile Glu
Gly Pro Thr Leu Arg Gln Trp Leu 1 5 10 15gct gct cgt gct ctc gag
66Ala Ala Arg Ala Leu Glu 203621PRTArtificial SequenceSynthesized
Peptide Sequence 36Met Gly Ala Gln Gly Ile Glu Gly Pro Thr Leu Arg
Gln Trp Leu Ala1 5 10 15Ala Arg Ala Leu Glu 203771PRTArtificial
SequenceSynthesized Peptide Sequence 37Thr Gly Cys Ala Cys Ala Ala
Gly Gly Thr Gly Gly Ala Gly Cys Ala1 5 10 15Cys Gly Thr Gly Ala Ala
Gly Gly Ala Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Cys Gly Thr
Cys Ala Ala Thr Gly Gly Cys Thr Thr Gly Ala 35 40 45Ala Thr Gly Gly
Gly Thr Thr Cys Gly Thr Gly Thr Thr Gly Gly Thr 50 55 60Cys Ala Thr
Thr Cys Thr Cys65 703871PRTArtificial SequenceSynthesized Peptide
Sequence 38Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Ala Cys Cys
Ala Ala1 5 10 15Cys Ala Cys Gly Ala Ala Cys Cys Cys Ala Thr Thr Cys
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Gly Ala Cys Gly Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Thr Cys Cys Thr Thr Cys Ala Cys Gly
Thr Gly Cys Thr Cys 50 55 60Cys Ala Cys Cys Thr Thr Gly65
703977DNAArtificial SequenceSynthesized Peptide Sequence 39gt gca
caa ggt gga gca cgt gaa gga cca act ctt cgt caa tgg ctt 47Ala Gln
Gly Gly Ala Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu1 5 10 15gaa tgg
gtt cgt gtt ggt cat tct ctc gag 77Glu Trp Val Arg Val Gly His Ser
Leu Glu 20 254025PRTArtificial SequenceSynthesized Peptide Sequence
40Ala Gln Gly Gly Ala Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu Glu1
5 10 15Trp Val Arg Val Gly His Ser Leu Glu 20 254171PRTArtificial
SequenceSynthesized Peptide Sequence 41Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Cys Gly Thr Gly Ala Thr1 5 10 15Cys Thr Thr Gly Ala Thr
Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Cys Gly Thr
Cys Ala Ala Thr Gly Gly Cys Thr Thr Cys Cys 35 40 45Ala Cys Thr Thr
Cys Cys Ala Thr Cys Thr Gly Thr Thr Cys Ala Ala 50 55 60Cys Ala Thr
Thr Cys Thr Cys65 704271PRTArtificial SequenceSynthesized Peptide
Sequence 42Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Gly
Ala Ala1
5 10 15Cys Ala Gly Ala Thr Gly Gly Ala Ala Gly Thr Gly Gly Ala Ala
Gly 20 25 30Cys Cys Ala Thr Thr Gly Ala Cys Gly Ala Ala Gly Ala Gly
Thr Thr 35 40 45Gly Gly Ala Cys Cys Ala Thr Cys Ala Ala Gly Ala Thr
Cys Ala Cys 50 55 60Gly Thr Cys Cys Thr Thr Gly65
704377DNAArtificial SequenceSynthesized Peptide Sequence 43gt gca
caa gga cgt gat ctt gat ggt cca act ctt cgt caa tgg ctt 47Ala Gln
Gly Arg Asp Leu Asp Gly Pro Thr Leu Arg Gln Trp Leu1 5 10 15cca ctt
cca tct gtt caa cat tct ctc gag 77Pro Leu Pro Ser Val Gln His Ser
Leu Glu 20 254425PRTArtificial SequenceSynthesized Peptide Sequence
44Ala Gln Gly Arg Asp Leu Asp Gly Pro Thr Leu Arg Gln Trp Leu Pro1
5 10 15Leu Pro Ser Val Gln His Ser Leu Glu 20 254571PRTArtificial
SequenceSynthesized Peptide Sequence 45Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Gly Cys Thr Thr Thr Ala1 5 10 15Cys Gly Thr Gly Ala Thr
Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Ala Ala Ala
Cys Ala Ala Thr Gly Gly Thr Thr Ala Gly Ala 35 40 45Ala Thr Ala Thr
Cys Gly Thr Cys Gly Thr Cys Ala Ala Gly Cys Thr 50 55 60Cys Ala Thr
Thr Cys Ala Cys65 704671PRTArtificial SequenceSynthesized Peptide
Sequence 46Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Ala Gly Cys
Thr Thr1 5 10 15Gly Ala Cys Gly Ala Cys Gly Ala Thr Ala Thr Thr Cys
Thr Ala Ala 20 25 30Cys Cys Ala Thr Thr Gly Thr Thr Thr Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Ala Thr Cys Ala Cys Gly
Thr Ala Ala Ala Gly 50 55 60Cys Thr Cys Cys Thr Thr Gly65
704777DNAArtificial SequenceSynthesized Peptide Sequence 47gt gca
caa gga gct tta cgt gat ggt cca act ctt aaa caa tgg tta 47Ala Gln
Gly Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu1 5 10 15gaa tat
cgt cgt caa gct cat tca ctc gag 77Glu Tyr Arg Arg Gln Ala His Ser
Leu Glu 20 254825PRTArtificial SequenceSynthesized Peptide Sequence
48Ala Gln Gly Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu1
5 10 15Tyr Arg Arg Gln Ala His Ser Leu Glu 20 254971PRTArtificial
SequenceSynthesized Peptide Sequence 49Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Gly Cys Ala Cys Gly Thr1 5 10 15Cys Ala Ala Gly Ala Ala
Gly Gly Ala Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Ala Ala Ala
Gly Ala Ala Thr Gly Gly Thr Thr Ala Thr Thr 35 40 45Thr Thr Gly Gly
Gly Thr Thr Cys Gly Thr Ala Thr Gly Gly Gly Thr 50 55 60Cys Ala Thr
Thr Cys Ala Cys65 705071PRTArtificial SequenceSynthesized Peptide
Sequence 50Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Ala Cys Cys
Cys Ala1 5 10 15Thr Ala Cys Gly Ala Ala Cys Cys Cys Ala Ala Ala Ala
Thr Ala Ala 20 25 30Cys Cys Ala Thr Thr Cys Thr Thr Thr Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Thr Cys Cys Thr Thr Cys Thr Thr Gly
Ala Cys Gly Thr Gly 50 55 60Cys Thr Cys Cys Thr Thr Gly65
705177DNAArtificial SequenceSynthesized Peptide Sequence 51gt gca
caa gga gca cgt caa gaa gga cca act ctt aaa gaa tgg tta 47Ala Gln
Gly Ala Arg Gln Glu Gly Pro Thr Leu Lys Glu Trp Leu1 5 10 15ttt tgg
gtt cgt atg ggt cat tca ctc gag 77Phe Trp Val Arg Met Gly His Ser
Leu Glu 20 255225PRTArtificial SequenceSynthesized Peptide Sequence
52Ala Gln Gly Ala Arg Gln Glu Gly Pro Thr Leu Lys Glu Trp Leu Phe1
5 10 15Trp Val Arg Met Gly His Ser Leu Glu 20 255371PRTArtificial
SequenceSynthesized Peptide Sequence 53Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Gly Ala Ala Gly Cys Thr1 5 10 15Thr Thr Ala Thr Thr Ala
Gly Gly Thr Cys Cys Ala Ala Cys Thr Thr 20 25 30Thr Ala Cys Gly Thr
Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys 35 40 45Thr Thr Gly Gly
Cys Gly Thr Cys Gly Thr Gly Cys Ala Cys Ala Ala 50 55 60Cys Ala Thr
Thr Cys Thr Cys65 705471PRTArtificial SequenceSynthesized Peptide
Sequence 54Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Gly
Thr Gly1 5 10 15Cys Ala Cys Gly Ala Cys Gly Cys Cys Ala Ala Gly Cys
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Cys Ala Cys Gly Thr Ala Ala
Ala Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Thr Ala Ala Thr Ala Ala
Ala Gly Cys Thr Thr 50 55 60Cys Thr Cys Cys Thr Thr Gly65
705577DNAArtificial SequenceSynthesized Peptide Sequence 55gt gca
caa gga gaa gct tta tta ggt cca act tta cgt gaa tgg ctt 47Ala Gln
Gly Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu1 5 10 15gct tgg
cgt cgt gca caa cat tct ctc gag 77Ala Trp Arg Arg Ala Gln His Ser
Leu Glu 20 255625PRTArtificial SequenceSynthesized Peptide Sequence
56Ala Gln Gly Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala1
5 10 15Trp Arg Arg Ala Gln His Ser Leu Glu 20 255771PRTArtificial
SequenceSynthesized Peptide Sequence 57Thr Gly Cys Ala Cys Ala Ala
Gly Gly Thr Ala Thr Gly Gly Cys Ala1 5 10 15Cys Gly Thr Gly Ala Thr
Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Cys Gly Thr
Gly Ala Ala Thr Gly Gly Cys Thr Thr Cys Gly 35 40 45Thr Ala Cys Thr
Thr Ala Thr Cys Gly Thr Ala Thr Gly Ala Thr Gly 50 55 60Cys Ala Thr
Thr Cys Thr Cys65 705871PRTArtificial SequenceSynthesized Peptide
Sequence 58Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Cys Ala Thr
Cys Ala1 5 10 15Thr Ala Cys Gly Ala Thr Ala Ala Gly Thr Ala Cys Gly
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Ala Thr Cys Ala Cys Gly
Thr Gly Cys Cys Ala 50 55 60Thr Ala Cys Cys Thr Thr Gly65
705977DNAArtificial SequenceSynthesized Peptide Sequence 59gt gca
caa ggt atg gca cgt gat ggt cca act ctt cgt gaa tgg ctt 47Ala Gln
Gly Met Ala Arg Asp Gly Pro Thr Leu Arg Glu Trp Leu1 5 10 15cgt act
tat cgt atg atg cat tct ctc gag 77Arg Thr Tyr Arg Met Met His Ser
Leu Glu 20 256025PRTArtificial SequenceSynthesized Peptide Sequence
60Ala Gln Gly Met Ala Arg Asp Gly Pro Thr Leu Arg Glu Trp Leu Arg1
5 10 15Thr Tyr Arg Met Met His Ser Leu Glu 20 256171PRTArtificial
SequenceSynthesized Peptide Sequence 61Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Thr Gly Gly Ala Thr Gly1 5 10 15Cys Cys Ala Gly Ala Ala
Gly Gly Ala Cys Cys Ala Ala Cys Ala Thr 20 25 30Thr Ala Ala Ala Ala
Cys Ala Ala Thr Gly Gly Cys Thr Thr Thr Thr 35 40 45Thr Cys Ala Thr
Gly Gly Thr Cys Gly Thr Gly Gly Thr Cys Ala Ala 50 55 60Cys Ala Thr
Thr Cys Thr Cys65 706271PRTArtificial SequenceSynthesized Peptide
Sequence 62Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Gly
Ala Cys1 5 10 15Cys Ala Cys Gly Ala Cys Cys Ala Thr Gly Ala Ala Ala
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Gly Thr Thr Thr Thr Ala Ala
Thr Gly Thr Thr 35 40 45Gly Gly Thr Cys Cys Thr Thr Cys Thr Gly Gly
Cys Ala Thr Cys Cys 50 55 60Ala Thr Cys Cys Thr Thr Gly65
706377DNAArtificial SequenceSynthesized Peptide Sequence 63gt gca
caa gga tgg atg cca gaa gga cca aca tta aaa caa tgg ctt 47Ala Gln
Gly Trp Met Pro Glu Gly Pro Thr Leu Lys Gln Trp Leu1 5 10 15ttt cat
ggt cgt ggt caa cat tct ctc gag 77Phe His Gly Arg Gly Gln His Ser
Leu Glu 20 256425PRTArtificial SequenceSynthesized Peptide Sequence
64Ala Gln Gly Trp Met Pro Glu Gly Pro Thr Leu Lys Gln Trp Leu Phe1
5 10 15His Gly Arg Gly Gln His Ser Leu Glu 20 256571PRTArtificial
SequenceSynthesized Peptide Sequence 65Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Cys Ala Thr Ala Thr Thr1 5 10 15Cys Gly Thr Gly Ala Ala
Gly Gly Thr Cys Cys Ala Ala Cys Ala Thr 20 25 30Thr Ala Cys Gly Thr
Cys Ala Ala Thr Gly Gly Cys Thr Thr Gly Thr 35 40 45Thr Gly Cys Thr
Cys Thr Thr Cys Gly Thr Ala Thr Gly Gly Thr Thr 50 55 60Cys Ala Thr
Thr Cys Thr Cys65 706671PRTArtificial SequenceSynthesized Peptide
Sequence 66Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Ala Ala Cys
Cys Ala1 5 10 15Thr Ala Cys Gly Ala Ala Gly Ala Gly Cys Ala Ala Cys
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Gly Ala Cys Gly Thr Ala Ala
Thr Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Thr Thr Cys Ala Cys Gly
Ala Ala Thr Ala Thr 50 55 60Gly Thr Cys Cys Thr Thr Gly65
706777DNAArtificial SequenceSynthesized Peptide Sequence 67gt gca
caa gga cat att cgt gaa ggt cca aca tta cgt caa tgg ctt 47Ala Gln
Gly His Ile Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu1 5 10 15gtt gct
ctt cgt atg gtt cat tct ctc gag 77Val Ala Leu Arg Met Val His Ser
Leu Glu 20 256825PRTArtificial SequenceSynthesized Peptide Sequence
68Ala Gln Gly His Ile Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu Val1
5 10 15Ala Leu Arg Met Val His Ser Leu Glu 20 256971PRTArtificial
SequenceSynthesized Peptide Sequence 69Thr Gly Cys Ala Cys Ala Ala
Gly Gly Thr Cys Ala Ala Thr Thr Ala1 5 10 15Gly Gly Ala Cys Ala Thr
Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Cys Gly Thr
Cys Ala Ala Thr Gly Gly Cys Thr Thr Thr Cys 35 40 45Thr Thr Gly Gly
Thr Ala Thr Cys Gly Thr Gly Gly Thr Ala Thr Gly 50 55 60Cys Ala Thr
Thr Cys Thr Cys65 707071PRTArtificial SequenceSynthesized Peptide
Sequence 70Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Cys Ala Thr
Ala Cys1 5 10 15Cys Ala Cys Gly Ala Thr Ala Cys Cys Ala Ala Gly Ala
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Gly Ala Cys Gly Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Ala Thr Gly Thr Cys Cys
Thr Ala Ala Thr Thr 50 55 60Gly Ala Cys Cys Thr Thr Gly65
707177DNAArtificial SequenceSynthesized Peptide Sequence 71gt gca
caa ggt caa tta gga cat ggt cca act ctt cgt caa tgg ctt 47Ala Gln
Gly Gln Leu Gly His Gly Pro Thr Leu Arg Gln Trp Leu1 5 10 15tct tgg
tat cgt ggt atg cat tct ctc gag 77Ser Trp Tyr Arg Gly Met His Ser
Leu Glu 20 257225PRTArtificial SequenceSynthesized Peptide Sequence
72Ala Gln Gly Gln Leu Gly His Gly Pro Thr Leu Arg Gln Trp Leu Ser1
5 10 15Trp Tyr Arg Gly Met His Ser Leu Glu 20 257371PRTArtificial
SequenceSynthesized Peptide Sequence 73Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Gly Ala Ala Thr Thr Ala1 5 10 15Cys Gly Thr Cys Ala Ala
Gly Gly Ala Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Cys Ala Thr
Gly Ala Ala Thr Gly Gly Cys Thr Thr Cys Ala 35 40 45Ala Cys Ala Thr
Thr Thr Ala Gly Cys Ala Ala Gly Cys Ala Ala Ala 50 55 60Cys Ala Thr
Thr Cys Thr Cys65 707471PRTArtificial SequenceSynthesized Peptide
Sequence 74Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Thr
Gly Cys1 5 10 15Thr Thr Gly Cys Thr Ala Ala Ala Thr Gly Thr Thr Gly
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Cys Ala Thr Gly Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Thr Cys Cys Thr Thr Gly Ala Cys Gly
Thr Ala Ala Thr Thr 50 55 60Cys Thr Cys Cys Thr Thr Gly65
707577DNAArtificial SequenceSynthesized Peptide Sequence 75gt gca
caa gga gaa tta cgt caa gga cca act ctt cat gaa tgg ctt 47Ala Gln
Gly Glu Leu Arg Gln Gly Pro Thr Leu His Glu Trp Leu1 5 10 15caa cat
tta gca agc aaa cat tct ctc gag 77Gln His Leu Ala Ser Lys His Ser
Leu Glu 20 257625PRTArtificial SequenceSynthesized Peptide Sequence
76Ala Gln Gly Glu Leu Arg Gln Gly Pro Thr Leu His Glu Trp Leu Gln1
5 10 15His Leu Ala Ser Lys His Ser Leu Glu 20 257771PRTArtificial
SequenceSynthesized Peptide Sequence 77Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Gly Thr Ala Gly Gly Thr1 5 10 15Ala Thr Thr Gly Ala Ala
Gly Gly Thr Cys Cys Ala Ala Cys Ala Thr 20 25 30Thr Ala Cys Gly Thr
Cys Ala Ala Thr Gly Gly Thr Thr Ala Gly Cys 35 40 45Thr Cys Ala Ala
Cys Gly Thr Cys Thr Thr Ala Ala Thr Cys Cys Ala 50 55 60Cys Ala Thr
Thr Cys Thr Cys65 707871PRTArtificial SequenceSynthesized Peptide
Sequence 78Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Gly Gly
Ala Thr1 5 10 15Thr Ala Ala Gly Ala Cys Gly Thr Thr Gly Ala Gly Cys
Thr Ala Ala 20 25 30Cys Cys Ala Thr Thr Gly Ala Cys Gly Thr Ala Ala
Thr Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Thr Thr Cys Ala Ala Thr
Ala Cys Cys Thr Ala 50 55 60Cys Thr Cys Cys Thr Thr Gly65
707977DNAArtificial SequenceSynthesized Peptide Sequence 79gt gca
caa gga gta ggt att gaa ggt cca aca tta cgt caa tgg tta 47Ala Gln
Gly Val Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu1 5 10 15gct caa
cgt ctt aat cca cat tct ctc gag 77Ala Gln Arg Leu Asn Pro His Ser
Leu Glu 20 258025PRTArtificial SequenceSynthesized Peptide Sequence
80Ala Gln Gly Val Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala1
5 10 15Gln Arg Leu Asn Pro His Ser Leu Glu 20 258171PRTArtificial
SequenceSynthesized Peptide Sequence 81Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Thr Gly Gly Thr Cys Ala1 5 10 15Cys Gly Thr Gly Ala Thr
Gly Gly Thr Cys Cys Ala Ala Cys Ala Cys 20 25 30Thr Thr Cys Gly Thr
Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys 35 40 45Thr Thr Gly Gly
Cys Gly Thr Gly Cys Thr Gly Thr Thr Gly Gly Ala 50 55 60Cys Ala Thr
Ala Gly Thr Cys65 708271PRTArtificial SequenceSynthesized Peptide
Sequence 82Thr Cys Gly Ala Gly Ala Cys Thr Ala Thr Gly Thr Cys Cys
Ala Ala1 5 10 15Cys Ala Gly Cys Ala Cys Gly Cys Cys Ala Ala Gly Cys
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly
Thr Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Ala Thr Cys Ala Cys Gly
Thr Gly Ala Cys Cys 50 55 60Ala Thr Cys Cys Thr Thr Gly65
708377DNAArtificial SequenceSynthesized Peptide Sequence 83gt gca
caa gga tgg tca cgt gat ggt cca aca ctt cgt gaa tgg ctt 47Ala Gln
Gly Trp Ser Arg Asp Gly Pro Thr Leu Arg Glu Trp Leu1 5 10 15gct tgg
cgt gct gtt gga cat agt ctc gag 77Ala Trp Arg Ala Val Gly His Ser
Leu Glu 20 258425PRTArtificial SequenceSynthesized Peptide Sequence
84Ala Gln Gly Trp Ser Arg Asp Gly Pro Thr Leu Arg Glu Trp Leu Ala1
5 10 15Trp Arg Ala Val Gly His Ser Leu Glu 20 258571PRTArtificial
SequenceSynthesized Peptide Sequence 85Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Gly Cys Ala Gly Thr Thr1 5 10 15Cys Cys Ala Cys Ala Ala
Gly Gly Ala Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Ala Ala Ala
Cys Ala Gly Thr Gly Gly Thr Thr Ala Thr Thr 35 40 45Ala Thr Gly Gly
Cys Gly Thr Cys Gly Thr Thr Gly Thr Gly Cys Ala 50 55 60Cys Ala Thr
Thr Cys Thr Cys65 708671PRTArtificial SequenceSynthesized Peptide
Sequence 86Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Gly Cys
Ala Cys1 5 10 15Ala Ala Cys Gly Ala Cys Gly Cys Cys Ala Thr Ala Ala
Thr Ala Ala 20 25 30Cys Cys Ala Cys Thr Gly Thr Thr Thr Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Thr Cys Cys Thr Thr Gly Thr Gly Gly
Ala Ala Cys Thr Gly 50 55 60Cys Thr Cys Cys Thr Thr Gly65
708777DNAArtificial SequenceSynthesized Peptide Sequence 87gt gca
caa gga gca gtt cca caa gga cca act ctt aaa cag tgg tta 47Ala Gln
Gly Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu1 5 10 15tta tgg
cgt cgt tgt gca cat tct ctc gag 77Leu Trp Arg Arg Cys Ala His Ser
Leu Glu 20 258825PRTArtificial SequenceSynthesized Peptide Sequence
88Ala Gln Gly Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu Leu1
5 10 15Trp Arg Arg Cys Ala His Ser Leu Glu 20 258971PRTArtificial
SequenceSynthesized Peptide Sequence 89Thr Gly Cys Ala Cys Ala Ala
Gly Gly Thr Cys Gly Thr Ala Thr Thr1 5 10 15Cys Gly Thr Gly Ala Ala
Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Ala Ala Ala
Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys 35 40 45Thr Cys Ala Ala
Cys Gly Thr Cys Gly Thr Gly Gly Thr Thr Thr Thr 50 55 60Cys Ala Thr
Ala Gly Thr Cys65 709071PRTArtificial SequenceSynthesized Peptide
Sequence 90Thr Cys Gly Ala Gly Ala Cys Thr Ala Thr Gly Ala Ala Ala
Ala Cys1 5 10 15Cys Ala Cys Gly Ala Cys Gly Thr Thr Gly Ala Gly Cys
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Cys Thr Thr Thr Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Thr Thr Cys Ala Cys Gly
Ala Ala Thr Ala Cys 50 55 60Gly Ala Cys Cys Thr Thr Gly65
709177DNAArtificial SequenceSynthesized Peptide Sequence 91gt gca
caa ggt cgt att cgt gaa ggt cca act ctt aaa gaa tgg ctt 47Ala Gln
Gly Arg Ile Arg Glu Gly Pro Thr Leu Lys Glu Trp Leu1 5 10 15gct caa
cgt cgt ggt ttt cat agt ctc gag 77Ala Gln Arg Arg Gly Phe His Ser
Leu Glu 20 259225PRTArtificial SequenceSynthesized Peptide Sequence
92Ala Gln Gly Arg Ile Arg Glu Gly Pro Thr Leu Lys Glu Trp Leu Ala1
5 10 15Gln Arg Arg Gly Phe His Ser Leu Glu 20 259371PRTArtificial
SequenceSynthesized Peptide Sequence 93Thr Gly Cys Ala Cys Ala Ala
Gly Gly Thr Cys Gly Thr Thr Thr Cys1 5 10 15Gly Cys Thr Gly Ala Ala
Gly Gly Thr Cys Cys Ala Ala Cys Ala Cys 20 25 30Thr Thr Cys Gly Thr
Gly Ala Ala Thr Gly Gly Thr Thr Ala Gly Ala 35 40 45Ala Cys Ala Ala
Cys Gly Thr Ala Ala Ala Cys Thr Thr Gly Thr Thr 50 55 60Cys Ala Thr
Ala Gly Thr Cys65 709471PRTArtificial SequenceSynthesized Peptide
Sequence 94Thr Cys Gly Ala Gly Ala Cys Thr Ala Thr Gly Ala Ala Cys
Ala Ala1 5 10 15Gly Thr Thr Thr Ala Cys Gly Thr Thr Gly Thr Thr Cys
Thr Ala Ala 20 25 30Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly
Thr Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Thr Thr Cys Ala Gly Cys
Gly Ala Ala Ala Cys 50 55 60Gly Ala Cys Cys Thr Thr Gly65
709577DNAArtificial SequenceSynthesized Peptide Sequence 95gt gca
caa ggt cgt ttc gct gaa ggt cca aca ctt cgt gaa tgg tta 47Ala Gln
Gly Arg Phe Ala Glu Gly Pro Thr Leu Arg Glu Trp Leu1 5 10 15gaa caa
cgt aaa ctt gtt cat agt ctc gag 77Glu Gln Arg Lys Leu Val His Ser
Leu Glu 20 259625PRTArtificial SequenceSynthesized Peptide Sequence
96Ala Gln Gly Arg Phe Ala Glu Gly Pro Thr Leu Arg Glu Trp Leu Glu1
5 10 15Gln Arg Lys Leu Val His Ser Leu Glu 20 259771PRTArtificial
SequenceSynthesized Peptide Sequence 97Thr Gly Cys Ala Cys Ala Ala
Gly Gly Thr Gly Ala Thr Cys Gly Thr1 5 10 15Thr Thr Cys Cys Ala Ala
Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Cys Gly Thr
Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys 35 40 45Thr Gly Cys Ala
Ala Thr Cys Cys Gly Thr Ala Gly Cys Gly Thr Ala 50 55 60Cys Ala Thr
Ala Gly Thr Cys65 709871PRTArtificial SequenceSynthesized Peptide
Sequence 98Thr Cys Gly Ala Gly Ala Cys Thr Ala Thr Gly Thr Ala Cys
Gly Cys1 5 10 15Thr Ala Cys Gly Gly Ala Thr Thr Gly Cys Ala Gly Cys
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Thr Thr Gly Gly Ala Ala
Ala Cys Gly Ala Thr 50 55 60Cys Ala Cys Cys Thr Thr Gly65
709977DNAArtificial SequenceSynthesized Peptide Sequence 99gt gca
caa ggt gat cgt ttc caa ggt cca act ctt cgt gaa tgg ctt 47Ala Gln
Gly Asp Arg Phe Gln Gly Pro Thr Leu Arg Glu Trp Leu1 5 10 15gct gca
atc cgt agc gta cat agt ctc gag 77Ala Ala Ile Arg Ser Val His Ser
Leu Glu 20 2510025PRTArtificial SequenceSynthesized Peptide
Sequence 100Ala Gln Gly Asp Arg Phe Gln Gly Pro Thr Leu Arg Glu Trp
Leu Ala1 5 10 15Ala Ile Arg Ser Val His Ser Leu Glu 20
2510171PRTArtificial SequenceSynthesized Peptide Sequence 101Thr
Gly Cys Ala Cys Ala Ala Gly Gly Thr Gly Cys Thr Gly Gly Thr1 5 10
15Cys Gly Thr Gly Ala Ala Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys
20 25 30Thr Ala Cys Gly Thr Gly Ala Ala Thr Gly Gly Cys Thr Thr Ala
Ala 35 40 45Thr Ala Thr Gly Cys Gly Thr Gly Thr Thr Thr Gly Gly Cys
Ala Ala 50 55 60Cys Ala Thr Thr Cys Thr Cys65 7010271PRTArtificial
SequenceSynthesized Peptide Sequence 102Thr Cys Gly Ala Gly Ala Gly
Ala Ala Thr Gly Thr Thr Gly Cys Cys1 5 10 15Ala Ala Ala Cys Ala Cys
Gly Cys Ala Thr Ala Thr Thr Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr
Cys Ala Cys Gly Thr Ala Gly Ala Gly Thr Thr 35 40 45Gly Gly Ala Cys
Cys Thr Thr Cys Ala Cys Gly Ala Cys Cys Ala Gly 50 55 60Cys Ala Cys
Cys Thr Thr Gly65 7010377DNAArtificial SequenceSynthesized Peptide
Sequence 103gt gca caa ggt gct ggt cgt gaa ggt cca act cta cgt gaa
tgg ctt 47Ala Gln Gly Ala Gly Arg Glu Gly Pro Thr Leu Arg Glu Trp
Leu1 5 10 15aat atg cgt gtt tgg caa cat tct ctc gag 77Asn Met Arg
Val Trp Gln His Ser Leu Glu 20 2510425PRTArtificial
SequenceSynthesized Peptide Sequence 104Ala Gln Gly Ala Gly Arg Glu
Gly Pro Thr Leu Arg Glu Trp Leu Asn1 5 10 15Met Arg Val Trp Gln His
Ser Leu Glu 20 2510571PRTArtificial SequenceSynthesized Peptide
Sequence 105Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Cys Thr Thr
Thr Ala1 5 10 15Cys Ala Ala Gly Ala Ala Gly Gly Ala Cys Cys Ala Ala
Cys Ala Thr 20 25 30Thr Ala Cys Gly Thr Cys Ala Ala Thr Gly Gly Thr
Thr Ala Gly Gly 35 40 45Ala Thr Gly Gly Gly Gly Thr Cys Ala Ala Thr
Gly Gly Gly Gly Ala 50 55 60Cys Ala Cys Thr Cys Thr Cys65
7010671PRTArtificial SequenceSynthesized Peptide Sequence 106Thr
Cys Gly Ala Gly Ala Gly Ala Gly Thr Gly Thr Cys Cys Cys Cys1 5 10
15Ala Thr Thr Gly Ala Cys Cys Cys Cys Ala Thr Cys Cys Thr Ala Ala
20 25 30Cys Cys Ala Thr Thr Gly Ala Cys Gly Thr Ala Ala Thr Gly Thr
Thr 35 40 45Gly Gly Thr Cys Cys Thr Thr Cys Thr Thr Gly Thr Ala Ala
Ala Gly 50 55 60Cys Thr Cys Cys Thr Thr Gly65 7010777DNAArtificial
SequenceSynthesized Peptide Sequence 107gt gca caa gga gct tta caa
gaa gga cca aca tta cgt caa tgg tta 47Ala Gln Gly Ala Leu Gln Glu
Gly Pro Thr Leu Arg Gln Trp Leu1 5 10 15gga tgg ggt caa tgg gga cac
tct ctc gag 77Gly Trp Gly Gln Trp Gly His Ser Leu Glu 20
2510825PRTArtificial SequenceSynthesized Peptide Sequence 108Ala
Gln Gly Ala Leu Gln Glu Gly Pro Thr Leu Arg Gln Trp Leu Gly1 5 10
15Trp Gly Gln Trp Gly His Ser Leu Glu 20 2510971PRTArtificial
SequenceSynthesized Peptide Sequence 109Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Thr Ala Cys Thr Gly Thr1 5 10 15Gly Ala Thr Gly Ala Ala
Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Ala Ala Ala
Cys Ala Ala Thr Gly Gly Thr Thr Ala Gly Thr 35 40 45Ala Thr Gly Thr
Cys Thr Thr Gly Gly Thr Thr Thr Ala Cys Ala Ala 50 55 60Cys Ala Thr
Ala Gly Thr Cys65 7011071PRTArtificial SequenceSynthesized Peptide
Sequence 110Thr Cys Gly Ala Gly Ala Cys Thr Ala Thr Gly Thr Thr Gly
Thr Ala1 5 10 15Ala Ala Cys Cys Ala Ala Gly Ala Cys Ala Thr Ala Cys
Thr Ala Ala 20 25 30Cys Cys Ala Thr Thr Gly Thr Thr Thr Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Thr Thr Cys Ala Thr Cys
Ala Cys Ala Gly Thr 50 55 60Ala Thr Cys Cys Thr Thr Gly65
7011177DNAArtificial SequenceSynthesized Peptide Sequence 111gt gca
caa gga tac tgt gat gaa ggt cca act ctt aaa caa tgg tta 47Ala Gln
Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu1 5 10 15gta tgt
ctt ggt tta caa cat agt ctc gag 77Val Cys Leu Gly Leu Gln His Ser
Leu Glu 20 2511225PRTArtificial SequenceSynthesized Peptide
Sequence 112Ala Gln Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp
Leu Val1 5 10 15Cys Leu Gly Leu Gln His Ser Leu Glu 20
2511371PRTArtificial SequenceSynthesized Peptide Sequence 113Thr
Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Gly Thr Ala Gly Thr1 5 10
15Thr Cys Ala Gly Gly Ala Gly Gly Thr Cys Cys Ala Ala Cys Thr Thr
20 25 30Thr Ala Cys Gly Thr Gly Ala Ala Thr Gly Gly Thr Thr Ala Cys
Ala 35 40 45Ala Thr Gly Thr Cys Gly Thr Cys Gly Thr Ala Thr Gly Cys
Ala Ala 50 55 60Cys Ala Thr Thr Cys Thr Cys65 7011471PRTArtificial
SequenceSynthesized Peptide Sequence 114Thr Cys Gly Ala Gly Ala Gly
Ala Ala Thr Gly Thr Thr Gly Cys Ala1 5 10 15Thr Ala Cys Gly Ala Cys
Gly Ala Cys Ala Thr Thr Gly Thr Ala Ala 20 25 30Cys Cys Ala Thr Thr
Cys Ala Cys Gly Thr Ala Ala Ala Gly Thr Thr 35 40 45Gly Gly Ala Cys
Cys Thr Cys Cys Thr Gly Ala Ala Cys Thr Ala Cys 50 55 60Ala Thr Cys
Cys Thr Thr Gly65 7011577DNAArtificial SequenceSynthesized Peptide
Sequence 115gt gca caa gga tgt agt tca gga ggt cca act tta cgt gaa
tgg tta 47Ala Gln Gly Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp
Leu1 5 10 15caa tgt cgt cgt atg caa cat tct ctc gag 77Gln Cys Arg
Arg Met Gln His Ser Leu Glu 20 2511625PRTArtificial
SequenceSynthesized Peptide Sequence 116Ala Gln Gly Cys Ser Ser Gly
Gly Pro Thr Leu Arg Glu Trp Leu Gln1 5 10 15Cys Arg Arg Met Gln His
Ser Leu Glu 20 2511771PRTArtificial SequenceSynthesized Peptide
Sequence 117Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Gly Thr Thr
Cys Ala1 5 10 15Thr Gly Gly Gly Gly Thr Gly Gly Thr Cys Cys Ala Ala
Cys Thr Cys 20 25 30Thr Thr Ala Ala Ala Cys Ala Ala Thr Gly Gly Thr
Thr Ala Cys Ala 35 40 45Ala Thr Gly Thr Gly Thr Thr Cys Gly Thr Gly
Cys Thr Ala Ala Ala 50 55 60Cys Ala Thr Thr Cys Thr Cys65
7011871PRTArtificial SequenceSynthesized Peptide Sequence 118Thr
Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Thr Ala Gly1 5 10
15Cys Ala Cys Gly Ala Ala Cys Ala Cys Ala Thr Thr Gly Thr Ala Ala
20 25 30Cys Cys Ala Thr Thr Gly Thr Thr Thr Ala Ala Gly Ala Gly Thr
Thr 35 40 45Gly Gly Ala Cys Cys Ala Cys Cys Cys Cys Ala Thr Gly Ala
Ala Cys 50 55 60Ala Thr Cys Cys Thr Thr Gly65 7011977DNAArtificial
SequenceSynthesized Peptide Sequence 119gt gca caa gga tgt tca tgg
ggt ggt cca act ctt aaa caa tgg tta 47Ala Gln Gly Cys Ser Trp Gly
Gly Pro Thr Leu Lys Gln Trp Leu1 5 10 15caa tgt gtt cgt gct aaa cat
tct ctc gag 77Gln Cys Val Arg Ala Lys His Ser Leu Glu 20
2512025PRTArtificial SequenceSynthesized Peptide Sequence 120Ala
Gln Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln1 5 10
15Cys Val Arg Ala Lys His Ser Leu Glu 20 2512171PRTArtificial
SequenceSynthesized Peptide Sequence 121Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Thr Gly Thr Cys Ala Ala1 5 10 15Thr Thr Ala Gly Gly Thr
Gly Gly Thr Cys Cys Gly Ala Cys Thr Cys 20 25 30Thr Thr Cys Gly Thr
Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys 35 40 45Thr Thr Gly Thr
Cys Gly Thr Cys Thr Thr Gly Gly Thr Gly Cys Thr 50 55 60Cys Ala Thr
Thr Cys Ala Cys65 7012271PRTArtificial SequenceSynthesized Peptide
Sequence 122Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Ala Gly Cys
Ala Cys1 5 10 15Cys Ala Ala Gly Ala Cys Gly Ala Cys Ala Ala Gly Cys
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly
Ala Gly Thr Cys 35 40 45Gly Gly Ala Cys Cys Ala Cys Cys Thr Ala Ala
Thr Thr Gly Ala Cys 50 55 60Ala Thr Cys Cys Thr Thr Gly65
7012377DNAArtificial SequenceSynthesized Peptide Sequence 123gt gca
caa gga tgt caa tta ggt ggt ccg act ctt cgt gaa tgg ctt 47Ala Gln
Gly Cys Gln Leu Gly Gly Pro Thr Leu Arg Glu Trp Leu1 5
10 15gct tgt cgt ctt ggt gct cat tca ctc gag 77Ala Cys Arg Leu Gly
Ala His Ser Leu Glu 20 2512425PRTArtificial SequenceSynthesized
Peptide Sequence 124Ala Gln Gly Cys Gln Leu Gly Gly Pro Thr Leu Arg
Glu Trp Leu Ala1 5 10 15Cys Arg Leu Gly Ala His Ser Leu Glu 20
2512571PRTArtificial SequenceSynthesized Peptide Sequence 125Thr
Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Gly Thr Thr Gly Gly1 5 10
15Gly Ala Ala Gly Gly Thr Gly Gly Thr Cys Cys Thr Ala Cys Ala Cys
20 25 30Thr Thr Ala Ala Ala Gly Ala Ala Thr Gly Gly Cys Thr Thr Cys
Ala 35 40 45Ala Thr Gly Thr Cys Thr Thr Gly Thr Ala Gly Ala Ala Cys
Gly Thr 50 55 60Cys Ala Thr Thr Cys Ala Cys65 7012671PRTArtificial
SequenceSynthesized Peptide Sequence 126Thr Cys Gly Ala Gly Thr Gly
Ala Ala Thr Gly Ala Cys Gly Thr Thr1 5 10 15Cys Thr Ala Cys Ala Ala
Gly Ala Cys Ala Thr Thr Gly Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr
Cys Thr Thr Thr Ala Ala Gly Thr Gly Thr Ala 35 40 45Gly Gly Ala Cys
Cys Ala Cys Cys Thr Thr Cys Cys Cys Ala Ala Cys 50 55 60Ala Thr Cys
Cys Thr Thr Gly65 7012777DNAArtificial SequenceSynthesized Peptide
Sequence 127gt gca caa gga tgt tgg gaa ggt ggt cct aca ctt aaa gaa
tgg ctt 47Ala Gln Gly Cys Trp Glu Gly Gly Pro Thr Leu Lys Glu Trp
Leu1 5 10 15caa tgt ctt gta gaa cgt cat tca ctc gag 77Gln Cys Leu
Val Glu Arg His Ser Leu Glu 20 2512825PRTArtificial
SequenceSynthesized Peptide Sequence 128Ala Gln Gly Cys Trp Glu Gly
Gly Pro Thr Leu Lys Glu Trp Leu Gln1 5 10 15Cys Leu Val Glu Arg His
Ser Leu Glu 20 2512971PRTArtificial SequenceSynthesized Peptide
Sequence 129Thr Gly Cys Ala Cys Ala Ala Gly Gly Thr Thr Gly Thr Cys
Gly Thr1 5 10 15Gly Gly Thr Gly Gly Thr Gly Gly Thr Cys Cys Ala Ala
Cys Thr Cys 20 25 30Thr Thr Cys Ala Thr Cys Ala Ala Thr Gly Gly Cys
Thr Thr Thr Cys 35 40 45Thr Thr Gly Thr Thr Thr Thr Cys Gly Thr Thr
Gly Gly Cys Ala Ala 50 55 60Cys Ala Thr Thr Cys Ala Cys65
7013071PRTArtificial SequenceSynthesized Peptide Sequence 130Thr
Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Thr Thr Gly Cys Cys1 5 10
15Ala Ala Cys Gly Ala Ala Ala Ala Cys Ala Ala Gly Ala Ala Ala Gly
20 25 30Cys Cys Ala Thr Thr Gly Ala Thr Gly Ala Ala Gly Ala Gly Thr
Thr 35 40 45Gly Gly Ala Cys Cys Ala Cys Cys Ala Cys Cys Ala Cys Gly
Ala Cys 50 55 60Ala Ala Cys Cys Thr Thr Gly65 7013177DNAArtificial
SequenceSynthesized Peptide Sequence 131gt gca caa ggt tgt cgt ggt
ggt ggt cca act ctt cat caa tgg ctt 47Ala Gln Gly Cys Arg Gly Gly
Gly Pro Thr Leu His Gln Trp Leu1 5 10 15tct tgt ttt cgt tgg caa cat
tca ctc gag 77Ser Cys Phe Arg Trp Gln His Ser Leu Glu 20
2513225PRTArtificial SequenceSynthesized Peptide Sequence 132Ala
Gln Gly Cys Arg Gly Gly Gly Pro Thr Leu His Gln Trp Leu Ser1 5 10
15Cys Phe Arg Trp Gln His Ser Leu Glu 20 2513371PRTArtificial
SequenceSynthesized Peptide Sequence 133Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Thr Gly Thr Cys Gly Thr1 5 10 15Gly Ala Thr Gly Gly Thr
Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Thr Ala Gly Ala
Cys Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys 35 40 45Thr Thr Gly Thr
Cys Thr Thr Cys Ala Ala Cys Ala Ala Ala Ala Ala 50 55 60Cys Ala Thr
Thr Cys Ala Cys65 7013471PRTArtificial SequenceSynthesized Peptide
Sequence 134Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Thr Thr Thr
Thr Thr1 5 10 15Gly Thr Thr Gly Ala Ala Gly Ala Cys Ala Ala Gly Cys
Ala Ala Gly 20 25 30Cys Cys Ala Thr Thr Gly Thr Cys Thr Ala Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Ala Cys Cys Ala Cys Cys Ala Thr Cys
Ala Cys Gly Ala Cys 50 55 60Ala Thr Cys Cys Thr Thr Gly65
7013577DNAArtificial SequenceSynthesized Peptide Sequence 135gt gca
caa gga tgt cgt gat ggt ggt cca act ctt aga caa tgg ctt 47Ala Gln
Gly Cys Arg Asp Gly Gly Pro Thr Leu Arg Gln Trp Leu1 5 10 15gct tgt
ctt caa caa aaa cat tca ctc gag 77Ala Cys Leu Gln Gln Lys His Ser
Leu Glu 20 2513625PRTArtificial SequenceSynthesized Peptide
Sequence 136Ala Gln Gly Cys Arg Asp Gly Gly Pro Thr Leu Arg Gln Trp
Leu Ala1 5 10 15Cys Leu Gln Gln Lys His Ser Leu Glu 20
2513771PRTArtificial SequenceSynthesized Peptide Sequence 137Thr
Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Thr Thr Gly Ala Gly1 5 10
15Cys Ala Ala Gly Ala Cys Gly Cys Cys Ala Ala Ala Cys Ala Ala Gly
20 25 30Cys Cys Ala Thr Thr Cys Thr Thr Thr Thr Ala Ala Ala Gly Thr
Thr 35 40 45Gly Gly Ala Cys Cys Ala Gly Ala Thr Cys Thr Thr Ala Ala
Thr Thr 50 55 60Cys Thr Cys Cys Thr Thr Gly65 7013871PRTArtificial
SequenceSynthesized Peptide Sequence 138Thr Gly Cys Ala Cys Ala Ala
Gly Gly Ala Gly Ala Ala Thr Thr Ala1 5 10 15Ala Gly Ala Thr Cys Thr
Gly Gly Thr Cys Cys Ala Ala Cys Thr Thr 20 25 30Thr Ala Ala Ala Ala
Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Thr 35 40 45Thr Thr Gly Gly
Cys Gly Thr Cys Thr Thr Gly Cys Thr Cys Ala Ala 50 55 60Cys Ala Thr
Thr Cys Ala Cys65 7013977DNAArtificial SequenceSynthesized Peptide
Sequence 139gt gca caa gga gaa tta aga tct ggt cca act tta aaa gaa
tgg ctt 47Ala Gln Gly Glu Leu Arg Ser Gly Pro Thr Leu Lys Glu Trp
Leu1 5 10 15gtt tgg cgt ctt gct caa cat tca ctc gag 77Val Trp Arg
Leu Ala Gln His Ser Leu Glu 20 2514025PRTArtificial
SequenceSynthesized Peptide Sequence 140Ala Gln Gly Glu Leu Arg Ser
Gly Pro Thr Leu Lys Glu Trp Leu Val1 5 10 15Trp Arg Leu Ala Gln His
Ser Leu Glu 20 2514171PRTArtificial SequenceSynthesized Peptide
Sequence 141Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Gly Ala Thr
Gly Thr1 5 10 15Ala Gly Ala Thr Cys Thr Gly Gly Thr Cys Cys Ala Ala
Cys Ala Cys 20 25 30Thr Thr Cys Gly Thr Gly Ala Ala Thr Gly Gly Thr
Thr Ala Gly Cys 35 40 45Thr Thr Gly Thr Ala Gly Ala Gly Ala Gly Gly
Thr Thr Cys Ala Ala 50 55 60Cys Ala Cys Thr Cys Thr Cys65
7014271PRTArtificial SequenceSynthesized Peptide Sequence 142Thr
Cys Gly Ala Gly Ala Gly Ala Gly Thr Gly Thr Thr Gly Ala Ala1 5 10
15Cys Cys Thr Cys Thr Cys Thr Ala Cys Ala Ala Gly Cys Thr Ala Ala
20 25 30Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly Thr Gly Thr
Thr 35 40 45Gly Gly Ala Cys Cys Ala Gly Ala Thr Cys Thr Ala Cys Ala
Thr Cys 50 55 60Cys Thr Cys Cys Thr Thr Gly65 7014377DNAArtificial
SequenceSynthesized Peptide Sequence 143gt gca caa gga gga tgt aga
tct ggt cca aca ctt cgt gaa tgg tta 47Ala Gln Gly Gly Cys Arg Ser
Gly Pro Thr Leu Arg Glu Trp Leu1 5 10 15gct tgt aga gag gtt caa cac
tct ctc gag 77Ala Cys Arg Glu Val Gln His Ser Leu Glu 20
2514425PRTArtificial SequenceSynthesized Peptide Sequence 144Ala
Gln Gly Gly Cys Arg Ser Gly Pro Thr Leu Arg Glu Trp Leu Ala1 5 10
15Cys Arg Glu Val Gln His Ser Leu Glu 20 2514571PRTArtificial
SequenceSynthesized Peptide Sequence 145Thr Gly Cys Ala Cys Ala Ala
Gly Gly Thr Ala Cys Ala Thr Gly Cys1 5 10 15Gly Ala Ala Cys Ala Ala
Gly Gly Ala Cys Cys Ala Ala Cys Thr Cys 20 25 30Thr Ala Ala Gly Ala
Cys Ala Ala Thr Gly Gly Cys Thr Ala Cys Thr 35 40 45Ala Thr Gly Thr
Ala Gly Ala Cys Ala Ala Gly Gly Ala Ala Gly Ala 50 55 60Cys Ala Cys
Thr Cys Ala Cys65 7014671PRTArtificial SequenceSynthesized Peptide
Sequence 146Thr Cys Gly Ala Gly Thr Gly Ala Gly Thr Gly Thr Cys Thr
Thr Cys1 5 10 15Cys Thr Thr Gly Thr Cys Thr Ala Cys Ala Thr Ala Gly
Thr Ala Gly 20 25 30Cys Cys Ala Thr Thr Gly Thr Cys Thr Thr Ala Gly
Ala Gly Thr Thr 35 40 45Gly Gly Thr Cys Cys Thr Thr Gly Thr Thr Cys
Gly Cys Ala Thr Gly 50 55 60Thr Ala Cys Cys Thr Thr Gly65
7014777DNAArtificial SequenceSynthesized Peptide Sequence 147gt gca
caa ggt aca tgc gaa caa gga cca act cta aga caa tgg cta 47Ala Gln
Gly Thr Cys Glu Gln Gly Pro Thr Leu Arg Gln Trp Leu1 5 10 15cta tgt
aga caa gga aga cac tca ctc gag 77Leu Cys Arg Gln Gly Arg His Ser
Leu Glu 20 2514825PRTArtificial SequenceSynthesized Peptide
Sequence 148Ala Gln Gly Thr Cys Glu Gln Gly Pro Thr Leu Arg Gln Trp
Leu Leu1 5 10 15Cys Arg Gln Gly Arg His Ser Leu Glu 20
2514977DNAArtificial SequenceSynthesized Peptide Sequence 149gt gca
cag ggt tgg tgt aag gag ggt cct act ctg cgt gag tgg ctg 47Ala Gln
Gly Trp Cys Lys Glu Gly Pro Thr Leu Arg Glu Trp Leu1 5 10 15cgg tgg
ggt ttt ctg tgt cat tct ctc gag 77Arg Trp Gly Phe Leu Cys His Ser
Leu Glu 20 2515025PRTArtificial SequenceSynthesized Peptide
Sequence 150Ala Gln Gly Trp Cys Lys Glu Gly Pro Thr Leu Arg Glu Trp
Leu Arg1 5 10 15Trp Gly Phe Leu Cys His Ser Leu Glu 20
25151837DNAArtificial SequenceSynthesized Peptide Sequence
151tcgattaatc gatttgattc tagatttgtt ttaactaatt aaaggaggaa taacat
atg 59 Met 1gac aaa act cac aca tgt cca cct tgt cca gct ccg gaa ctc
ctg ggg 107Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly 5 10 15gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac
acc ctc atg 155Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 20 25 30atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg
gac gtg agc cac 203Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45gaa gac cct gag gtc aag ttc aac tgg tac gtg
gac ggc gtg gag gtg 251Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val50 55 60 65cat aat gcc aag aca aag ccg cgg gag
gag cag tac aac agc acg tac 299His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 70 75 80cgt gtg gtc agc gtc ctc acc gtc
ctg cac cag gac tgg ctg aat ggc 347Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95aag gag tac aag tgc aag gtc
tcc aac aaa gcc ctc cca gcc ccc atc 395Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110gag aaa acc atc tcc
aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 443Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125tac acc ctg
ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 491Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser130 135 140
145ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag
539Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
150 155 160tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg
cct ccc 587Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 165 170 175gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc
aag ctc acc gtg 635Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 180 185 190gac aag agc agg tgg cag cag ggg aac gtc
ttc tca tgc tcc gtg atg 683Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 195 200 205cat gag gct ctg cac aac cac tac
acg cag aag agc ctc tcc ctg tct 731His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser210 215 220 225ccg ggt aaa ggt gga
ggt ggt ggt gca cag aaa gcg gcc gca aaa aaa 779Pro Gly Lys Gly Gly
Gly Gly Gly Ala Gln Lys Ala Ala Ala Lys Lys 230 235 240ctc gag
taatggatcc gcggaaagaa gaagaagaag aagaaagccc gaaaggaagc tg 837Leu
Glu152243PRTArtificial SequenceSynthesized Peptide Sequence 152Met
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10
15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 35 40 45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro 100 105 110Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170
175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val 195 200 205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 210 215 220Ser Pro Gly Lys Gly Gly Gly Gly Gly Ala
Gln Lys Ala Ala Ala Lys225 230 235 240Lys Leu Glu15345PRTArtificial
SequenceSynthesized Peptide Sequence 153His Ile Arg Glu Gly Pro Thr
Leu Arg Gln Trp Leu Val Ala Leu Arg1 5 10 15Met Val Gly Gly Gly Pro
Glu Gly Gly Gly Gly His Ile Arg Glu Gly 20 25 30Pro Thr Leu Arg Gln
Trp Leu Val Ala Leu Arg Met Val 35 40 4515443PRTArtificial
SequenceSynthesized Peptide Sequence 154Thr Cys Glu Gln Gly Pro Thr
Leu Arg Gln Trp Leu Leu Cys Arg Gln1 5 10 15Gly Arg Gly Gly Gly Lys
Gly Gly Gly Thr Cys Glu Gln Gly Pro Thr 20 25 30Leu Arg Gln Trp Leu
Leu Cys Arg Gln Gly Arg 35 4015540PRTArtificial SequenceSynthesized
Peptide Sequence 155Gln Leu Gly His Gly Pro Thr Leu Arg Gln Trp Leu
Ser Trp Tyr Arg1 5 10 15Gly Met Gly Pro Asn Gly Glu Leu Arg Ser Gly
Pro Thr Leu Lys Glu 20 25 30Trp Leu Val Trp Arg Leu Ala Gln 35
4015618PRTArtificial SequenceSynthesized Peptide Sequence 156Cys
Ser Trp
Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg1 5 10 15Ala
Lys15725PRTArtificial SequenceSynthesized Peptide Sequence 157Gly
Gly Gly Lys Gly Gly Gly Ala Val Pro Gln Gly Pro Thr Leu Lys1 5 10
15Gln Trp Leu Leu Trp Arg Arg Cys Ala 20 2515818PRTArtificial
SequenceSynthesized Peptide Sequence 158Cys Ser Ser Gly Gly Pro Thr
Leu Arg Glu Trp Leu Gln Cys Arg Arg1 5 10 15Met
Gln15946PRTArtificial SequenceSynthesized Peptide Sequence 159Gly
Gly Gly Gly Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp1 5 10
15Leu Val Cys Leu Gly Leu Gln Gly Gly Gly Gly Gly Tyr Cys Asp Glu
20 25 30Gly Pro Thr Leu Lys Gln Trp Leu Val Cys Leu Gly Leu Gln 35
40 4516075PRTArtificial SequenceSynthesized Peptide Sequence 160Cys
Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg1 5 10
15Ala Lys Gly Gly Gly Ala Gly Gly Gly Cys Ser Trp Gly Gly Pro Thr
20 25 30Leu Lys Gln Trp Leu Gln Cys Val Arg Ala Lys Gly Gly Gly Ala
Gly 35 40 45Gly Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu
Gln Cys 50 55 60Val Arg Ala Lys Gly Gly Gly Ala Gly Gly Gly65 70
7516143PRTArtificial SequenceSynthesized Peptide Sequence 161Val
Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Gln Arg Leu1 5 10
15Asn Pro Gly Gly Gly Cys Gly Gly Gly Val Gly Ile Glu Gly Pro Thr
20 25 30Leu Arg Gln Trp Leu Ala Gln Arg Leu Asn Pro 35
4016240PRTArtificial SequenceSynthesized Peptide Sequence 162Glu
Leu Arg Ser Gly Pro Thr Leu Lys Glu Trp Leu Val Trp Arg Leu1 5 10
15Ala Gln Gly Gly Gly Gly Glu Leu Arg Ser Gly Pro Thr Leu Lys Glu
20 25 30Trp Leu Val Trp Arg Leu Ala Gln 35 4016343PRTArtificial
SequenceSynthesized Peptide Sequence 163Ala Leu Arg Asp Gly Pro Thr
Leu Lys Gln Trp Leu Glu Tyr Arg Arg1 5 10 15Gln Ala Gly Gly Gly Lys
Gly Gly Gly Ala Leu Arg Asp Gly Pro Thr 20 25 30Leu Lys Gln Trp Leu
Glu Tyr Arg Arg Gln Ala 35 4016436PRTArtificial SequenceSynthesized
Peptide Sequence 164Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu
Glu Tyr Arg Arg1 5 10 15Gln Ala Ala Leu Arg Asp Gly Pro Thr Leu Lys
Gln Trp Leu Glu Tyr 20 25 30Arg Arg Gln Ala 3516536PRTArtificial
SequenceSynthesized Peptide Sequence 165Glu Ala Leu Leu Gly Pro Thr
Leu Arg Glu Trp Leu Ala Trp Arg Arg1 5 10 15Ala Gln Glu Ala Leu Leu
Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp 20 25 30Arg Arg Ala Gln
3516636PRTArtificial SequenceSynthesized Peptide Sequence 166Ala
Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu Leu Trp Arg Arg1 5 10
15Cys Ala Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu Leu Trp
20 25 30Arg Arg Cys Ala 3516736PRTArtificial SequenceSynthesized
Peptide Sequence 167Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu
Val Cys Leu Gly1 5 10 15Leu Gln Tyr Cys Asp Glu Gly Pro Thr Leu Lys
Gln Trp Leu Val Cys 20 25 30Leu Gly Leu Gln 3516836PRTArtificial
SequenceSynthesized Peptide Sequence 168Cys Ser Ser Gly Gly Pro Thr
Leu Arg Glu Trp Leu Gln Cys Arg Arg1 5 10 15Met Gln Cys Ser Ser Gly
Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys 20 25 30Arg Arg Met Gln
3516936PRTArtificial SequenceSynthesized Peptide Sequence 169Cys
Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg1 5 10
15Ala Lys Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys
20 25 30Val Arg Ala Lys 3517041PRTArtificial SequenceSynthesized
Peptide Sequence 170Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu
Glu Tyr Arg Arg1 5 10 15Gln Ala Gly Gly Gly Gly Gly Ala Leu Arg Asp
Gly Pro Thr Leu Lys 20 25 30Gln Trp Leu Glu Tyr Arg Arg Gln Ala 35
4017141PRTArtificial SequenceSynthesized Peptide Sequence 171Glu
Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg1 5 10
15Ala Gln Gly Gly Gly Gly Gly Glu Ala Leu Leu Gly Pro Thr Leu Arg
20 25 30Glu Trp Leu Ala Trp Arg Arg Ala Gln 35 4017241PRTArtificial
SequenceSynthesized Peptide Sequence 172Ala Val Pro Gln Gly Pro Thr
Leu Lys Gln Trp Leu Leu Trp Arg Arg1 5 10 15Cys Ala Gly Gly Gly Gly
Gly Ala Val Pro Gln Gly Pro Thr Leu Lys 20 25 30Gln Trp Leu Leu Trp
Arg Arg Cys Ala 35 4017341PRTArtificial SequenceSynthesized Peptide
Sequence 173Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu Val Cys
Leu Gly1 5 10 15Leu Gln Gly Gly Gly Gly Gly Tyr Cys Asp Glu Gly Pro
Thr Leu Lys 20 25 30Gln Trp Leu Val Cys Leu Gly Leu Gln 35
4017441PRTArtificial SequenceSynthesized Peptide Sequence 174Cys
Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg1 5 10
15Met Gln Gly Gly Gly Gly Gly Cys Ser Ser Gly Gly Pro Thr Leu Arg
20 25 30Glu Trp Leu Gln Cys Arg Arg Met Gln 35 4017541PRTArtificial
SequenceSynthesized Peptide Sequence 175Cys Ser Trp Gly Gly Pro Thr
Leu Lys Gln Trp Leu Gln Cys Val Arg1 5 10 15Ala Lys Gly Gly Gly Gly
Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys 20 25 30Gln Trp Leu Gln Cys
Val Arg Ala Lys 35 4017623PRTArtificial SequenceSynthesized Peptide
Sequence 176Gly Gly Gly Gly Gly Ala Leu Arg Asp Gly Pro Thr Leu Lys
Gln Trp1 5 10 15Leu Glu Tyr Arg Arg Gln Ala 2017723PRTArtificial
SequenceSynthesized Peptide Sequence 177Gly Gly Gly Gly Gly Glu Ala
Leu Leu Gly Pro Thr Leu Arg Glu Trp1 5 10 15Leu Ala Trp Arg Arg Ala
Gln 2017823PRTArtificial SequenceSynthesized Peptide Sequence
178Gly Gly Gly Gly Gly Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp1
5 10 15Leu Leu Trp Arg Arg Cys Ala 2017923PRTArtificial
SequenceSynthesized Peptide Sequence 179Gly Gly Gly Gly Gly Tyr Cys
Asp Glu Gly Pro Thr Leu Lys Gln Trp1 5 10 15Leu Val Cys Leu Gly Leu
Gln 2018023PRTArtificial SequenceSynthesized Peptide Sequence
180Gly Gly Gly Gly Gly Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp1
5 10 15Leu Gln Cys Arg Arg Met Gln 2018123PRTArtificial
SequenceSynthesized Peptide Sequence 181Gly Gly Gly Gly Gly Cys Ser
Trp Gly Gly Pro Thr Leu Lys Gln Trp1 5 10 15Leu Gln Cys Val Arg Ala
Lys 2018246PRTArtificial SequenceSynthesized Peptide Sequence
182Gly Gly Gly Gly Gly Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp1
5 10 15Leu Glu Tyr Arg Arg Gln Ala Gly Gly Gly Gly Gly Ala Leu Arg
Asp 20 25 30Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr Arg Arg Gln Ala
35 40 4518346PRTArtificial SequenceSynthesized Peptide Sequence
183Gly Gly Gly Gly Gly Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp1
5 10 15Leu Ala Trp Arg Arg Ala Gln Gly Gly Gly Gly Gly Glu Ala Leu
Leu 20 25 30Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg Ala Gln
35 40 4518446PRTArtificial SequenceSynthesized Peptide Sequence
184Gly Gly Gly Gly Gly Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp1
5 10 15Leu Leu Trp Arg Arg Cys Ala Gly Gly Gly Gly Gly Ala Val Pro
Gln 20 25 30Gly Pro Thr Leu Lys Gln Trp Leu Leu Trp Arg Arg Cys Ala
35 40 4518546PRTArtificial SequenceSynthesized Peptide Sequence
185Gly Gly Gly Gly Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp1
5 10 15Leu Val Cys Leu Gly Leu Gln Gly Gly Gly Gly Gly Tyr Cys Asp
Glu 20 25 30Gly Pro Thr Leu Lys Gln Trp Leu Val Cys Leu Gly Leu Gln
35 40 4518646PRTArtificial SequenceSynthesized Peptide Sequence
186Gly Gly Gly Gly Gly Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp1
5 10 15Leu Gln Cys Arg Arg Met Gln Gly Gly Gly Gly Gly Cys Ser Ser
Gly 20 25 30Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg Met Gln
35 40 4518746PRTArtificial SequenceSynthesized Peptide Sequence
187Gly Gly Gly Gly Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp1
5 10 15Leu Gln Cys Val Arg Ala Lys Gly Gly Gly Gly Gly Cys Ser Trp
Gly 20 25 30Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg Ala Lys
35 40 4518846PRTArtificial SequenceSynthesized Peptide Sequence
188Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr Arg Arg1
5 10 15Gln Ala Gly Gly Gly Gly Gly Ala Leu Arg Asp Gly Pro Thr Leu
Lys 20 25 30Gln Trp Leu Glu Tyr Arg Arg Gln Ala Gly Gly Gly Gly Gly
35 40 4518946PRTArtificial SequenceSynthesized Peptide Sequence
189Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg1
5 10 15Ala Gln Gly Gly Gly Gly Gly Glu Ala Leu Leu Gly Pro Thr Leu
Arg 20 25 30Glu Trp Leu Ala Trp Arg Arg Ala Gln Gly Gly Gly Gly Gly
35 40 4519046PRTArtificial SequenceSynthesized Peptide Sequence
190Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu Leu Trp Arg Arg1
5 10 15Cys Ala Gly Gly Gly Gly Gly Ala Val Pro Gln Gly Pro Thr Leu
Lys 20 25 30Gln Trp Leu Leu Trp Arg Arg Cys Ala Gly Gly Gly Gly Gly
35 40 4519146PRTArtificial SequenceSynthesized Peptide Sequence
191Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu Val Cys Leu Gly1
5 10 15Leu Gln Gly Gly Gly Gly Gly Tyr Cys Asp Glu Gly Pro Thr Leu
Lys 20 25 30Gln Trp Leu Val Cys Leu Gly Leu Gln Gly Gly Gly Gly Gly
35 40 4519246PRTArtificial SequenceSynthesized Peptide Sequence
192Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg1
5 10 15Met Gln Gly Gly Gly Gly Gly Cys Ser Ser Gly Gly Pro Thr Leu
Arg 20 25 30Glu Trp Leu Gln Cys Arg Arg Met Gln Gly Gly Gly Gly Gly
35 40 4519346PRTArtificial SequenceSynthesized Peptide Sequence
193Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg1
5 10 15Ala Lys Gly Gly Gly Gly Gly Cys Ser Trp Gly Gly Pro Thr Leu
Lys 20 25 30Gln Trp Leu Gln Cys Val Arg Ala Lys Gly Gly Gly Gly Gly
35 40 4519423PRTArtificial SequenceSynthesized Peptide Sequence
194Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr Arg Arg1
5 10 15Gln Ala Gly Gly Gly Gly Gly 2019523PRTArtificial
SequenceSynthesized Peptide Sequence 195Glu Ala Leu Leu Gly Pro Thr
Leu Arg Glu Trp Leu Ala Trp Arg Arg1 5 10 15Ala Gln Gly Gly Gly Gly
Gly 2019623PRTArtificial SequenceSynthesized Peptide Sequence
196Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg1
5 10 15Ala Gln Gly Gly Gly Gly Gly 2019723PRTArtificial
SequenceSynthesized Peptide Sequence 197Tyr Cys Asp Glu Gly Pro Thr
Leu Lys Gln Trp Leu Val Cys Leu Gly1 5 10 15Leu Gln Gly Gly Gly Gly
Gly 2019823PRTArtificial SequenceSynthesized Peptide Sequence
198Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg1
5 10 15Met Gln Gly Gly Gly Gly Gly 2019923PRTArtificial
SequenceSynthesized Peptide Sequence 199Cys Ser Trp Gly Gly Pro Thr
Leu Lys Gln Trp Leu Gln Cys Val Arg1 5 10 15Ala Lys Gly Gly Gly Gly
Gly 20
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