U.S. patent application number 13/666579 was filed with the patent office on 2013-09-19 for novel peptides that bind to the erythropoietin receptor.
This patent application is currently assigned to AFFYMAX, INC.. The applicant listed for this patent is AFFYMAX, INC.. Invention is credited to Christopher P. Holmes, Guy Lalonde, Balu Palani, Peter J. Schatz, David Tumelty, Qun Yin, Genet Zemede.
Application Number | 20130244939 13/666579 |
Document ID | / |
Family ID | 33452385 |
Filed Date | 2013-09-19 |
United States Patent
Application |
20130244939 |
Kind Code |
A1 |
Holmes; Christopher P. ; et
al. |
September 19, 2013 |
NOVEL PEPTIDES THAT BIND TO THE ERYTHROPOIETIN RECEPTOR
Abstract
The present invention relates to peptide compounds that are
agonists of the erythropoietin receptor (EPO-R). The invention also
relates to therapeutic methods using such peptide compounds to
treat disorders associated with insufficient or defective red blood
cell production. Pharmaceutical compositions, which comprise the
peptide compounds of the invention, are also provided.
Inventors: |
Holmes; Christopher P.;
(Saratoga, CA) ; Yin; Qun; (Palo Alto, CA)
; Lalonde; Guy; (Woodside, CA) ; Schatz; Peter
J.; (Cupertino, CA) ; Tumelty; David; (San
Diego, CA) ; Palani; Balu; (Cupertino, CA) ;
Zemede; Genet; (San Jose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AFFYMAX, INC. |
Palo Alto |
CA |
US |
|
|
Assignee: |
AFFYMAX, INC.
Palo Alto
CA
|
Family ID: |
33452385 |
Appl. No.: |
13/666579 |
Filed: |
November 1, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12405599 |
Mar 17, 2009 |
8304391 |
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13666579 |
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10555868 |
Nov 2, 2005 |
7528104 |
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PCT/US2004/014886 |
May 12, 2004 |
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12405599 |
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60470245 |
May 12, 2003 |
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Current U.S.
Class: |
514/13.5 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
43/00 20180101; A61K 38/10 20130101; A61P 29/00 20180101; A61P 7/00
20180101; A61P 7/04 20180101; A61P 1/00 20180101; A61P 7/08
20180101; A61P 15/00 20180101; A61P 9/10 20180101; A61P 17/02
20180101; A61K 38/16 20130101; A61P 31/18 20180101; A61K 38/00
20130101; A61P 25/00 20180101; A61P 13/12 20180101; A61P 35/00
20180101; C07K 14/505 20130101; A61P 7/06 20180101; A61P 37/02
20180101; A61P 19/02 20180101; C07K 14/001 20130101; C07K 7/08
20130101 |
Class at
Publication: |
514/13.5 |
International
Class: |
C07K 14/00 20060101
C07K014/00; C07K 7/08 20060101 C07K007/08 |
Claims
1. A method for treating a patient, comprising administering to a
patient having a disorder characterized by a deficiency of
erythropoietin or a low or defective red blood cell population, a
therapeutically effective amount of a peptide comprising about 17
to about 40 amino acid residues in length and comprising the amino
acid sequence: TABLE-US-00010 (SEQ ID NO: 1)
LYACHX.sub.0GPITX.sub.1VCQPLR
wherein X.sub.0 is a residue selected from the group consisting of
methionine (M) and homoserine methylether (Hsm), and X.sub.1 is a
residue selected from tryptophan (W), 1-naphthylalanine (1-nal),
and 2-naphthylalanine (2-nal).
2-29. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 10/555,868, filed Nov. 2, 2005, which is a 371 National Phase
Application of International Application No. PCT/US2004/014886,
filed May 12, 2004, which claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application No. 60/470,245, filed
May 12, 2003. The International Application was published in
English under PCT Article 21(2) as International Publication No. WO
2004/11016 on Nov. 25, 2004. The entire contents of each of these
applications is incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to peptide compounds that are
agonists of the erythropoietin receptor (EPO-R). The invention also
relates to therapeutic methods using such peptide compounds to
treat disorders associated with insufficient or defective red blood
cell production. Pharmaceutical compositions, which comprise the
peptide compounds of the invention, are also provided.
BACKGROUND OF THE INVENTION
[0003] Erythropoietin (EPO) is a glycoprotein hormone of 165 amino
acids, with a molecular weight of about 34 kilodaltons (kD) and
preferred glycosylation sites on amino-acid positions 24, 38, 83,
and 126. It is initially produced as a precursor protein with a
signal peptide of 23 amino acids. EPO can occur in three forms:
.alpha., .beta., and asialo. The .alpha. and .beta. forms differ
slightly in their carbohydrate components, but have the same
potency, biological activity, and molecular weight. The asialo form
is an .alpha. or .beta. form with the terminal carbohydrate (sialic
acid) removed. The DNA sequences encoding EPO have been reported
[U.S. Pat. No. 4,703,008 to Lin].
[0004] EPO stimulates mitotic division and differentiation of
erythrocyte precursor cells, and thus ensures the production of
erythrocytes. It is produced in the kidney when hypoxic conditions
prevail. During EPO-induced differentiation of erythrocyte
precursor cells, globin synthesis is induced; heme complex
synthesis is stimulated; and the number of ferritin receptors
increases. These changes allow the cell to take on more iron and
synthesize functional hemoglobin, which in mature erythrocytes
binds oxygen. Thus, erythrocytes and their hemoglobin play a key
role in supplying the body with oxygen. These changes are initiated
by the interaction of EPO with an appropriate receptor on the cell
surface of the erythrocyte precursor cells [See, e.g., Graber and
Krantz (1978) Ann. Rev. Med. 29.51-66].
[0005] EPO is present in very low concentrations in plasma when the
body is in a healthy state wherein tissues receive sufficient
oxygenation from the existing number of erythrocytes. This normal
low concentration is sufficient to stimulate replacement of red
blood cells which are lost normally through aging.
[0006] The amount of EPO in the circulation is increased under
conditions of hypoxia when oxygen transport by blood cells in the
circulation is reduced. Hypoxia may be caused, for example, by
substantial blood loss through hemorrhage, destruction of red blood
cells by over-exposure to radiation, reduction in oxygen intake due
to high altitude or prolonged unconsciousness, or various forms of
anemia. In response to such hypoxic stress, elevated EPO levels
increase red blood cell production by stimulating the proliferation
of erythroid progenitor cells. When the number of red blood cells
in circulation is greater than needed for normal tissue oxygen
requirements, EPO levels in circulation are decreased.
[0007] Because EPO is essential in the process of red blood cell
formation, this hormone has potentially useful applications in both
the diagnosis and the treatment of blood disorders characterized by
low or defective red blood cell production. Recent studies have
provided a basis for the projection of EPO therapy efficacy for a
variety of disease states, disorders, and states of hematologic
irregularity, including: beta-thalassemia [see, Vedovato, et al.
(1984) Acta. Haematol. 71:211-213]; cystic fibrosis [see,
Vichinsky, et al. (1984) J. Pediatric 105:15-21]; pregnancy and
menstrual disorders [see, Cotes, et al. (193) Brit. J. Ostet.
Gyneacol. 90:304-311]; early anemia of prematurity [see, Haga, et
al. (1983) Acta Pediatr. Scand. 72; 827-831]; spinal cord injury
[see, Claus-Walker, et al. (1984) Arch. Phys. Med. Rehabil.
65:370-374]; space flight [see, Dunn, et al. (1984) Eur. J. Appl.
Physiol. 52:178-182]; acute blood loss [see, Miller, et al., (1982)
Brit. J. Haematol. 52:545-590]; aging [see, Udupa, et al. (1984) J.
Lab. Clin. Med. 103:574-580 and 581-588 and Lipschitz, et al.
(1983) Blood 63:502-509]; various neoplastic disease states
accompanied by abnormal erythropoiesis [see, Dainiak, et al. (1983)
Cancer 5:1101-1106 and Schwartz, et al. (1983) Otolaryngol.
109:269-272]; and renal insufficiency [see, Eschbach. et al. (1987)
N. Eng. J. Med. 316:73-78].
[0008] Purified, homogeneous EPO has been characterized [U.S. Pat.
No. 4,677,195 to Hewick]. A DNA sequence encoding EPO was purified,
cloned, and expressed to produce recombinant polypeptides with the
same biochemical and immunological properties as natural EPO. A
recombinant EPO molecule with oligosaccharides identical to those
on natural EPO has also been produced [See, Sasaki, et al. (1987)
J. Biol. Chem. 262:12059-12076].
[0009] The biological effect of EPO appears to be mediated, in
part, through interaction with a cell membrane bound receptor.
Initial studies, using immature erythroid cells isolated from mouse
spleen, suggested that the EPO-binding cell surface proteins
comprise two polypeptides having approximate molecular weights of
85,000 Daltons and 100,000 Daltons, respectively [Sawyer, et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3690-3694]. The number of
EPO-binding sites was calculated to average from 800 to 1000 per
cell surface. Of these binding sites, approximately 300 bound EPO
with a K.sub.d of approximately 90 pM (picomolar), while the
remaining bound EPO with a reduced affinity of approximately 570 pM
[Sawyer, et al. (1987) J. Biol. Chem. 262:5554-5562]. An
independent study suggested that EPO-responsive splenic
erythroblasts, prepared from mice injected with the anemic strain
(FVA) of the Friend leukemia virus, possess a total of
approximately 400 high and low affinity EPO binding sites with
K.sub.d values of approximately 100 pM and 800 pM, respectively
[Landschulz, et al. (1989) Blood 73:1476-1486].
[0010] Subsequent work indicated that the two forms of EPO receptor
(EPO-R) were encoded by a single gene. This gene has been cloned
[See, e.g., Jones, et al. (1990) Blood 76, 31-35; Noguchi, et al.
(1991) Blood 78:2548-2556; Maouche, et al. (1991) Blood
78:2557-2563]. For example, the DNA sequences and encoded peptide
sequences for murine and human EPO-R proteins are described in PCT
Pub. No. WO 90/08822 to D'Andrea, et al. Current models suggest
that binding of EPO to EPO-R results in the dimerization and
activation of two EPO-R molecules, which results in subsequent
steps of signal transduction [See, e.g., Watowich, et al. (1992)
Proc. Natl. Acad. Sci. USA 89:2140-2144].
[0011] The availability of cloned genes for EPO-R facilitates the
search for agonists and antagonists of this important receptor. The
availability of the recombinant receptor protein allows the study
of receptor-ligand interaction in a variety of random and
semi-random peptide diversity generation systems. These systems
include the "peptides on plasmids" system [described in U.S. Pat.
No. 6,270,170]; the "peptides on phage" system [described in U.S.
Pat. No. 5,432,018 and Cwirla, et al. (1990) Proc. Natl. Acad. Sci.
USA 87:6378-6382]; the "encoded synthetic library" (ESL) system
[described in U.S. patent application Ser. No. 946,239, filed Sep.
16, 1992]; and the "very large scale immobilized polymer synthesis"
system [described in U.S. Pat. No. 5,143,854; PCT Pub. No.
90/15070; Fodor, et al. (1991) Science 251:767-773; Dower and Fodor
(1991) Ann. Rep. Med. Chem. 26:271-180; and U.S. Pat. No.
5,424,186].
[0012] Peptides that interact to a least some extent with EPO-R
have been identified and are described, for example in U.S. Pat.
Nos. 5,773,569; 5,830,851; and 5,986,047 to Wrighton, et al.; PCT
Pub. No. WO 96/40749 to Wrighton, et al.; U.S. Pat. No. 5,767,078
and PCT Pub. No. 96/40772 to Johnson and Zivin; PCT Pub. No. WO
01/38342 to Balu; and WO 01/91780 to Smith-Swintosky, et al. In
particular, a group of peptides containing a peptide motif has been
identified, members of which bind to EPO-R and stimulate
EPO-dependent cell proliferation. Yet, peptides identified to date
that contain the motif stimulate EPO-dependent cell proliferation
in vitro with EC50 values between about 20 nanomolar (DM) and 250
nM. Thus, peptide concentrations of 20 nM to 250 nM are required to
stimulate 50% of the maximal cell proliferation stimulated by
EPO.
[0013] Given the immense potential of EPO-R agonists, both for
studies of the important biological activities mediated by this
receptor and for treatment of disease, there remains a need for the
identification of peptide EPO-R agonists of enhanced potency and
activity. The present invention provides such compounds.
[0014] The citation and/or discussion of cited references in this
section and throughout the specification is provided merely to
clarify the description of the present invention and is not an
admission that any such reference is "prior art" to the present
invention.
SUMMARY OF THE INVENTION
[0015] The present invention provides EPO-R agonist peptides of
dramatically enhanced potency and activity. These agonists include
monomeric peptide agonists of 17 to about 40 amino acids in length
that comprise the core amino acid sequence
LYACHX.sub.0GPITX.sub.1VCQPLR (SEQ ID NO: 1), where each amino acid
is indicated by standard one letter abbreviation, X.sub.0 is
methionine (M) or homoserine methylether (Hsm), and X.sub.1 is
tryptophan (W), 1-naphthylalanine (1-nal), or 2-naphthylalanine
(2-nal); as well as dimeric peptide agonists that comprise two
peptide monomers, wherein each peptide monomer is of 17 to about 40
amino acids in length and comprises the core amino acid sequence
LYACHX.sub.0GPITX.sub.1VCQPLR (SEQ ID NO:1), where each amino acid
is indicated by standard one letter abbreviation, X.sub.0 is
methionine (M) or homoserine methylether (Hsm), and X.sub.1 is
tryptophan (W), 1-naphthylalanine (1-nal), or 2-naphthylalanine
(2-nal). The potency of these novel peptide agonists may be further
enhanced by one or more modifications, including: acetylation,
intramolecular disulfide bond formation, and covalent attachment of
one or more polyethylene glycol (PEG) moieties. The invention
further provides pharmaceutical compositions comprised of such
peptide agonists, and methods to treat various medical conditions
using such peptide agonists.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0016] Amino acid residues in peptides are abbreviated as follows:
Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile
or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or
S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A;
Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q;
Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or
D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is
Trp or W; Arginine is Arg or R; and Glycine is Gly or G. The
unconventional amino acids in peptides are abbreviated as follows:
1-naphthylalanine is 1-nal or Np; 2-naphthylalanine is 2-nal;
N-methylglycine (also known as sarcosine) is MeG or Sc; homoserine
methylether is Hsm; and acetylated glycine (N-acetylglycine) is
AcG.
[0017] As used herein, the term "polypeptide" or "protein" refers
to a polymer of amino acid monomers that are alpha amino acids
joined together through amide bonds. Polypeptides are therefore at
least two amino acid residues in length, and are usually longer.
Generally, the term "peptide" refers to a polypeptide that is only
a few amino acid residues in length. The novel EPO-R agonist
peptides of the present invention are preferably no more than about
50 amino acid residues in length. They are more preferably from
about 17 to about 40 amino acid residues in length. A polypeptide,
in contrast with a peptide, may comprise any number of amino acid
residues. Hence, the term polypeptide included peptides as well as
longer sequences of amino acids.
[0018] As used herein, the phrase "pharmaceutically acceptable"
refers to molecular entities and compositions that are "generally
regarded as safe", e.g., that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water or aqueous solution
saline solutions and aqueous dextrose and glycerol solutions are
preferably employed as carriers, particularly for injectable
solutions. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin. As used
herein the term "agonist" refers to a biologically active ligand
which binds to its complementary biologically active receptor and
activates the latter either to cause a biological response in the
receptor, or to enhance preexisting biological activity of the
receptor.
Novel Peptides that are EPO-R Agonists
[0019] The present invention relates to peptides that are agonists
of the EPO-R and show dramatically enhanced potency and activity.
These peptide agonists are preferably of 17 to about 40 amino acids
in length and comprise the core amino acid sequence
LYACHX.sub.0GPITX.sub.1VCQPLR (SEQ ID NO: 1), where each amino acid
is indicated by standard one letter abbreviation, X.sub.0 is
methionine (M) or homoserine methylether (Hsm), and X.sub.1 is
tryptophan (W), 1-naphthylalanine (1-nal), or 2-naphthylalanine
(2-nal).
[0020] The peptides of this invention may be monomers, dimers, or
other multimers. The peptide multimers of the invention may be
trimers, tetramers, pentamers, or other higher order structures.
Moreover, such dimers and other multimers may be heterodimers or
heteromultimers. The peptide monomers of the present invention may
be degradation products (e.g., oxidation products of methionine or
deamidated glutamine, arganine, and C-terminus amide). Such
degradation products may be used in and are therefore considered
part of the present invention. In preferred embodiments, the
heteromultimers of the invention comprise multiple peptides that
are all EPO-R agonist peptides. In highly preferred embodiments,
the multimers of the invention are homomultimers: i.e., they
comprise multiple EPO-R agonist peptides of the same amino acid
sequence.
[0021] Accordingly, the present invention also relates to dimeric
peptide agonists of EPO-R, which show dramatically enhanced potency
and activity. In preferred embodiments, the dimers of the invention
comprise two peptides that are both EPO-R agonist peptides. These
preferred dimeric peptide agonists comprise two peptide monomers,
wherein each peptide monomer is of 17 to about 40 amino acids in
length and comprises the core amino acid sequence
LYACHX.sub.0GPITX.sub.1VCQPLR (SEQ ID NO: 1), where each amino acid
is indicated by standard one letter abbreviation, X.sub.0 is
methionine (M) or homoserine methylether (Hsm), and X.sub.1 is
tryptophan (W), 1-naphthylalanine (1-nal), or 2-naphthylalanine
(2-nal). In particularly preferred embodiments, the dimers of the
invention comprise two EPO-R agonist peptides of the same amino
acid sequence.
[0022] According to some embodiments of the invention, two or more,
and preferably from two to six amino acid residues, independently
selected from any of the 20 genetically encoded L-amino acids or
the stereoisomeric D-amino acids, will be coupled to either or both
ends of the core sequence described above. For example, the
sequence GG will often be appended to either terminus or both
termini of the core sequence.
[0023] Stereoisomers (e.g., D-amino acids) of the twenty
conventional amino acids, unnatural amino acids such as
a,a-disubstituted amino acids, N-alkyl amino acids, lactic acid,
and other unconventional amino acids may also be suitable
components for compounds of the present invention. Examples of
unconventional amino acids include, but are not limited to:
.beta.-alanine, 3-pyridylalanine, 4-hydroxyproline,
O-phosphoserine, N-methylglycine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
nor-leucine, and other similar amino acids and imino acids.
[0024] Other modifications are also possible, including
modification of the amino terminus, modification of the carboxy
terminus, replacement of one or more of the naturally occurring
genetically encoded amino acids with an unconventional amino acid,
modification of the side chain of one or more amino acid residues,
peptide phosphorylation, and the like. A preferred amino terminal
modification is acetylation (e.g., with acetic acid or a halogen
substituted acetic acid). In preferred embodiments an N-terminal
glycine is acetylated to N-acetylglycine (AcG). In preferred
embodiments, a the C-terminal glycine is N-methylglycine (MeG, also
known as sarcosine).
[0025] Preferred peptide monomers of the present invention include,
but are not limited to:
TABLE-US-00001 (SEQ ID NO: 2) LYACHMGPITWVCQPLR; (SEQ ID NO: 3)
LYACHMGPIT(1-nal)VCQPLR; (SEQ ID NO: 4) LYACHMGPIT(2-nal)VCQPLR;
(SEQ ID NO: 5) GGLYACHMGPITWVCQPLRG; (SEQ ID NO: 6)
GGLYACHMGPIT(1-nal)VCQPLRG; (SEQ ID NO: 7)
GGLYACHMGPIT(2-nal)VCQPLRG; (SEQ ID NO: 8)
(AcG)GLYACHMGPITWVCQPLRG; (SEQ ID NO: 9)
(AcG)GLYACHMGPIT(1-nal)VCQPLRG; (SEQ ID NO: 10)
(AcG)GLYACHMGPIT(2-nal)VCQPLRG; (SEQ ID NO: 11)
GGLYACHMGPITWVCQPLR(MeG); (SEQ ID NO: 12)
GGLYACHMGPIT(1-nal)VCQPLR(MeG); (SEQ ID NO: 13)
GGLYACHMGPIT(2-nal)VCQPLR(MeG); (SEQ ID NO: 14)
(AcG)GLYACHMGPITWVCQPLRG(MeG); (SEQ ID NO: 15)
(AcG)GLYACHMGPIT(1-nal)VCQPLRG(MeG); (SEQ ID NO: 16)
(AcG)GLYACHMGPIT(2-nal)VCQPLRG(MeG); (SEQ ID NO: 17)
LYACH(Hsm)GPITWVCQPLR; (SEQ ID NO: 18) LYACH(Hsm)GPIT(1-nal)VCQPLR;
(SEQ ID NO: 19) LYACH(Hsm)GPIT(2-nal)VCQPLR; (SEQ ID NO: 20)
GGLYACH(Hsm)GPITWVCQPLRG; (SEQ ID NO: 21)
GGLYACH(Hsm)GPIT(1-nal)VCQPLRG; (SEQ ID NO: 22)
GGLYACH(Hsm)GPIT(2-nal)VCQPLRG; (SEQ ID NO: 23)
(AcG)GLYACH(Hsm)GPITWVCQPLRG; (SEQ ID NO: 24)
(AcG)GLYACH(Hsm)GPIT(1-nal)VCQPLRG; (SEQ ID NO: 25)
(AcG)GLYACH(Hsm)GPIT(2-nal)VCQPLRG; (SEQ ID NO: 26)
GGLYACH(Hsm)GPITWVCQPLR(MeG); (SEQ ID NO: 27)
GGLYACH(Hsm)GPIT(1-nal)VCQPLR(MeG); (SEQ ID NO: 28)
GGLYACH(Hsm)GPIT(2-nal)VCQPLR(MeG); (SEQ ID NO: 29)
(AcG)GLYACH(Hsm)GPITWVCQPLRG(MeG); (SEQ ID NO: 30)
(AcG)GLYACH(Hsm)GPIT(1-nal)VCQPLRG(MeG); and. (SEQ ID NO: 31)
(AcG)GLYACH(Hsm)GPIT(2-nal)VCQPLRG(MeG).
[0026] In preferred embodiments, the peptide monomers of the
invention contain an intramolecular disulfide bond between the two
cysteine residues of the core sequence. Such monomers may be
represented schematically as follows:
##STR00001##
[0027] The present invention also provides conjugates of these
peptide monomers. Thus, according to a preferred embodiment, the
monomeric peptides of the present invention are dimerized or
oligomerized, thereby enhancing EPO-R agonist activity.
[0028] In one embodiment, the peptide monomers of the invention may
be oligomerized using the biotin/streptavidin system. Biotinylated
analogs of peptide monomers may be synthesized by standard
techniques. For example, the peptide monomers may be C-terminally
biotinylated. These biotinylated monomers are then oligomerized by
incubation with streptavidin [e.g., at a 4:1 molar ratio at room
temperature in phosphate buffered saline (PBS) or HEPES-buffered
RPMI medium (Invitrogen) for 1 hour]. In a variation of this
embodiment, biotinylated peptide monomers may be oligomerized by
incubation with any one of a number of commercially available
anti-biotin antibodies [e.g., goat anti-biotin IgG from Kirkegaard
& Perry Laboratories, Inc. (Washington, D.C.)].
[0029] In preferred embodiments, the peptide monomers of the
invention are dimerized by covalent attachment to at least one
linker moiety. The linker (L.sub.K) moiety is preferably, although
not necessarily, a C.sub.1-12 linking moiety optionally terminated
with one or two --NH-- linkages and optionally substituted at one
or more available carbon atoms with a lower alkyl substituent.
Preferably the linker L.sub.K comprises --NH--R--NH-- wherein R is
a lower (C.sub.1-6) alkylene substituted with a functional group
such as a carboxyl group or an amino group that enables binding to
another molecular moiety (e.g., as may be present on the surface of
a solid support). Most preferably the linker is a lysine residue or
a lysine amide (a lysine residue wherein the carboxyl group has
been converted to an amide moiety --CONH.sub.2). In preferred
embodiments, the linker bridges the C-termini of two peptide
monomers, by simultaneous attachment to the C-terminal amino acid
of each monomer.
[0030] For example, when the C-terminal linker L.sub.K is a lysine
amide the dimer may be illustrated structurally as shown in Formula
I, and summarized as shown in Formula II:
##STR00002##
In Formula I and Formula II, N.sup.2 represents the nitrogen atom
of lysine's .epsilon.-amino group and N.sup.1 represents the
nitrogen atom of lysine's .alpha.-amino group. The dimeric
structure can be written as [peptide].sub.2Lys-amide to denote a
peptide bound to both the .alpha. and .epsilon. amino groups of
lysine, or [Ac-peptide].sub.2Lys-amide to denote an N-terminally
acetylated peptide bound to both the a and c amino groups of
lysine, or [Ac-peptide, disulfide].sub.2Lys-amide to denote an
N-terminally acetylated peptide bound to both the a and E amino
groups of lysine with each peptide containing an intramolecular
disulfide loop, or [Ac-peptide, disulfide].sub.2Lys-spacer-PEG to
denote an N-terminally acetylated peptide bound to both the .alpha.
and .epsilon. amino groups of lysine with each peptide containing
an intramolecular disulfide loop and a spacer molecule forming a
covalent linkage between the C-terminus of lysine and a PEG moiety,
or [Ac-peptide-Lys*-NH.sub.2].sub.2-Iminodiacetic-N-(Boc-.beta.Ala)
to denote a homodimer of an N-terminally acetylated peptide bearing
a C-terminal lysineamide residue where the c amine of lysine is
bound to each of the two carboxyl groups of iminodiacetic acid and
where Boc-beta-alanine is covalently bound to the nitrogen atom of
iminodiacetic acid via an amide bond.
[0031] In an additional embodiment, polyethylene glycol (PEG) may
serve as the linker L.sub.K that dimerizes two peptide monomers:
for example, a single PEG moiety may be simultaneously attached to
the N-termini of both peptide chains of a peptide dimer.
[0032] In yet another additional embodiment, the linker (L.sub.K)
moiety is preferably, but not necessarily, a molecule containing
two carboxylic acids and optionally substituted at one or more
available atoms with an additional functional group such as an
amine capable of being bound to one or more PEG molecules. Such a
molecule can be depicted as:
--CO--(CH.sub.2).sub.n--X--(CH.sub.2).sub.m--CO--
where n is an integer from 0 to 10, m is an integer from 1 to 10, X
is selected from O, S, N(CH.sub.2).sub.pNR.sub.1,
NCO(CH.sub.2).sub.pNR.sub.1, and CHNR.sub.1, R.sub.1 is selected
from H, Boc, Cbz, etc., and p is an integer from 1 to 10.
[0033] In preferred embodiments, one amino group of each of the
peptides form an amide bond with the linker L.sub.K. In
particularly preferred embodiments, the amino group of the peptide
bound to the linker L.sub.K is the epsilon amine of a lysine
residue or the alpha amine of the N-terminal residue, or an amino
group of the optional spacer molecule. In particularly preferred
embodiments, both n and m are one, X is
NCO(CH.sub.2).sub.pNR.sub.1, p is two, and R.sub.1 is Boc. A
dimeric EPO peptide containing such a preferred linker may be
structurally illustrated as shown in Formula III.
##STR00003##
Optionally, the Boc group can be removed to liberate a reactive
amine group capable of forming a covalent bond with a suitably
activated water soluble polymer species, for example, a PEG species
such as mPEG-para-nitrophenylcarbonate (mPEG-NPC),
mPEG-succinimidyl propionate (mPEG-SPA), and
N-hydroxysuccinimide-PEG (NHS-PEG) (see, e.g., U.S. Pat. No.
5,672,662). A dimeric EPO peptide containing such a preferred
linker may be structurally illustrated as shown in Formula IV.
##STR00004##
[0034] Generally, although not necessarily, peptide dimers will
also contain one or more intramolecular disulfide bonds between
cysteine residues of the peptide monomers. Preferably, the two
monomers contain at least one intramolecular disulfide bond. Most
preferably, both monomers of a peptide dimer contain an
intramolecular disulfide bond, such that each monomer contains a
cyclic group.
[0035] A peptide monomer or dimer may further comprise one or more
spacer moieties. Such spacer moieties may be attached to a peptide
monomer or to a peptide dimer. Preferably, such spacer moieties are
attached to the linker L.sub.K moiety that connects the monomers of
a peptide dimer. For example, such spacer moieties may be attached
to a peptide dimer via the carbonyl carbon of a lysine linker, or
via the nitrogen atom of an iminodiacetic acid linker. For example,
such a spacer may connect the linker of a peptide dimer to an
attached water soluble polymer moiety or a protecting group. In
another example, such a spacer may connect a peptide monomer to an
attached water soluble polymer moiety.
[0036] In one embodiment, the spacer moiety is a C.sub.1-12 linking
moiety optionally terminated with --NH-linkages or carboxyl
(--COOH) groups, and optionally substituted at one or more
available carbon atoms with a lower alkyl substituent. In one
embodiment, the spacer is R--COOH wherein R is a lower (C.sub.1-6)
alkylene optionally substituted with a functional group such as a
carboxyl group or an amino group that enables binding to another
molecular moiety. For example, the spacer may be a glycine (G)
residue, or an amino hexanoic acid. In preferred embodiments the
amino hexanoic acid is 6-amino hexanoic acid (Ahx). For example,
where the spacer 6-amino hexanoic acid (Ahx) is bound to the
N-terminus of a peptide, the peptide terminal amine group may be
linked to the carboxyl group of Ahx via a standard amide coupling.
In another example, where Ahx is bound to the C-terminus of a
peptide, the amine of Ahx may be linked to the carboxyl group of
the terminal amino acid via a standard amide coupling. The
structure of such a peptide may be depicted as shown in Formula V,
and summarized as shown in Formula VI.
##STR00005##
[0037] In other embodiments, the spacer is --NH--R--NH-- wherein R
is a lower (C.sub.1-6) alkylene substituted with a functional group
such as a carboxyl group or an amino group that enables binding to
another molecular moiety. For example, the spacer may be a lysine
(K) residue or a lysine amide (K--NH.sub.2, a lysine residue
wherein the carboxyl group has been converted to an amide moiety
--CONH.sub.2).
[0038] In preferred embodiments, the spacer moiety has the
following structure:
--NH--(CH.sub.2).sub..alpha.--[O--(CH.sub.2).sub..beta.].sub..gamma.--O.-
sub..delta.--(CH.sub.2).sub..epsilon.--Y--
where .alpha., .beta., .gamma., .delta., and .epsilon. are each
integers whose values are independently selected. In preferred
embodiments, .alpha., .beta., and .epsilon. are each integers whose
values are independently selected from one to about six, .delta. is
zero or one, .gamma. is an integer selected from zero to about ten,
except that when .gamma. is greater than one, .beta. is two, and Y
is selected from NH or CO. In particularly preferred embodiments
.alpha., .beta., and .epsilon. are each equal to two, both .gamma.
and .delta. are equal to 1, and Y is NH. For example, a peptide
dimer containing such a spacer is illustrated schematically in
Formula VII, where the linker is a lysine and the spacer joins the
linker to a Boc protecting group.
##STR00006##
In another particularly preferred embodiment .gamma. and .delta.
are zero, .alpha. and .epsilon. together equal five, and Y is
CO.
[0039] In particularly preferred embodiments, the linker plus
spacer moiety has the structure shown in Formula VIII or Formula
IX.
##STR00007##
[0040] The peptide monomers, dimers, or multimers of the invention
may further comprise one or more water soluble polymer moieties.
Preferably, these polymers are covalently attached to the peptide
compounds of the invention. Preferably, for therapeutic use of the
end-product preparation, the polymer will be pharmaceutically
acceptable. One skilled in the art will be able to select the
desired polymer based on such considerations as whether the
polymer-peptide conjugate will be used therapeutically, and if so,
the desired dosage, circulation time, resistance to proteolysis,
and other considerations. The water soluble polymer may be, for
example, polyethylene glycol (PEG), copolymers of ethylene
glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl
alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers),
poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol
homopolymers, polypropylene oxide/ethylene oxide copolymers, and
polyoxyethylated polyols. A preferred water soluble polymer is
PEG.
[0041] The polymer may be of any molecular weight, and may be
branched or unbranched. A preferred PEG for use in the present
invention comprises linear, unbranched PEG having a molecular
weight that is greater than 10 kilodaltons (kD) and is more
preferably between about 20 and 60 kD in molecular weight. Still
more preferably, the linear unbranched PEG moiety should have a
molecular weight of between about 20 and 40 kD, with 20 kD PEG
being particularly preferred. It is understood that in a given
preparation of PEG, the molecular weights will typically vary among
individual molecules. Some molecules will weight more, and some
less, than the stated molecular weight. Such variation is generally
reflect by use of the word "about" to describe molecular weights of
the PEG molecules.
[0042] The number of polymer molecules attached may vary; for
example, one, two, three, or more water soluble polymers may be
attached to an EPO-R agonist peptide of the invention. The multiple
attached polymers may be the same or different chemical moieties
(e.g., PEGs of different molecular weight). Thus, in a preferred
embodiment the invention contemplates EPO-R agonist peptides having
two or more PEG moieities attached thereto. Preferably, both of the
PEG moietieis are linear, unbranched PEG each preferably having a
molecular weight of between about 10 and about 60 kD. More
preferably, each linear unbranched PEG moiety has a molecular
weight that is between about 20 and 40 kD, and still more
preferably between about 20 and 30 kD with a molecular weight of
about 20 kD for each linear PEG moiety being particularly
preferred. However, other molecular weights for PEG are also
contemplated in such embodiments. For example, the invention
contemplates and encompasses EPO-R agonist peptides having two or
more linear unbranched PEG moieties attached thereto, at least one
or both of which has a molecular weight between about 20 and 40 kD
or between about 20 and 30 kD. In other embodiments the invention
contemplates and encompasses EPO-R agonist peptides having two or
more linear unbranched PEG moieties attached thereto, at least one
of which has a molecular weight between about 40 and 60 kD.
[0043] In one embodiment, PEG may serve as a linker that dimerizes
two peptide monomers. In one embodiment, PEG is attached to at
least one terminus (N-terminus or C-teiminus) of a peptide monomer
or dimer. In another embodiment, PEG is attached to a spacer moiety
of a peptide monomer or dimer. In a preferred embodiment PEG is
attached to the linker moiety of a peptide dimer. In a highly
preferred embodiment, PEG is attached to a spacer moiety, where
said spacer moiety is attached to the linker L.sub.K moiety that
connects the monomers of a peptide dimer. In particularly preferred
embodiments, PEG is attached to a spacer moiety, where said spacer
moiety is attached to a peptide dimer via the carbonyl carbon of a
lysine linker, or the amide nitrogen of a lysine amide linker.
[0044] Preferred peptide dimers of the present invention include,
but are not limited to:
TABLE-US-00002 Compound designation Peptide dimer AF33065
##STR00008## AF34602 ##STR00009## AF34395 ##STR00010## AF34601
##STR00011## AF32579 ##STR00012## AF33068 ##STR00013## AF33131
##STR00014## AF34351 ##STR00015## AF34350 ##STR00016## AF34753
##STR00017## AF34757 ##STR00018## AF35062 ##STR00019## AF35218
##STR00020## AF35462 ##STR00021## AF35464 ##STR00022## AF33197
##STR00023## AF34994 ##STR00024## AF35083 ##STR00025## AF35525
##STR00026## AF35526 ##STR00027## AF35563 ##STR00028## AF35575
##STR00029## AF35592 ##STR00030## AF35593 ##STR00031## AF35594
##STR00032## AF35219 ##STR00033## AF32876 ##STR00034## AF32881
##STR00035## AF35179 ##STR00036## AF35180 ##STR00037## AF35463
##STR00038## AF35090 ##STR00039## AF35148 ##STR00040## AF35149
##STR00041## AF35168 ##STR00042## AF35361 ##STR00043## AF35595
##STR00044## AF35564 ##STR00045##
Still other peptides of the present invention, including peptide
monomers and dimers, are as follows:
TABLE-US-00003 SEQ ID PEG NO: size 34 Ac G G L Y A C H M G P I T
1Nal V 35 [ Ac G G L Y A C H M G P I T 1Nal V 36 [ Ac G G L Y A C H
M G P I T 1Nal V 37 40 KDa [ Ac G G L Y A C H M G P I T 1Nal V 38
39 [ Ac G G L Y A C H M(O2 G P I T 1Nal V 40 40 KDa [ Ac G G L Y A
C H M(O2 G P I T 1Nal V 41 [ Ac G G L Y A C H M(O) G P I T 1Nal V
42 [ Ac G G L Y A C H M(O) G P I T 1Nal V 43 [ Ac G G L Y A C H MXX
G P I T 1Nal V 44 [ Ac G G L Y A C H MXX G P I T 1Nal V 45 [ Ac G G
L Y A C H MXX G P I T 1Nal V 46 [ ##STR00046## G L Y A C H M G P I
T 1Nal V 47 [ ##STR00047## G G L Y A C H M G P I T 1Nal V 48 [ Ac G
G L Y A C H M G P I T 1Nal V 49 [ Ac G G L Y A C H M(O) G P I T
1Nal V 50 [ Ac G G L Y A C H M G P O T 1Nal V 51 [ Ac S R T R Y R C
E M G P L T W V 52 [ Ac L T R L Y S C H M G P S T W V 53 [ Ac R G Q
L Y A C H F G P V T W V 54 [ Ac S G I L Y E C H M G P L T W V 55 [
Ac L G R R Y S C H F G A L T W V 56 [ Ac G S R T Y S C Q L G P V O
W V 57 Ac A R G R Y Q C Q F G P L T W E 58 Ac V T R M Y R C R M G P
L T W V 59 Ac R P S L Y E C H L G P L T W E 60 Ac R G H M Y S C Q L
G P V T W V 61 Ac I T P T Y H C R F G P Q T W V 62 Ac G N R M Y Q C
H M G P L T W V 63 [ Ac A R G R Y Q C Q F G P L T W E 64 [ Ac V T R
M Y R C R M G P L T W V 65 [ Ac R P S L Y E C H L G P L T W E 66 [
Ac R G H M Y S C Q L G P V T W V 67 [ Ac I T P T Y H C R F G P Q T
W V 68 [ Ac G N R M Y Q C H M G P L T W V 69 [ Ac R N H L Y Q C R M
G P L T W V 70 [ Ac P D L A Y S C R M G P L T W V 71 [ Ac L G R R Y
S C H F G P L T W V 72 [ Ac L L R G Y E C Y M G P L T W V 73 [ Ac M
R T R Y R C Y M G P L T W V 74 [ Ac H L G R Y D C S F G P Q T W V
75 [ Ac I R G R N R C R F G P Q T W V 76 Ac Q R R H V F L S D C A A
Y V G T W V 77 [ Ac V L P L Y R C R M G R E T W E 78 [ Ac P G N S Y
R C H M G P L T W V 79 Ac R N H L Y G C R M G P L T W V 80 Ac P D L
A Y S C R M G P L T W V 81 Ac L G R R Y S C H F G P L T W V 82 Ac L
L R G Y E C Y M G P L T W V 83 Ac M R T R Y R C Y M G P L T W V 84
Ac H L G R Y D C S F G P Q T W V 85 Ac I R G R N R C R F G P Q T W
V 86 Ac R P R P Y S C T M G P R T W V 87 Ac V L P L Y R C R M G R E
T W E 88 Ac P G N S Y R C M G P L T W V C 89 40 KDa [ Ac G G L Y A
C H M(x) G P I T 1Nal V 90 40 KDa [ Ac G G L Y A C H M(x) G P I T
1Nal V 91 [ Ac G G I Y A C H M G P I T 1Nal V 92 [ Ac G G L Y A C H
m G P I T 1Nal V 93 [ Ac G G L Y A C H M G P I T 1Nal V 94 [ Ac G G
L Y A C H M G P I T 1Nal V 95 [ Ac G G L Y A C H M G P I T 1Nal V
96 [ Ac G G L Y A C H M G P I T 1Nal V 97 [ Ac E Y L C R M G P I T
W V 98 [ Ac T Y S C H F G P L T W V 99 [ Ac D Y H C R M G P L T W V
100 [ Ac L Y E C R M G P M T W V 101 [ Ac L Y L C R M G P V T W E
102 [ Ac D Y N C R F G P L T W V 103 [ Ac S Y L C R R G P I T W L
104 [ Ac E Y S C R M G P I T W V 105 [ Ac L Y L C R F G P I T W D
106 [ Ac I Y R C L M G P I T W V 107 [ Ac G G L Y A C H M G P I T
1Nal V 108 40 KDa [ ##STR00048## G G L Y A C H M G P I T 1Nal V 109
40 [ G G L Y A C H M G P I T 1Nal V KDa 110 [ Ac G G L Y A C H M G
p I T 1Nal V 111 [ Ac G G I Y A C h M G P I T 1Nal V 112 [ Ac G G L
Y a C H M G P I T 1Nal V 113 [ Ac G G L Y A Cxxx H M G P I T 1Nal V
114 [ Ac G G L Y A C(Acm) H M G P I T 1Nal V 115 40 KDa [
##STR00049## G L Y A C H M G P I T 1Nal V 116 40 KDa [ Ac G G L Y A
C H M G P I T 1Nal V 117 [ Ac G G L Y A CSH H M G P I T 1Nal V 118
[ Ac G G L Y A C(Ase) H M G P I T 1Nal V 119 [ Ac R T R E Y S C Q M
G P L T W T 120 [ Ac S R A R Y M C H M G P L T W V 121 [ Ac G G R A
Y M C R L G P V T W V 122 [ Ac T I A Q Y I C Y M G P E T W E 123 [
Ac N G R T Y S C Q L G P V T W V 122 [ Ac T I A Q Y I C Y M G P E T
W E 123 [ Ac N G R T Y S C Q L G P V T W V 124 [ Ac M R T R Y R C Y
M G P L T W V 125 [ Ac S R T R Y R C E M G P L T W V 126 [ Ac G S R
T Y S C Q L G P V T W V 127 [ Ac R P R P Y S C T M G P R T W V 128
[ Ac G G T Y S C H F G P L T W V 129 [ Ac G G D Y H C R M G P L T W
V 130 [ Ac G G V Y A C R M G P I T W V 131 [ Ac G G L Y A C H M G P
I Y 1Nal V 132 [ Ac G G L Y A C H M G P I Y 1Nal V 133 [ Ac G G L Y
A C H M G P I T 1Nal V 134 [ Ac G G L Y A C H M G P I T 1Nal V 135
[ Ac G G L Y A C H M G P I t 1Nal V 136 [ Ac G G L y A C H M G P I
T 1Nal V 137 [ Ac G G L Y A C H M G P I T 1Nal V 138 [ Ac G G L Y A
C H M G P I T 1Nal v 139 [ Ac G G L Y A C H M G P I T 1Nal V 140 [
Ac G G G L Y A C H M G P I T 1Nal V 141 [ Ac G G L L Y A C H M G P
I T 1Nal V 142 [ Ac G G L Y Y A C H M G P I T 1Nal V 143 [ Ac G G L
Y A A C H M G P I T 1Nal V 144 [ Ac G G L Y A C H H M G P I T 1Nal
145 [ Ac G G L Y A C H M M G P I T 1Nal 146 [ Ac N Y T C R F G P L
T W E 147 [ Ac S W D C R I G P I T W V 148 [ Ac N Y M C H F G P I T
W V 149 [ Ac L Y L C R M G P Q T W M 150 [ Ac W Y S C L M G P M T W
V 151 [ Ac E Y F C R M G P I T W V 152 [ Ac G G L Y A C H M G G P I
T 1Nal 153 [ Ac G G L Y A C H M G P I I T 1Nal 154 [ Ac G G L Y A C
H M G P I T T 1Nal 155 [ Ac G G L Y A C H M G P I T 1Nal V 156 [ Ac
G G L Y A C H M G P I T 1Nal V 157 [ Ac G G L Y A C H M G P I T
1Nal V 158 [ Ac G G L Y A C H M G P I T 1Nal V 159 [ Ac G G L Y A C
H M G P I T 1Nal V 160 [ Ac G G L Y A C H M G P I T 1Nal V SEQ ID
NO: Linker 34 C Q P L R Sar K NH.sub.2 Monomer 35 C Q P L R Sar
##STR00050## NH.sub.2 ]2 IDA H2 Dimer 36 C Q P L R Sar ##STR00051##
NH.sub.2 ]2 IDA + Boc Boc on IDA 37 C Q P L R Sar ##STR00052##
NH.sub.2 ]2 IDA PEG 38 39 C Q P L R Sar ##STR00053## NH.sub.2 ]2
IDA Bis sulfone 40 C Q P L R Sar ##STR00054## NH.sub.2 ]2 IDA Bis
sulfone 41 C Q P L R Sar ##STR00055## NH.sub.2 ]2 IDA Bis
sulfoxide, asym- metric 42 C Q P L R Sar ##STR00056## NH.sub.2 ]2
IDA Bis sulfoxide, asym- metric 43 C Q P L R Sar ##STR00057##
NH.sub.2 ]2 IDA Mono sulfoxide, asym- metric 44 C Q P L R Sar
##STR00058## NH.sub.2 ]2 IDA Mono sulfoxide, asym- metric 45 C Q P
L R Sar ##STR00059## NH.sub.2 ]2 IDA Mono sulfone, asym- metric 46
C Q P L R Sar NH.sub.2 ]2 IDA N- terminal dimer 47 C Q P L R Sar
NH.sub.2 ]2 IDA N- terminal dimer 48 C Q P L R Sar ##STR00060##
NH.sub.2 ]2 IDA Parallel disulfide 49 C Q P L R Sar ##STR00061##
NH.sub.2 ]2 IDA Bis sulfoxide 50 C Q P L R Sar ##STR00062##
NH.sub.2 ]2 IDA Criss- cross disulfide 51 C R R W ##STR00063##
NH.sub.2 ]2 IDA + Boc 52 C S T A L R ##STR00064## NH.sub.2 ]2 IDA +
Boc 53 C R R R R R V ##STR00065## NH.sub.2 ]2 IDA + Boc 54 C T P S
R R R ##STR00066## NH.sub.2 ]2 IDA + Boc 55 E Q P A R R D
##STR00067## NH.sub.2 ]2 IDA + Boc 56 C G R R R ##STR00068##
NH.sub.2 ]2 IDA + Boc 57 C A P I R P R K NH.sub.2 Monomer 58 C E R
K NH.sub.2 Monomer 59 C R P R R R E K NH.sub.2 Monomer 60 C R P L S
G R K NH.sub.2 Monomer 61 C A P R R S A L T K NH.sub.2 Monomer 62 C
Q P T R I H K NH.sub.2 Monomer 63 C L P I R P R ##STR00069##
NH.sub.2 ]2 IDA + Boc 64 C E R ##STR00070## NH.sub.2 ]2 IDA + Boc
65 C R P R R R E ##STR00071## NH.sub.2 ]2 IDA + Boc 66 C R P L S G
R ##STR00072## NH.sub.2 ]2 IDA + Boc 67 c A P R R S A L T
##STR00073## NH.sub.2 ]2 IDA + Boc 68 C Q P T R I H ##STR00074##
NH.sub.2 ]2 IDA + Boc 69 C S S R G T Q ##STR00075## NH.sub.2 ]2 IDA
+ Boc 70 C A P N R ##STR00076## NH.sub.2 ]2 IDA + Boc 71 C Q P A R
R D ##STR00077## NH.sub.2 ]2 IDA + Boc 72 C R S S R P R
##STR00078## NH.sub.2 ]2 IDA + Boc 73 C E G S R L ##STR00079##
NH.sub.2 ]2 IDA + Boc 74 C R P R R S L ##STR00080## NH.sub.2 ]2 IDA
+ Boc 75 C P D S Y E R ##STR00081## NH.sub.2 ]2 IDA + Boc 76 E C D
D I S K NH.sub.2 Monomer 77 C M R A A G V T ##STR00082## NH.sub.2
]2 IDA + Boc 78 C G R D R H L ##STR00083## NH.sub.2 ]2 IDA + Boc 79
C S S R G T Q K NH.sub.2 Monomer 80 C A P N R K NH.sub.2 Monomer 81
C Q P A R R D K NH.sub.2 Monomer 82 C R S S R P R K NH.sub.2
Monomer 83 C E G S R L K NH.sub.2 Monomer 84 C R P R R S L K
NH.sub.2 Monomer 85 C P D S Y E F K NH.sub.2 Monomer 86 C G G V R A
G K NH.sub.2 Monomer 87 C M R A A G V T K NH.sub.2 Monomer 88 G R D
R H L K NH.sub.2 Monomer 89 C Q P L R Sar ##STR00084## NH.sub.2 ]2
IDA Mono- sulfoxide 90 C Q P L R Sar ##STR00085## NH.sub.2 ]2 IDA
Mono- sulfoxide
91 C Q P L R Sar ##STR00086## NH.sub.2 ]2 IDA D Leu 92 C Q P L R
Sar ##STR00087## NH.sub.2 ]2 IDA D Met 93 c Q P L R Sar
##STR00088## NH.sub.2 ]2 IDA 2 D cys 94 C Q P L R Sar ##STR00089##
NH.sub.2 ]2 IDA D Cys 95 c Q P L R Sar ##STR00090## NH.sub.2 ]2 IDA
D Cys 96 C Q P L R Sar ##STR00091## NH.sub.2 ]2 IDA D Pro 97 C E R
Y ##STR00092## NH.sub.2 ]2 IDA + Boc 98 C R P Q ##STR00093##
NH.sub.2 ]2 IDA + Boc 99 C R P L ##STR00094## NH.sub.2 ]2 IDA + Boc
100 C R P G ##STR00095## NH.sub.2 ]2 IDA + Boc 101 C Q P R
##STR00096## NH.sub.2 ]2 IDA + Boc 102 C R P S ##STR00097##
NH.sub.2 ]2 IDA + Boc 103 C T A Q ##STR00098## NH.sub.2 ]2 IDA 104
C S P T ##STR00099## NH.sub.2 ]2 IDA 105 C G Y ##STR00100##
NH.sub.2 ]2 IDA 106 C T P D ##STR00101## NH.sub.2 ]2 IDA 107 C Q P
L R Sar ##STR00102## NH.sub.2 ]2 IDA D Leu 108 C Q P L R Sar
NH.sub.2 ]2 IDA N-termi- nal dimer 109 C Q P L R Sar NH.sub.2 ]2
IDA Parallel disulfide 110 C Q P L R Sar ##STR00103## NH.sub.2 ]2
IDA D Pro 111 C Q P L R Sar ##STR00104## NH.sub.2 ]2 IDA D His 112
C Q P L R Sar ##STR00105## NH.sub.2 ]2 IDA D Ala 113 Cxx Q P L R
Sar ##STR00106## NH.sub.2 ]2 IDA 1/2 Criss- cross disulfide 114
C(Acm) Q P L R Sar ##STR00107## NH.sub.2 ]2 IDA 1/2 Parallel
disulfide 115 C Q P L R Sar NH.sub.2 ]2 IDA N-Termi- nal dimer 116
C Q P L R Sar ##STR00108## NH.sub.2 ]2 IDA Criss- cross disulfide
117 CSH Q P L R Sar ##STR00109## NH.sub.2 ]2 IDA Reduced disulfide
118 C(Ace) Q P L R Sar ##STR00110## NH.sub.2 ]2 IDA Capped
Cysteine, no SS 119 C V P R S ##STR00111## NH.sub.2 ]2 IDA + Boc
120 C R P E V ##STR00112## NH.sub.2 ]2 IDA + Boc 121 C S P R I R I
##STR00113## NH.sub.2 ]2 IDA + Boc 122 C R P S P R A ##STR00114##
NH.sub.2 ]2 IDA + Boc 123 C S P G V R R ##STR00115## NH.sub.2 ]2
IDA + Boc 122 C R P S P R A ##STR00116## NH.sub.2 ]2 IDA + Boc 123
C S P G V R R ##STR00117## NH.sub.2 ]2 IDA + Boc 124 C E G S R L
##STR00118## NH.sub.2 ]2 IDA + Boc 125 C E R W ##STR00119##
NH.sub.2 ]2 IDA + Boc 126 C G R R R ##STR00120## NH.sub.2 ]2 IDA +
Boc 127 C G G V R A G ##STR00121## NH.sub.2 ]2 IDA + Boc 128 C R P
Q G G ##STR00122## NH.sub.2 ]2 IDA + Boc 129 C R P L G G
##STR00123## NH.sub.2 ]2 IDA + Boc 130 C S P L G G ##STR00124##
NH.sub.2 ]2 IDA 131 C E P L R Sar ##STR00125## NH.sub.2 ]2 IDA
Deami- dated 132 C E P L R Sar ##STR00126## OH ]2 IDA Bis- Deami-
dated 133 C Q P L R Sar k NH2 ]2 IDA D Lys 134 C Q P L R Sar
##STR00127## NH.sub.2 ]2 IDA D Thr 135 C q P L R Sar ##STR00128##
NH.sub.2 ]2 IDA D Gln 136 C Q P L R Sar ##STR00129## NH.sub.2 ]2
IDA D Tyr 137 C Q P L R Sar ##STR00130## NH.sub.2 ]2 IDA D Ile 138
C Q P L R Sar ##STR00131## NH.sub.2 ]2 IDA D Val 139 C Q P L R Sar
##STR00132## NH.sub.2 ]2 IDA D 1Nal 140 C Q P L R Sar ##STR00133##
NH.sub.2 ]2 IDA Gly Insertion 141 C Q P L R Sar ##STR00134##
NH.sub.2 ]2 IDA Leu Insertion 142 C Q P L R Sar ##STR00135##
NH.sub.2 ]2 IDA Tyr Insertion 143 C Q P L R Sar ##STR00136##
NH.sub.2 ]2 IDA Ala Insertion 144 V C Q P L R Sar ##STR00137##
NH.sub.2 ]2 IDA His Insertion 145 V C Q P L R Sar ##STR00138##
NH.sub.2 ]2 IDA Met Insertion 146 C T P Q ##STR00139## NH.sub.2 ]2
IDA - Boc 147 C R W S ##STR00140## NH.sub.2 ]2 IDA - Boc 148 C R P
G ##STR00141## NH.sub.2 ]2 IDA - Boc 149 C Q P G ##STR00142##
NH.sub.2 ]2 IDA - Boc 150 C R A H ##STR00143## NH.sub.2 ]2 IDA -
Boc 151 C Q R S ##STR00144## NH.sub.2 ]2 IDA - Boc 152 V C Q P L R
Sar ##STR00145## NH.sub.2 ]2 IDA Gly Insertion 153 V C Q P L R Sar
##STR00146## NH.sub.2 ]2 IDA Ile Insertion 154 V C Q P L R Sar
##STR00147## NH.sub.2 ]2 IDA Thr Insertion 155 V C Q P L R Sar
##STR00148## NH.sub.2 ]2 IDA Val Insertion 156 C Q Q P L R Sar
##STR00149## NH.sub.2 ]2 IDA Gln Insertion 157 C Q P P L R Sar
##STR00150## NH.sub.2 ]2 IDA Pro Insertion 158 C Q P L L R Sar
##STR00151## NH.sub.2 ]2 IDA Leu Insertion 159 C Q P L R R Sar
##STR00152## NH.sub.2 ]2 IDA Arg Insertion 160 C Q P L R Sar Sar
##STR00153## NH.sub.2 ]2 IDA Sar Insertion Branching amino acid to
linker shown shaded IDA is imino diacetic acid (BetaAfa
conjugate)
[0045] The peptide sequences of the present invention can be
present alone or in conjunction with N-terminal and/or C-terminal
extensions of the peptide chain. Such extensions may be naturally
encoded peptide sequences optionally with or substantially without
non-naturally occurring sequences; the extensions may include any
additions, deletions, point mutations, or other sequence
modifications or combinations as desired by those skilled in the
art. For example and not limitation, naturally-occurring sequences
may be full-length or partial length and may include amino acid
substitutions to provide a site for attachment of carbohydrate,
PEG, other polymer, or the like via side chain conjugation. In a
variation, the amino acid substitution results in humanization of a
sequence to make in compatible with the human immune system. Fusion
proteins of all types are provided, including immunoglobulin
sequences adjacent to or in near proximity to the EPO-R activating
sequences of the present invention with or without a
non-immunoglobulin spacer sequence. One type of embodiment is an
immunoglobulin chain having the EPO-R activating sequence in place
of the variable (V) region of the heavy and/or light chain.
Preparation of the Peptide Compounds of the Invention:
Peptide Synthesis
[0046] The peptides of the invention may be prepared by classical
methods known in the art. These standard methods include exclusive
solid phase synthesis, partial solid phase synthesis methods,
fragment condensation, classical solution synthesis, and
recombinant DNA technology [See, e.g., Merrifield J. Am. Chem. Soc.
1963 85:2149].
[0047] In one embodiment, the peptide monomers of a peptide dimer
are synthesized individually and dimerized subsequent to synthesis.
In preferred embodiments the peptide monomers of a dimer have the
same amino acid sequence.
[0048] In particularly preferred embodiments, the peptide monomers
of a dimer are linked via their C-termini by a linker L.sub.K
moiety having two functional groups capable of serving as
initiation sites for peptide synthesis and a third functional group
(e.g., a carboxyl group or an amino group) that enables binding to
another molecular moiety (e.g., as may be present on the surface of
a solid support). In this case, the two peptide monomers may be
synthesized directly onto two reactive nitrogen groups of the
linker L.sub.K moiety in a variation of the solid phase synthesis
technique. Such synthesis may be sequential or simultaneous.
[0049] Where sequential synthesis of the peptide chains of a dimer
onto a linker is to be performed, two amine functional groups on
the linker molecule are protected with two different orthogonally
removable amine protecting groups. In preferred embodiments, the
protected diamine is a protected lysine. The protected linker is
coupled to a solid support via the linker's third functional group.
The first amine protecting group is removed, and the first peptide
of the dimer is synthesized on the first deprotected amine moiety.
Then the second amine protecting group is removed, and the second
peptide of the dimer is synthesized on the second deprotected amine
moiety. For example, the first amino moiety of the linker may be
protected with Alloc, and the second with Fmoc. In this case, the
Fmoc group (but not the Alloc group) may be removed by treatment
with a mild base [e.g., 20% piperidine in dimethyl formamide
(DMF)], and the first peptide chain synthesized. Thereafter the
Alloc group may be removed with a suitable reagent [e.g.,
Pd(PPh.sub.3)/4-methyl morpholine and chloroform], and the second
peptide chain synthesized. This technique may be used to generate
dimers wherein the sequences of the two peptide chains are
identical or different. Note that where different thiol-protecting
groups for cysteine are to be used to control disulfide bond
formation (as discussed below) this technique must be used even
where the final amino acid sequences of the peptide chains of a
dimer are identical.
[0050] Where simultaneous synthesis of the peptide chains of a
dimer onto a linker is to be performed, two amine functional groups
of the linker molecule are protected with the same removable amine
protecting group. In preferred embodiments, the protected diamine
is a protected lysine. The protected linker is coupled to a solid
support via the linker's third functional group. In this case the
two protected functional groups of the linker molecule are
simultaneously deprotected, and the two peptide chains
simultaneously synthesized on the deprotected amines. Note that
using this technique, the sequences of the peptide chains of the
dimer will be identical, and the thiol-protecting groups for the
cysteine residues are all the same.
[0051] A preferred method for peptide synthesis is solid phase
synthesis. Solid phase peptide synthesis procedures are well-known
in the art [see, e.g., Stewart Solid Phase Peptide Syntheses
(Freeman and Co.: San Francisco) 1969; 2002/2003 General Catalog
from Novabiochem Corp, San Diego, USA; Goodman Synthesis of
Peptides and Peptidomimetics (Houben-Weyl, Stuttgart) 2002]. In
solid phase synthesis, synthesis is typically commenced from the
C-terminal end of the peptide using an .alpha.-amino protected
resin. A suitable starting material can be prepared, for instance,
by attaching the required .alpha.-amino acid to a chloromethylated
resin, a hydroxymethyl resin, a polystyrene resin, a
benzhydrylamine resin, or the like. One such chloromethylated resin
is sold under the trade name BIO-BEADS SX-1 by Bio Rad Laboratories
(Richmond, Calif.). The preparation of the hydroxymethyl resin has
been described [Bodonszky, et al. (1966) Chem. Ind. London
38:1597]. The benzhydrylamine (BHA) resin has been described
[Pietta and Marshall (1970) Chem. Commun. 650], and the
hydrochloride form is commercially available from Beckman
Instruments, Inc. (Palo Alto, Calif.). For example, an
.alpha.-amino protected amino acid may be coupled to a
chloromethylated resin with the aid of a cesium bicarbonate
catalyst, according to the method described by Gisin (1973) Helv.
Chim. Acta 56:1467.
[0052] After initial coupling, the .alpha.-amino protecting group
is removed, for example, using trifluoroacetic acid (TFA) or
hydrochloric acid (HCl) solutions in organic solvents at room
temperature. Thereafter, .alpha.-amino protected amino acids are
successively coupled to a growing support-bound peptide chain. The
.alpha.-amino protecting groups are those known to be useful in the
art of stepwise synthesis of peptides, including: acyl-type
protecting groups (e.g., formyl, trifluoroacetyl, acetyl), aromatic
urethane-type protecting groups [e.g., benzyloxycarboyl (Cbz) and
substituted Cbz], aliphatic urethane protecting groups [e.g.,
t-butyloxycarbonyl (Boc), isopropyloxycarbonyl,
cyclohexyloxycarbonyl], and alkyl type protecting groups (e.g.,
benzyl, triphenylmethyl), fluorenylmethyl oxycarbonyl (Fmoc),
allyloxycarbonyl (Alloc), and
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde).
[0053] The side chain protecting groups (typically ethers, esters,
trityl, PMC, and the like) remain intact during coupling and is not
split off during the deprotection of the amino-terminus protecting
group or during coupling. The side chain protecting group must be
removable upon the completion of the synthesis of the final peptide
and under reaction conditions that will not alter the target
peptide. The side chain protecting groups for Tyr include
tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z--Br--Cbz, and
2,5-dichlorobenzyl. The side chain protecting groups for Asp
include benzyl, 2,6-dichlorobenzyl, methyl, ethyl, and cyclohexyl.
The side chain protecting groups for Thr and Ser include acetyl,
benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and
Cbz. The side chain protecting groups for Arg include nitro, Tosyl
(Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts),
2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf),
4-mthoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr), or Boc. The side
chain protecting groups for Lys include Cbz,
2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl
(2-Br--Cbz), Tos, or Boc.
[0054] After removal of the .alpha.-amino protecting group, the
remaining protected amino acids are coupled stepwise in the desired
order. Each protected amino acid is generally reacted in about a
3-fold excess using an appropriate carboxyl group activator such as
2-(1H-benzotriazol-1-yl)-1,1,3,3 tetramethyluronium
hexafluorophosphate (HBTU) or dicyclohexylcarbodimide (DCC) in
solution, for example, in methylene chloride (CH.sub.2Cl.sub.2),
N-methylpyrrolidone, dimethyl formamide (DMF), or mixtures
thereof.
[0055] After the desired amino acid sequence has been completed,
the desired peptide is decoupled from the resin support by
treatment with a reagent, such as trifluoroacetic acid (TFA) or
hydrogen fluoride (HF), which not only cleaves the peptide from the
resin, but also cleaves all remaining side chain protecting groups.
When a chloromethylated resin is used, hydrogen fluoride treatment
results in the formation of the free peptide acids. When the
benzhydrylamine resin is used, hydrogen fluoride treatment results
directly in the free peptide amide. Alternatively, when the
chloromethylated resin is employed, the side chain protected
peptide can be decoupled by treatment of the peptide resin with
ammonia to give the desired side chain protected amide or with an
alkylamine to give a side chain protected alkylamide or
dialkylamide. Side chain protection is then removed in the usual
fashion by treatment with hydrogen fluoride to give the free
amides, alkylamides, or dialkylamides. In preparing the esters of
the invention, the resins used to prepare the peptide acids are
employed, and the side chain protected peptide is cleaved with base
and the appropriate alcohol (e.g., methanol). Side chain protecting
groups are then removed in the usual fashion by treatment with
hydrogen fluoride to obtain the desired ester.
[0056] These procedures can also be used to synthesize peptides in
which amino acids other than the 20 naturally occurring,
genetically encoded amino acids are substituted at one, two, or
more positions of any of the compounds of the invention. Synthetic
amino acids that can be substituted into the peptides of the
present invention include, but are not limited to, N-methyl,
L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, .delta. amino acids
such as L-.delta.-hydroxylysyl and D-.delta.-methylalanyl,
L-.alpha.-methylalanyl, .beta. amino acids, and isoquinolyl.
D-amino acids and non-naturally occurring synthetic amino acids can
also be incorporated into the peptides of the present
invention.
Peptide Modifications
[0057] One can also modify the amino and/or carboxy termini of the
peptide compounds of the invention to produce other compounds of
the invention. Amino terminus modifications include methylation
(e.g., --NHCH.sub.3 or --N(CH.sub.3).sub.2), acetylation (e.g.,
with acetic acid or a halogenated derivative thereof such as
.alpha.-chloroacetic acid, .alpha.-bromoacetic acid, or
.alpha.-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group,
or blocking the amino terminus with any blocking group containing a
carboxylate functionality defined by RCOO-- or sulfonyl
functionality defined by R--SO.sub.2--, where R is selected from
alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar
groups. One can also incorporate a desamino acid at the N-terminus
(so that there is no N-terminal amino group) to decrease
susceptibility to proteases or to restrict the conformation of the
peptide compound. In preferred embodiments, the N-terminus is
acetylated. In particularly preferred embodiments an N-terminal
glycine is acetylated to yield N-acetylglycine (AcG).
[0058] Carboxy terminus modifications include replacing the free
acid with a carboxamide group or forming a cyclic lactam at the
carboxy terminus to introduce structural constraints. One can also
cyclize the peptides of the invention, or incorporate a desamino or
descarboxy residue at the termini of the peptide, so that there is
no terminal amino or carboxyl group, to decrease susceptibility to
proteases or to restrict the conformation of the peptide.
C-terminal functional groups of the compounds of the present
invention include amide, amide lower alkyl, amide di(lower alkyl),
lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives
thereof, and the pharmaceutically acceptable salts thereof.
[0059] One can replace the naturally occurring side chains of the
20 genetically encoded amino acids (or the stereoisomeric D amino
acids) with other side chains, for instance with groups such as
alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide,
amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,
carboxy and the lower ester derivatives thereof, and with 4-, 5-,
6-, to 7-membered heterocyclic. In particular, proline analogues in
which the ring size of the proline residue is changed from 5
members to 4, 6, or 7 members can be employed. Cyclic groups can be
saturated or unsaturated, and if unsaturated, can be aromatic or
non-aromatic. Heterocyclic groups preferably contain one or more
nitrogen, oxygen, and/or sulfur heteroatoms. Examples of such
groups include the furazanyl, furyl, imidazolidinyl, imidazolyl,
imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g.
morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl
(e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,
pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,
thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g.,
thiomorpholino), and triazolyl. These heterocyclic groups can be
substituted or unsubstituted. Where a group is substituted, the
substituent can be alkyl, alkoxy, halogen, oxygen, or substituted
or unsubstituted phenyl.
[0060] One can also readily modify peptides by phosphorylation, and
other methods [e.g., as described in Hruby, et al. (1990) Biochem
J. 268:249-262].
[0061] The peptide compounds of the invention also serve as
structural models for non-peptidic compounds with similar
biological activity. Those of skill in the art recognize that a
variety of techniques are available for constructing compounds with
the same or similar desired biological activity as the lead peptide
compound, but with more favorable activity than the lead with
respect to solubility, stability, and susceptibility to hydrolysis
and proteolysis [See, Morgan and Gainor (1989) Ann. Rep. Med. Chem.
24:243-252]. These techniques include replacing the peptide
backbone with a backbone composed of phosphonates, amidates,
carbamates, sulfonamides, secondary amines, and N-methylamino
acids.
Formation of Disulfide Bonds
[0062] The compounds of the present invention may contain one or
more intramolecular disulfide bonds. In one embodiment, embodiment,
a peptide monomer or dimer comprises at least one intramolecular
disulfide bond. In preferred embodiments, a peptide dimer comprises
two intramolecular disulfide bonds.
[0063] Such disulfide bonds may be formed by oxidation of the
cysteine residues of the peptide core sequence. In one embodiment
the control of cysteine bond formation is exercised by choosing an
oxidizing agent of the type and concentration effective to optimize
formation of the desired isomer. For example, oxidation of a
peptide dimer to form two intramolecular disulfide bonds (one on
each peptide chain) is preferentially achieved (over formation of
intermolecular disulfide bonds) when the oxidizing agent is
DMSO.
[0064] In preferred embodiments, the formation of cysteine bonds is
controlled by the selective use of thiol-protecting groups during
peptide synthesis. For example, where a dimer with two
intramolecular disulfide bonds is desired, the first monomer
peptide chain is synthesized with the two cysteine residues of the
core sequence protected with a first thiol protecting group [e.g.,
trityl(Trt), allyloxycarbonyl (Alloc), and
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) or the
like], then the second monomer peptide is synthesized the two
cysteine residues of the core sequence protected with a second
thiol protecting group different from the first thiol protecting
group [e.g., acetamidomethyl (Acm), t-butyl (tBu), or the like].
Thereafter, the first thiol protecting groups are removed effecting
bisulfide cyclization of the first monomer, and then the second
thiol protecting groups are removed effecting bisulfide cyclization
of the second monomer.
[0065] Other embodiments of this invention provide for analogues of
these disulfide derivatives in which one of the sulfurs has been
replaced by a CH.sub.2 group or other isotere for sulfur. These
analogues can be prepared from the compounds of the present
invention, wherein each core sequence contains at least one C or
homocysteine residue and an .alpha.-amino-.gamma.-butyric acid in
place of the second C residue, via an intramolecular or
intermolecular displacement, using methods known in the art [See,
e.g., Barker, et al. (1992) J. Med. Chem. 35:2040-2048 and Or, et
al. (1991) J. Org. Chem. 56:3146-3149]. One of skill in the art
will readily appreciate that this displacement can also occur using
other homologs of .alpha.-amino-.gamma.-butyric acid and
homocysteine.
[0066] In addition to the foregoing cyclization strategies, other
non-disulfide peptide cyclization strategies can be employed. Such
alternative cyclization strategies include, for example,
amide-cyclization strategies as well as those involving the
formation of thio-ether bonds. Thus, the compounds of the present
invention can exist in a cyclized form with either an
intramolecular amide bond or an intramolecular thio-ether bond. For
example, a peptide may be synthesized wherein one cysteine of the
core sequence is replaced with lysine and the second cysteine is
replaced with glutamic acid. Thereafter a cyclic monomer may be
formed through an amide bond between the side chains of these two
residues. Alternatively, a peptide may be synthesized wherein one
cysteine of the core sequence is replaced with lysine. A cyclic
monomer may then be formed through a thio-ether linkage between the
side chains of the lysine residue and the second cysteine residue
of the core sequence. As such, in addition to disulfide cyclization
strategies, amide-cyclization strategies and thio-ether cyclization
strategies can both be readily used to cyclize the compounds of the
present invention. Alternatively, the amino-terminus of the peptide
can be capped with an .alpha.-substituted acetic acid, wherein the
.alpha.-substituent is a leaving group, such as an
.alpha.-haloacetic acid, for example, .alpha.-chloroacetic acid,
.alpha.-bromoacetic acid, or .alpha.-iodoacetic acid.
Addition of Linkers
[0067] In embodiments where a peptide dimer is dimerized by a
linker L.sub.K moiety, said linker may be incorporated into the
peptide during peptide synthesis. For example, where a linker
L.sub.K moiety contains two functional groups capable of serving as
initiation sites for peptide synthesis and a third functional group
(e.g., a carboxyl group or an amino group) that enables binding to
another molecular moiety, the linker may be conjugated to a solid
support. Thereafter, two peptide monomers may be synthesized
directly onto the two reactive nitrogen groups of the linker
L.sub.K moiety in a variation of the solid phase synthesis
technique.
[0068] In alternate embodiments where a peptide dimer is dimerized
by a linker L.sub.K moiety, said linker may be conjugated to the
two peptide monomers of a peptide dimer after peptide synthesis.
Such conjugation may be achieved by methods well established in the
art. In one embodiment, the linker contains at least two functional
groups suitable for attachment to the target functional groups of
the synthesized peptide monomers. For example, a linker with two
free amine groups may be reacted with the C-terminal carboxyl
groups of each of two peptide monomers. In another example, linkers
containing two carboxyl groups, either preactivated or in the
presence of a suitable coupling reagent, may be reacted with the
N-terminal or side chain amine groups, or C-terminal lysine amides,
of each of two peptide monomers.
Addition of Spacers
[0069] In embodiments where the peptide compounds contain a spacer
moiety, said spacer may be incorporated into the peptide during
peptide synthesis. For example, where a spacer contains a free
amino group and a second functional group (e.g., a carboxyl group
or an amino group) that enables binding to another molecular
moiety, the spacer may be conjugated to the solid support.
Thereafter, the peptide may be synthesized directly onto the
spacer's free amino group by standard solid phase techniques.
[0070] In a preferred embodiment, a spacer containing two
functional groups is first coupled to the solid support via a first
functional group. Next a linker L.sub.K moiety having two
functional groups capable of serving as initiation sites for
peptide synthesis and a third functional group (e.g., a carboxyl
group or an amino group) that enables binding to another molecular
moiety is conjugated to the spacer via the spacer's second
functional group and the linker's third functional group.
Thereafter, two peptide monomers may be synthesized directly onto
the two reactive nitrogen groups of the linker L.sub.K moiety in a
variation of the solid phase synthesis technique. For example, a
solid support coupled spacer with a free amine group may be reacted
with a lysine linker via the linker's free carboxyl group.
[0071] In alternate embodiments where the peptide compounds contain
a spacer moiety, said spacer may be conjugated to the peptide after
peptide synthesis. Such conjugation may be achieved by methods well
established in the art. In one embodiment, the linker contains at
least one functional group suitable for attachment to the target
functional group of the synthesized peptide. For example, a spacer
with a free amine group may be reacted with a peptide's C-terminal
carboxyl group. In another example, a linker with a free carboxyl
group may be reacted with the free amine group of a peptide's
N-terminus or of a lysine residue. In yet another example, a spacer
containing a free sulfhydryl group may be conjugated to a cysteine
residue of a peptide by oxidation to form a disulfide bond.
Attachment of Water Soluble Polymers
[0072] In recent years, water-soluble polymers, such as
polyethylene glycol (PEG), have been used for the covalent
modification of peptides of therapeutic and diagnostic importance.
Attachment of such polymers is thought to enhance biological
activity, prolong blood circulation time, reduce immunogenicity,
increase aqueous solubility, and enhance resistance to protease
digestion. For example, covalent attachment of PEG to therapeutic
polypeptides such as interleukins [Knauf, et al. (1988) J. Biol.
Chem. 263; 15064; Tsutsumi, et al. (1995) J. Controlled Release
33:447), interferons (Kita, et al. (1990) Drug Des. Delivery
6:157), catalase (Abuchowski, et al. (1977) J. Biol. Chem.
252:582), superoxide dismutase (Beauchamp, et al. (1983) Anal.
Biochem. 131:25), and adenosine deaminase (Chen, et al. (1981)
Biochim. Biophy. Acta 660:293), has been reported to extend their
half life in vivo, and/or reduce their immunogenicity and
antigenicity.
[0073] The peptide compounds of the invention may further comprise
one or more water soluble polymer moieties. Preferably, these
polymers are covalently attached to the peptide compounds. The
water soluble polymer may be, for example, polyethylene glycol
(PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
polypropylene oxide/ethylene oxide copolymers, and polyoxyethylated
polyols. A preferred water soluble polymer is PEG.
[0074] Peptides, peptide dimers and other peptide-based molecules
of the invention can be attached to water-soluble polymers (e.g.,
PEG) using any of a variety of chemistries to link the
water-soluble polymer(s) to the receptor-binding portion of the
molecule (e.g., peptide+spacer). A typical embodiment employs a
single attachment junction for covalent attachment of the water
soluble polymer(s) to the receptor-binding portion, however in
alternative embodiments multiple attachment junctions may be used,
including further variations wherein different species of
water-soluble polymer are attached to the receptor-binding portion
at distinct attachment junctions, which may include covalent
attachment junction(s) to the spacer and/or to one or both peptide
chains. In some embodiments, the dimer or higher order multimer
will comprise distinct species of peptide chain (i.e., a
heterodimer or other heteromultimer). By way of example and not
limitation, a dimer may comprise a first peptide chain having a PEG
attachment junction and the second peptide chain may either lack a
PEG attachment junction or utilize a different linkage chemistry
than the first peptide chain and in some variations the spacer may
contain or lack a PEG attachment junction and said spacer, if
PEGylated, may utilize a linkage chemistry different than that of
the first and/or second peptide chains. An alternative embodiment
employs a PEG attached to the spacer portion of the
receptor-binding portion and a different water-soluble polymer
(e.g., a carbohydrate) conjugated to a side chain of one of the
amino acids of the peptide portion of the molecule.
[0075] A wide variety of polyethylene glycol (PEG) species may be
used for PEGylation of the receptor-binding portion
(peptides+spacer). Substantially any suitable reactive PEG reagent
can be used. In preferred embodiments, the reactive PEG reagent
will result in formation of a carbamate or amide bond upon
conjugation to the receptor-binding portion. Suitable reactive PEG
species include, but are not limited to, those which are available
for sale in the Drug Delivery Systems catalog (2003) of NOF
Corporation (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome,
Shibuya-ku, Tokyo 150-6019) and the Molecular Engineering catalog
(2003) of Nektar Therapeutics (490 Discovery Drive, Huntsville,
Ala. 35806). For example and not limitation, the following PEG
reagents are often preferred in various embodiments: mPEG2-NHS,
mPEG2-ALD, multi-Arm PEG, mPEG(MAL).sub.2, mPEG2(MAL), mPEG-NH2,
mPEG-SPA, mPEG-SBA, mPEG-thioesters, mPEG-Double Esters, mPEG-BTC,
mPEG-ButyrALD, mPEG-ACET, heterofunctional PEGs (NH2-PEG-COOH,
Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-VS, NHS-PEG-MAL), PEG acrylates
(ACRL-PEG-NHS), PEG-phospholipids (e.g., mPEG-DSPE), multiarmed
PEGs of the SUNBRITE series including the GL series of
glycerine-based PEGs activated by a chemistry chosen by those
skilled in the art, any of the SUNBRITE activated PEGs (including
but not limited to carboxyl-PEGs, p-NP-PEGs, Tresyl-PEGs, aldehyde
PEGs, acetal-PEGs, amino-PEGs, thiol-PEGs, maleimido-PEGs,
hydroxyl-PEG-amine, amino-PEG-COOH, hydroxyl-PEG-aldehyde,
carboxylic anhydride type-PEG, functionalized PEG-phospholipid, and
other similar and/or suitable reactive PEGs as selected by those
skilled in the art for their particular application and usage.
[0076] The polymer may be of any molecular weight, and may be
branched or unbranched. A preferred PEG for use in the present
invention comprises linear, unbranched PEG having a molecular
weight of from about 20 kilodaltons (kD) to about 40 kD (the term
"about" indicating that in preparations of PEG, some molecules will
weigh more, some less, than the stated molecular weight). Most
preferably, the PEG has a molecular weight of from about 30 kD to
about 40 kD. Other sizes may be used, depending on the desired
therapeutic profile (e.g., duration of sustained release desired;
effects, if any, on biological activity; ease in handling; degree
or lack of antigenicity; and other known effects of PEG on a
therapeutic peptide).
[0077] The number of polymer molecules attached may vary; for
example, one, two, three, or more water soluble polymers may be
attached to an EPO-R agonist peptide of the invention. The multiple
attached polymers may be the same or different chemical moieties
(e.g., PEGs of different molecular weight). In some cases, the
degree of polymer attachment (the number of polymer moieties
attached to a peptide and/or the total number of peptides to which
a polymer is attached) may be influenced by the proportion of
polymer molecules versus peptide molecules in an attachment
reaction, as well as by the total concentration of each in the
reaction mixture. In general, the optimum polymer versus peptide
ratio (in terms of reaction efficiency to provide for no excess
unreacted peptides and/or polymer moieties) will be determined by
factors such as the desired degree of polymer attachment (e.g.,
mono, di-, tri-, etc.), the molecular weight of the polymer
selected, whether the polymer is branched or unbranched, and the
reaction conditions for a particular attachment method.
[0078] In preferred embodiments, the covalently attached water
soluble polymer is PEG. For illustrative purposes, examples of
methods for covalent attachment of PEG (PEGylation) are described
below. These illustrative descriptions are not intended to be
limiting. One of ordinary skill in the art will appreciate that a
variety of methods for covalent attachment of a broad range of
water soluble polymers is well established in the art. As such,
peptide compounds to which any of a number of water soluble
polymers known in the art have been attached by any of a number of
attachment methods known in the art are encompassed by the present
invention.
[0079] In one embodiment, PEG may serve as a linker that dimerizes
two peptide monomers. In one embodiment, PEG is attached to at
least one terminus (N-terminus or C-terminus) of a peptide monomer
or dimer. In another embodiment, PEG is attached to a spacer moiety
of a peptide monomer or dimer. In a preferred embodiment PEG is
attached to the linker moiety of a peptide dimer. In a highly
preferred embodiment, PEG is attached to a spacer moiety, where
said spacer moiety is attached to the linker L.sub.K moiety that
connects the monomers of a peptide dimer. Most preferably, PEG is
attached to a spacer moiety, where said spacer moiety is attached
to a peptide dimer via the carbonyl carbon of a lysine linker, or
the amide nitrogen of a lysine amide linker.
[0080] There are a number of PEG attachment methods available to
those skilled in the art [see, e.g., Goodson, et al. (1990)
Bio/Technology 8:343 (PEGylation of interleukin-2 at its
glycosylation site after site-directed mutagenesis); EP 0 401 384
(coupling PEG to G-CSF); Malik, et al., (1992) Exp. Hematol.
20:1028-1035 (PEGylation of GM-CSF using tresyl chloride); PCT Pub.
No. WO 90/12874 (PEGylation of erythropoietin containing a
recombinantly introduced cysteine residue using a cysteine-specific
mPEG derivative); U.S. Pat. No. 5,757,078 (PEGylation of EPO
peptides); and U.S. Pat. No. 6,077,939 (PEGylation of an N-terminal
.alpha.-carbon of a peptide)].
[0081] For example, PEG may be covalently bound to amino acid
residues via a reactive group. Reactive groups are those to which
an activated PEG molecule may be bound (e.g., a free amino or
carboxyl group). For example, N-terminal amino acid residues and
lysine (K) residues have a free amino group; and C-terminal amino
acid residues have a free carboxyl group. Sulfhydryl groups (e.g.,
as found on cysteine residues) may also be used as a reactive group
for attaching PEG. In addition, enzyme-assisted methods for
introducing activated groups (e.g., hydrazide, aldehyde, and
aromatic-amino groups) specifically at the C-terminus of a
polypeptide have been described [Schwarz, et al. (1990) Methods
Enzymol. 184:160; Rose, et al. (1991) Bioconjugate Chem. 2:154;
Gaertner, et al. (1994) J. Biol. Chem. 269:7224].
[0082] For example, PEG molecules may be attached to peptide amino
groups using methoxylated PEG ("mPEG") having different reactive
moieties. Such polymers include mPEG-succinimidyl succinate,
mPEG-succinimidyl carbonate, mPEG-imidate, mPEG-4-nitrophenyl
carbonate, and mPEG-cyanuric chloride. Similarly, PEG molecules may
be attached to peptide carboxyl groups using methoxylated PEG with
a free amine group (mPEG-NH.sub.2).
[0083] Where attachment of the PEG is non-specific and a peptide
containing a specific PEG attachment is desired, the desired
PEGylated compound may be purified from the mixture of PEGylated
compounds. For example, if an N-terminally PEGylated peptide is
desired, the N-terminally PEGylated form may be purified from a
population of randomly PEGylated peptides (i.e., separating this
moiety from other monoPEGylated moieties).
[0084] In preferred embodiments, PEG is attached site-specifically
to a peptide. Site-specific PEGylation at the N-terminus, side
chain, and C-terminus of a potent analog of growth
hormone-releasing factor has been performed through solid-phase
synthesis [Felix, et al. (1995) Int. J. Peptide Protein Res.
46:253]. Another site-specific method involves attaching a peptide
to extremities of liposomal surface-grafted PEG chains in a
site-specific manner through a reactive aldehyde group at the
N-terminus generated by sodium periodate oxidation of N-terminal
threonine [Zalipsky, et al. (1995) Bioconj. Chem. 6:705]. However,
this method is limited to polypeptides with N-terminal serine or
threonine residues. Another site-specific method for N-terminal
PEGylation of a peptide via a hydrazone, reduced hydrazone, oxime,
or reduced oxime bond is described in U.S. Pat. No. 6,077,939 to
Wei, et al.
[0085] In one method, selective N-terminal PEGylation may be
accomplished by reductive alkylation which exploits differential
reactivity of different types of primary amino groups (lysine
versus the N-terminal) available for derivatization in a particular
protein. Under the appropriate reaction conditions, a carbonyl
group containing PEG is selective attached to the N-terminus of a
peptide. For example, one may selectively N-terminally PEGylate the
protein by performing the reaction at a pH which exploits the
pK.sub.a differences between the s-amino groups of a lysine residue
and the .alpha.-amino group of the N-terminal residue of the
peptide. By such selective attachment, PEGylation takes place
predominantly at the N-terminus of the protein, with no significant
modification of other reactive groups (e.g., lysine side chain
amino groups). Using reductive alkylation, the PEG should have a
single reactive aldehyde for coupling to the protein (e.g., PEG
proprionaldehyde may be used).
[0086] Site-specific mutagenesis is a further approach which may be
used to prepare peptides for site-specific polymer attachment. By
this method, the amino acid sequence of a peptide is designed to
incorporate an appropriate reactive group at the desired position
within the peptide. For example, WO 90/12874 describes the
site-directed PEGylation of proteins modified by the insertion of
cysteine residues or the substitution of other residues for
cysteine residues. This publication also describes the preparation
of mPEG-erythropoietin ("mPEG-EPO") by reacting a cysteine-specific
mPEG derivative with a recombinantly introduced cysteine residue on
EPO.
[0087] Where PEG is attached to a spacer or linker moiety, similar
attachment methods may be used. In this case, the linker or spacer
contains a reactive group and an activated PEG molecule containing
the appropriate complementary reactive group is used to effect
covalent attachment. In preferred embodiments the linker or spacer
reactive group contains a terminal amino group (i.e., positioned at
the terminus of the linker or spacer) which is reacted with a
suitably activated PEG molecule to make a stable covalent bond such
as an amide or a carbamate. Suitable activated PEG species include,
but are not limited to, mPEG-para-nitrophenylcarbonate (mPEG-NPC),
mPEG-succinimidyl carbonate (mPEG-SC), and mPEG-succinimidyl
propionate (mPEG-SPA). In other preferred embodiments, the linker
or spacer reactive group contains a carboxyl group capable of being
activated to form a covalent bond with an amine-containing PEG
molecule under suitable reaction conditions. Suitable PEG molecules
include mPEG-NH.sub.2 and suitable reaction conditions include
carbodiimide-mediated amide formation or the like.
EPO-R Agonist Activity Assays:
In Vitro Functional Assays
[0088] In vitro competitive binding assays quantitate the ability
of a test peptide to compete with EPO for binding to EPO-R. For
example (see, e.g., as described in U.S. Pat. No. 5,773,569), the
extracellular domain of the human EPO-R (EPO binding protein, EBP)
may be recombinantly produced in E. coli and the recombinant
protein coupled to a solid support, such as a microtitre dish or a
synthetic bead [e.g., Sulfolink beads from Pierce Chemical Co.
(Rockford, Ill.)]. Immobilized EBP is then incubated with labeled
recombinant EPO, or with labeled recombinant EPO and a test
peptide. Serial dilutions of test peptide are employed for such
experiments. Assay points with no added test peptide define total
EPO binding to EBP. For reactions containing test peptide, the
amount of bound EPO is quantitated and expressed as a percentage of
the control (total=100%) binding. These values are plotted versus
peptide concentration. The IC50 value is defined as the
concentration of test peptide which reduces the binding of EPO to
EBP by 50% (i.e., 50% inhibition of EPO binding).
[0089] A different in vitro competitive binding assay measures the
light signal generated as a function of the proximity of two beads:
an EPO-conjugated bead and an EPO-R-conjugated bead. Bead proximity
is generated by the binding of EPO to EPO-R. A test peptide that
competes with EPO for binding to EPO-R will prevent this binding,
causing a decrease in light emission. The concentration of test
peptide that results in a 50% decrease in light emission is defined
as the IC50 value.
[0090] The peptides of the present invention compete very
efficiently with EPO for binding to the EPO-R. This enhanced
function is represented by their ability to inhibit the binding of
EPO at substantially lower concentrations of peptide (i.e., they
have very low IC50 values).
[0091] The biological activity and potency of monomeric and dimeric
peptide EPO-R agonists of the invention, which bind specifically to
the EPO-receptor, may be measured using in vitro cell-based
functional assays.
[0092] One assay is based upon a murine pre-B-cell line expressing
human EPO-R and further transfected with a fos promoter-driven
luciferase reporter gene construct. Upon exposure to EPO or another
EPO-R agonist, such cells respond by synthesizing luciferase.
Luciferase causes the emission of light upon addition of its
substrate luciferin. Thus, the level of EPO-R activation in such
cells may be quantitated via measurement of luciferase activity.
The activity of a test peptide is measured by adding serial
dilutions of the test peptide to the cells, which are then
incubated for 4 hours. After incubation, luciferin substrate is
added to the cells, and light emission is measured. The
concentration of test peptide that results in a half-maximal
emission of light is recorded as the EC50.
[0093] The peptides of the present invention show dramatically
enhanced ability to promote EPO-R signaling-dependent luciferase
expression in this assay. This enhanced function is represented by
their ability to yield half of the maximal luciferase activity at
substantially lower concentrations of peptide (i.e., they have very
low EC50 values). This assay is a preferred method for estimating
the potency and activity of an EPO-R agonist peptide of the
invention.
[0094] Another assay may be performed using FDC-P1/ER cells
[Dexter, et al. (1980) J. Exp. Med. 152:1036-1047], a well
characterized nontransformed murine bone marrow derived cell line
into which EPO-R has been stably transfected. These cells exhibit
EPO-dependent proliferation.
[0095] In one such assay, the cells are grown to half stationary
density in the presence of the necessary growth factors (see, e.g.,
as described in U.S. Pat. No. 5,773,569). The cells are then washed
in PBS and starved for 16-24 hours in whole media without the
growth factors. After determining the viability of the cells (e.g.,
by trypan blue staining), stock solutions (in whole media without
the growth factors) are made to give about 10.sup.5 cells per 50
.mu.L. Serial dilutions of the peptide EPO-R agonist compounds
(typically the free, solution phase peptide as opposed to a
phage-bound or other bound or immobilized peptide) to be tested are
made in 96-well tissue culture plates for a final volume of 50
.mu.L per well. Cells (50 .mu.L) are added to each well and the
cells are incubated 24-48 hours, at which point the negative
controls should die or be quiescent. Cell proliferation is then
measured by techniques known in the art, such as an MTT assay which
measures H.sup.3-thymidine incorporation as an indication of cell
proliferation [see, Mosmann (1983) J. Immunol. Methods 65:55-63].
Peptides are evaluated on both the EPO-R-expressing cell line and a
parental non-expressing cell line. The concentration of test
peptide necessary to yield one half of the maximal cell
proliferation is recorded as the EC50.
[0096] The peptides of the present invention show dramatically
enhanced ability to promote EPO-dependent cell growth in this
assay. This enhanced function is represented by their ability to
yield half of the maximal cell proliferation stimulation activity
at substantially lower concentrations of peptide (i.e., they have
very low EC50 values). This assay is a preferred method for
estimating the potency and activity of an EPO-R agonist peptide of
the invention.
[0097] In another assay, the cells are grown to stationary phase in
EPO-supplemented medium, collected, and then cultured for an
additional 18 hr in medium without EPO. The cells are divided into
three groups of equal cell density: one group with no added factor
(negative control), a group with EPO (positive control), and an
experimental group with the test peptide. The cultured cells are
then collected at various time points, fixed, and stained with a
DNA-binding fluorescent dye (e.g., propidium iodide or Hoechst dye,
both available from Sigma). Fluorescence is then measured, for
example, using a FACS Scan Flow cytometer. The percentage of cells
in each phase of the cell cycle may then be determined, for
example, using the SOBR model of CellFIT software (Becton
Dickinson). Cells treated with EPO or an active peptide will show a
greater proportion of cells in S phase (as determined by increased
fluorescence as an indicator of increased DNA content) relative to
the negative control group.
[0098] Similar assays may be performed using FDCP-1 [see, e.g.,
Dexter et al. (1980) J. Exp. Med. 152:1036-1047] or TF-1 [Kitamura,
et al. (1989) Blood 73:375-380] cell lines. FDCP-1 is a growth
factor dependent murine multi-potential primitive hematopoietic
progenitor cell line that can proliferate, but not differentiate,
when supplemented with WEHI-3-conditioned media (a medium that
contains IL-3, ATCC number TIB-68). For such experiments, the
FDCP-1 cell line is transfected with the human or murine EPO-R to
produce FDCP-1-hEPO-R or FDCP-1-mEPO-R cell lines, respectively,
that can proliferate, but not differentiate, in the presence of
EPO. TF-1, an EPO-dependent cell line, may also be used to measure
the effects of peptide EPO-R agonists on cellular
proliferation.
[0099] In yet another assay, the procedure set forth in Krystal
(1983) Exp. Hematol 11:649-660 for a microassay based on
H.sup.3-thymidine incorporation into spleen cells may be employed
to ascertain the ability of the compounds of the present invention
to serve as EPO agonists. In brief, B6C3F.sub.1 mice are injected
daily for two days with phenylhydrazine (60 mg/kg). On the third
day, spleen cells are removed and their ability to proliferate over
a 24 hour period ascertained using an MIT assay.
[0100] The binding of EPO to EPO-R in an erythropoietin-responsive
cell line induces tyrosine phosphorylation of both the receptor and
numerous intracellular proteins, including Shc, vav and JAK2
kinase. Therefore, another in vitro assay measures the ability of
peptides of the invention to induce tyrosine phosphorylation of
EPO-R and downstream intracellular signal transducer proteins.
Active peptides, as identified by binding and proliferation assays
described above, elicit a phosphorylation pattern nearly identical
to that of EPO in erythropoietin-responsive cells. For this assay,
FDC-P1/ER cells [Dexter, et al. (1980) J Exp Med 152:1036-47] are
maintained in EPO-supplemented medium and grown to stationary
phase. These cells are then cultured in medium without EPO for 24
hr. A defined number of such cells is then incubated with a test
peptide for approximately 10 min at 37.degree. C. A control sample
of cells with EPO is also run with each assay. The treated cells
are then collected by centrifugation, resuspended in SDS lysis
buffer, and subjected to SDS polyacrylamide gel electrophoresis.
The electrophoresed proteins in the gel are transferred to
nitrocellulose, and the phosphotyrosine containing proteins on the
blot visualized by standard immunological techniques. For example,
the blot may be probed with an anti-phosphotyrosine antibody (e.g.,
mouse anti-phosphotyrosine IgG from Upstate Biotechnology, Inc.),
washed, and then probed with a secondary antibody [e.g., peroxidase
labeled goat anti-mouse IgG from Kirkegaard & Perry
Laboratories, Inc. (Washington, D.C.)]. Thereafter,
phosphotyrosine-containing proteins may be visualized by standard
techniques including colorimetric, chemiluminescent, or fluorescent
assays. For example, a chemiluminescent assay may be performed
using the ECL Western Blotting System from Amersham.
[0101] Another cell-based in vitro assay that may be used to assess
the activity of the peptides of the present invention comprises a
colony assay, using murine bone marrow or human peripheral blood
cells. Murine bone marrow may be obtained from the femurs of mice,
while a sample of human peripheral blood may obtained from a
healthy donor. In the case of peripheral blood, mononuclear cells
are first isolated from the blood, for example, by centrifugation
through a Ficoll-Hypaque gradient [Stem Cell Technologies, Inc.
(Vancouver, Canada)]. For this assay a nucleated cell count is
performed to establish the number and concentration of nucleated
cells in the original sample. A defined number of cells is plated
on methyl cellulose as per manufacturer's instructions [Stem Cell
Technologies, Inc. (Vancouver, Canada)]. An experimental group is
treated with a test peptide, a positive control group is treated
with EPO, and a negative control group receives no treatment. The
number of growing colonies for each group is then scored after
defined periods of incubation, generally 10 days and 18 days. An
active peptide will promote colony formation.
[0102] Other in vitro biological assays that can be used to
demonstrate the activity of the compounds of the present invention
are disclosed in Greenberger, et al. (1983) Proc. Natl. Acad. Sci.
USA 80:2931-2935 (EPO-dependent hematopoietic progenitor cell
line); Quelle and Wojchowski (1991) J. Biol. Chem. 266:609-614
(protein tyrosine phosphorylation in B6SUt.EP cells);
Dusanter-Fourt, et al. (1992) J. Biol. Chem. 287:10670-10678
(tyrosine phosphorylation of EPO-receptor in human EPO-responsive
cells); Quelle, et al. (1992) J. Biol. Chem. 267:17055-17060
(tyrosine phosphorylation of a cytosolic protein, pp 100, in FDC-ER
cells); Worthington, et al. (1987) Exp. Hematol. 15:85-92
(colorimetric assay for hemoglobin); Kaiho and Miuno (1985) Anal.
Biochem. 149:117-120 (detection of hemoglobin with
2,7-diaminofluorene); Patel, et al. (1992) J. Biol. Chem.
267:21300-21302 (expression of c-myb); Witthuhn, et al. (1993) Cell
74:227-236 (association and tyrosine phosphorylation of JAK2);
Leonard, et al. (1993) Blood 82:1071-1079 (expression of GATA
transcription factors); and Ando, et al. (1993) Proc. Natl. Acad.
Sci. USA 90:9571-9575 (regulation of G.sub.1 transition by cycling
D2 and D3).
[0103] An instrument designed by Molecular Devices Corp., known as
a microphysiometer, has been reported to be successfully used for
measurement of the effect of agonists and antagonists on various
receptors. The basis for this apparatus is the measurement of the
alterations in the acidification rate of the extracellular media in
response to receptor activation.
In Vivo Functional Assays
[0104] One in vivo functional assay that may be used to assess the
potency of a test peptide is the polycythemic exhypoxic mouse
bioassay. For this assay, mice are subjected to an alternating
conditioning cycle for several days. In this cycle, the mice
alternate between periods of hypobaric conditions and ambient
pressure conditions. Thereafter, the mice are maintained at ambient
pressure for 2-3 days prior to administration of test samples. Test
peptide samples, or EPO standard in the case positive control mice,
are injected subcutaneously into the conditioned mice. Radiolabeled
iron (e.g., Fe.sup.59) is administered 2 days later, and blood
samples taken two days after administration of radiolabeled iron.
Hematocrits and radioactivity measurements are then determined for
each blood sample by standard techniques. Blood samples from mice
injected with active test peptides will show greater radioactivity
(due to binding of Fe.sup.59 by erythrocyte hemoglobin) than mice
that did not receive test peptides or EPO.
[0105] Another in vivo functional assay that may be used to assess
the potency of a test peptide is the reticulocyte assay. For this
assay, normal untreated mice are subcutaneously injected on three
consecutive days with either EPO or test peptide. On the third day,
the mice are also intraperitoneally injected with iron dextran. At
day five, blood samples are collected from the mice. The percent
(%) of reticulocytes in the blood is determined by thiazole orange
staining and flow cytometer analysis (retic-count program). In
addition, hematocrits are manually determined. The percent of
corrected reticulocytes is determined using the following
formula:
% RETIC.sub.CORRECTED=%
RETIC.sub.OBSERVED.times.(Hematocrit.sub.INDIVIDUAL/Hematocrit.sub.NORMAL-
)
Active test compounds will show an increased % RETIC.sub.CORRECTED
level relative to mice that did not receive test peptides or
EPO.
Use of EPO-R Agonist Peptides of the Invention
[0106] The peptide compounds of the invention are useful in vitro
as tools for understanding the biological role of EPO, including
the evaluation of the many factors thought to influence, and be
influenced by, the production of EPO and the binding of EPO to the
EPO-R (e.g., the mechanism of EPO/EPO-R signal
transduction/receptor activation). The present peptides are also
useful in the development of other compounds that bind to the
EPO-R, because the present compounds provide important
structure-activity-relationship information that facilitate that
development.
[0107] Moreover, based on their ability to bind to EPO-R, the
peptides of the present invention can be used as reagents for
detecting EPO-R on living cells; fixed cells; in biological fluids;
in tissue homogenates; in purified, natural biological materials;
etc. For example, by labeling such peptides, one can identify cells
having EPO-R on their surfaces. In addition, based on their ability
to bind EPO-R, the peptides of the present invention can be used in
in situ staining, FACS (fluorescence-activated cell sorting)
analysis, Western blotting, ELISA (enzyme-linked immunosorbent
assay), etc. In addition, based on their ability to bind to EPO-R,
the peptides of the present invention can be used in receptor
purification, or in purifying cells expressing EPO-R on the cell
surface (or inside permeabilized cells).
[0108] The peptides of the invention can also be utilized as
commercial reagents for various medical research and diagnostic
purposes. Such uses can include but are not limited to: (1) use as
a calibration standard for quantitating the activities of candidate
EPO-R agonists in a variety of functional assays; (2) use as
blocking reagents in random peptide screening, i.e., in looking for
new families of EPO-R peptide ligands, the peptides can be used to
block recovery of EPO peptides of the present invention; (3) use in
co-crystallization with EPO-R, i.e., crystals of the peptides of
the present invention bound to the EPO-R may be formed, enabling
determination of receptor/peptide structure by X-ray
crystallography; (4) use to measure the capacity of erythrocyte
precursor cells induce globin synthesis and heme complex synthesis,
and to increase the number of ferritin receptors, by initiating
differentiation; (5) use to maintain the proliferation and growth
of EPO-dependent cell lines, such as the FDCP-1-mEPO-R and the TF-1
cell lines; and (6) other research and diagnostic applications
wherein the EPO-R is preferably activated or such activation is
conveniently calibrated against a known quantity of an EPO-R
agonist, and the like.
[0109] In yet another aspect of the present invention, methods of
treatment and manufacture of a medicament are provided. The peptide
compounds of the invention may be administered to warm blooded
animals, including humans, to simulate the binding of EPO to the
EPO-R in vivo. Thus, the present invention encompasses methods for
therapeutic treatment of disorders associated with a deficiency of
EPO, which methods comprise administering a peptide of the
invention in amounts sufficient to stimulate the EPO-R and thus,
alleviate the symptoms associated with a deficiency of EPO in vivo.
For example, the peptides of this invention will find use in the
treatment of renal insufficiency and/or end-stage renal
failure/dialysis; anemia associated with AIDS; anemia associated
with chronic inflammatory diseases (for example, rheumatoid
arthritis and chronic bowel inflammation) and autoimmune disease;
and for boosting the red blood count of a patient prior to surgery.
Other disease states, disorders, and states of hematologic
irregularity that may be treated by administration of the peptides
of this invention include: beta-thalassemia; cystic fibrosis;
pregnancy and menstrual disorders; early anemia of prematurity;
spinal cord injury; space flight; acute blood loss; aging; and
various neoplastic disease states accompanied by abnormal
erythropoiesis.
[0110] In other embodiments, the peptide compounds of the invention
may be used for the treatment of disorders which are not
characterized by low or deficient red blood cells, for example as a
pretreatment prior to transfusions. In addition, administration of
the compounds of this invention can result in a decrease in
bleeding time and thus, will find use in the administration to
patients prior to surgery or for indications wherein bleeding is
expected to occur. In addition, the compounds of this invention
will find use in the activation of megakaryoctes.
[0111] Since EPO has been shown to have a mitogenic and chemotactic
effect on vascular endothelial cells as well as an effect on
central cholinergic neurons [see, e.g., Amagnostou, et al. (1990)
Proc. Natl. Acad. Sci. USA 87:5978-5982 and Konishi, et al. (1993)
Brain Res. 609:29-35], the compounds of this invention will also
find use for the treatment of a variety of vascular disorders, such
as: promoting wound healing; promoting growth of collateral
coronary blood vessels (such as those that may occur after
myocardial infarction); trauma treatment; and post-vascular graft
treatment. The compounds of this invention will also find use for
the treatment of a variety of neurological disorders, generally
characterized by low absolute levels of acetyl choline or low
relative levels of acetyl choline as compared to other neuroactive
substances e.g., neurotransmitters.
Pharmaceutical Compositions
[0112] In yet another aspect of the present invention,
pharmaceutical compositions of the above EPO-R agonist peptide
compounds are provided. Conditions alleviated or modulated by the
administration of such compositions include those indicated above.
Such pharmaceutical compositions may be for administration by oral,
parenteral (intramuscular, intraperitoneal, intravenous (IV) or
subcutaneous injection), transdermal (either passively or using
iontophoresis or electroporation), transmucosal (nasal, vaginal,
rectal, or sublingual) routes of administration or using
bioerodible inserts and can be formulated in dosage forms
appropriate for each route of administration. In general,
comprehended by the invention are pharmaceutical compositions
comprising effective amounts of an EPO-R agonist peptide, or
derivative products, 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 20, 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.
Hylauronic acid may also be used. 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 (e.g., lyophilized)
form.
Oral Delivery
[0113] 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, pellets, powders, or granules. 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. In: 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 EPO-R agonist peptides (or chemically modified
forms thereof) and inert ingredients which allow for protection
against the stomach environment, and release of the biologically
active material in the intestine.
[0114] Also contemplated for use herein are liquid dosage forms for
oral administration, including pharmaceutically acceptable
emulsions, solutions, suspensions, and syrups, which may contain
other components including inert diluents; adjuvants such as
wetting agents, emulsifying and suspending agents; and sweetening,
flavoring, and perfuming agents.
[0115] The peptides may be chemically modified so that oral
delivery of the derivative is efficacious. Generally, the chemical
modification contemplated is the attachment of at least one moiety
to the component 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 component or components and increase in
circulation time in the body. As discussed above, PEGylation is a
preferred chemical modification for pharmaceutical usage. Other
moieties that may be used include: propylene glycol, copolymers of
ethylene glycol and propylene glycol, carboxymethyl cellulose,
dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline,
poly-1,3-dioxolane and poly-1,3,6-tioxocane [see, e.g., Abuchowski
and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as
Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York,
N.Y.) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem.
4:185-189].
[0116] For oral formulations, the location of release may be the
stomach, the small intestine (the duodenum, the jejunem, or the
ileum), or the large intestine. One skilled in the art has
available formulations which will not dissolve in the stomach, yet
will release the material in the duodenum or elsewhere in the
intestine. Preferably, the release will avoid the deleterious
effects of the stomach environment, either by protection of the
peptide (or derivative) or by release of the peptide (or
derivative) beyond the stomach environment, such as in the
intestine.
[0117] To ensure full gastric resistance a coating impermeable to
at least pH 5.0 is essential. Examples of the more common inert
ingredients that are used as enteric coatings are cellulose acetate
trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP),
HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit
L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L,
Eudragit S, and Shellac. These coatings may be used as mixed
films.
[0118] A coating or mixture of coatings can also be used on
tablets, which are not intended for protection against the stomach.
This can include sugar coatings, or coatings which make the tablet
easier to swallow. Capsules may consist of a hard shell (such as
gelatin) for delivery of dry therapeutic (i.e. powder), for liquid
forms a soft gelatin shell may be used. The shell material of
cachets could be thick starch or other edible paper. For pills,
lozenges, molded tablets or tablet triturates, moist massing
techniques can be used.
[0119] The peptide (or derivative) 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. These therapeutics
could be prepared by compression.
[0120] Colorants and/or flavoring agents may also be included. For
example, the peptide (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.
[0121] One may dilute or increase the volume of the peptide (or
derivative) with an inert material. These diluents could include
carbohydrates, especially mannitol, .alpha.-lactose, anhydrous
lactose, cellulose, sucrose, modified dextrans and starch. Certain
inorganic salts may be 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.
[0122] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrates 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. The disintegrants may also be insoluble cationic exchange
resins. Powdered gums may be used as disintegrants and as binders.
and can include powdered gums such as agar, Karaya or tragacanth.
Alginic acid and its sodium salt are also useful as
disintegrants.
[0123] Binders may be used to hold the peptide (or derivative)
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 peptide (or derivative).
[0124] An antifrictional agent may be included in the formulation
of the peptide (or derivative) to prevent sticking during the
formulation process. Lubricants may be used as a layer between the
peptide (or derivative) 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.
[0125] 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.
[0126] To aid dissolution of the peptide (or derivative) 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 benzethomium 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 20, 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.
[0127] Additives which potentially enhance uptake of the peptide
(or derivative) are for instance the fatty acids oleic acid,
linoleic acid and linolenic acid.
[0128] Controlled release oral formulations may be desirable. The
peptide (or derivative) 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. Some enteric coatings also have a delayed
release effect. Another form of a controlled release 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.
[0129] Other coatings may be used for the formulation. These
include a variety of sugars which could be applied in a coating
pan. The peptide (or derivative) 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.
[0130] 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.
Parenteral Delivery
[0131] Preparations according to this invention for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil and corn oil, gelatin, and injectable organic
esters such as ethyl oleate. Such dosage forms may also contain
adjuvants such as preserving, wetting, emulsifying, and dispersing
agents. They may be sterilized by, for example, filtration through
a bacteria retaining filter, by incorporating sterilizing agents
into the compositions, by irradiating the compositions, or by
heating the compositions. They can also be manufactured using
sterile water, or some other sterile injectable medium, immediately
before use.
Rectal or Vaginal Delivery
[0132] Compositions for rectal or vaginal administration are
preferably suppositories which may contain, in addition to the
active substance, excipients such as cocoa butter or a suppository
wax. Compositions for nasal or sublingual administration are also
prepared with standard excipients well known in the art.
Pulmonary Delivery
[0133] Also contemplated herein is pulmonary delivery of the EPO-R
agonist peptides (or derivatives thereof). The peptide (or
derivative) is delivered to the lungs of a mammal while inhaling
and traverses across the lung epithelial lining to the blood stream
[see, e.g., Adjei, et al. (1990) Pharmaceutical Research 7:565-569;
Adjei, et al. (1990) Int. J. Pharmaceutics 63:135-144 (leuprolide
acetate); Braquet, et al. (1989) J. Cardiovascular Pharmacology
13(sup5):143-146 (endothelin-1); Hubbard, et al. (1989) Annals of
Internal Medicine, Vol. III, pp. 206-212 (.alpha.1-antitrypsin);
Smith, et al. (1989) J. Clin. Invest. 84:1145-1146
(.alpha.-1-proteinase); Oswein, et al. (1990) "Aerosolization of
Proteins", Proceedings of Symposium on Respiratory Drug Delivery II
Keystone, Colorado (recombinant human growth hormone); Debs, et al.
(1988) J. Immunol. 140:3482-3488 (interferon-.gamma. and tumor
necrosis factor .alpha.); and U.S. Pat. No. 5,284,656 to Platz, et
al. (granulocyte colony stimulating factor). A method and
composition for pulmonary delivery of drugs for systemic effect is
described in U.S. Pat. No. 5,451,569 to Wong, et al.
[0134] 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. Some specific examples of
commercially available devices suitable for the practice of this
invention are the Ultravent nebulizer (Mallinckrodt Inc., St.
Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products,
Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc.,
Research Triangle Park, N.C.); and the Spinhaler powder inhaler
(Fisons Corp., Bedford, Mass.).
[0135] All such devices require the use of formulations suitable
for the dispensing of peptide (or derivative). Typically, each
formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to the usual diluents, adjuvants and/or carriers useful in therapy.
Also, the use of liposomes, microcapsules or microspheres,
inclusion complexes, or other types of carriers is contemplated.
Chemically modified peptides may also be prepared in different
formulations depending on the type of chemical modification or the
type of device employed.
[0136] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise peptide (or derivative)
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 peptide (or derivative) caused
by atomization of the solution in forming the aerosol.
[0137] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the peptide
(or derivative) 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.
[0138] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing peptide (or
derivative) and may also include a bulking agent, such as lactose,
sorbitol, sucrose, or mannitol in amounts which facilitate
dispersal of the powder from the device, e.g., 50 to 90% by weight
of the formulation. The peptide (or derivative) should most
advantageously be prepared in particulate form with an average
particle size of less than 10 mm (or microns), most preferably 0.5
to 5 mm, for most effective delivery to the distal lung.
Nasal Delivery
[0139] Nasal delivery of the EPO-R agonist peptides (or
derivatives) is also contemplated. Nasal delivery allows the
passage of the peptide 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.
Dosages
[0140] For all of the peptide compounds, as further studies are
conducted, information will emerge regarding appropriate dosage
levels for treatment of various conditions in various patients, and
the ordinary skilled worker, considering the therapeutic context,
age, and general health of the recipient, will be able to ascertain
proper dosing. The selected dosage depends upon the desired
therapeutic effect, on the route of administration, and on the
duration of the treatment desired. Generally dosage levels of 0.001
to 10 mg/kg of body weight daily are administered to mammals.
Generally, for intravenous injection or infusion dosage may be
lower. The dosing schedule may vary, depending on the circulation
half-life, and the formulation used.
[0141] The peptides of the present invention (or their derivatives)
may be administered in conjunction with one or more additional
active ingredients or pharmaceutical compositions.
EXAMPLES
[0142] The present invention is next described by means of the
following examples. However, the use of these and other examples
anywhere in the specification is illustrative only, and in no way
limits the scope and meaning of the invention or of any exemplified
form. Likewise, the invention is not limited to any particular
preferred embodiments described herein. Indeed, many modifications
and variations of the invention may be apparent to those skilled in
the art upon reading this specification, and can be made without
departing from its spirit and scope. The invention is therefore to
be limited only by the terms of the appended claims, along with the
full scope of equivalents to which the claims are entitled.
Example 1
Synthesis of EPO-R Agonist Peptides
1. Peptide Monomer Synthesis
[0143] Various peptide monomers of the invention were synthesized
using the Merrifield solid phase synthesis technique [see, Stewart
and Young. Solid Phase Peptide Synthesis, 2.sup.nd edition (Pierce
Chemical, Rockford, Ill.) 1984] on an Applied Biosystems 433A
automated instrument. The resin used was PAL (Milligen/Biosearch),
which is cross-linked polystyrene with
5-(4'-Fmoc-aminomethyl-3,5'-dimethoxyphenoxy) valeric acid. Use of
PAL resin results in a carboxyl terminal amide function upon
cleavage of the peptide from the resin. Primary amine protection on
amino acids was achieved with Fmoc, and side chain protection
groups were t-butyl for serine, threonine, and tyrosine hydroxyls;
trityl for glutamine and asparagine amides; Trt or Acm for
cysteine; and Pmc (2,2,5,7,8-pentamethylchroman sulfonate) for the
arginine guanidino group. Each coupling was performed for either 1
hr or 2 hr with BOP (benzotriazolyl
N-oxtrisdimethylaminophosphonium hexafluorophosphate) and HOBt
(1-hydroxybenztriazole).
[0144] For the synthesis of peptides with an amidated carboxy
terminus, the fully assembled peptide was cleaved with a mixture of
90% trifluoroacetic acid, 5% ethanedithiol, and 5% water, initially
at 4.degree. C. and gradually increasing to room temperature over
1.5 hr. The deprotected product was filtered from the resin and
precipitated with diethyl ether. After thorough drying the product
was purified by C 18 reverse phase high performance liquid
chromatography with a gradient of acetonitrile/water in 0.1%
trifluoroacetic acid.
2. Peptide Dimer Synthesis
[0145] Various peptide dimers of the invention were synthesized
directly onto a lysine linker in a variation of the solid phase
technique.
[0146] For simultaneous synthesis of the two peptide chains,
Fmoc-Lys-Fmoc was coupled to a PAL resin (Milligen/Biosearch),
thereby providing an initial lysine residue to serve as the linker
between the two chains to be synthesized. The Fmoc protecting
groups were removed with mild base (20% piperidine in DMF), and the
peptide chains were synthesized using the resulting free amino
groups as starting points. Peptide chain synthesis was performed
using the solid phase synthesis technique described above. Trt was
used to protect all cysteine residues. Following dimer
deprotection, cleavage from the resin, and purification, oxidation
of the cysteine residues was performed by incubating the
deprotected dimer in 100% DMSO for 2-3 days at 5.degree. C. to
25.degree. C. This oxidation reaction yielded predominantly
(>75%) dimers with two intramolecular disulfide bonds.
[0147] For sequential synthesis of the two peptide chains,
Fmoc-Lys-Alloc was coupled to a PAL resin (Milligen/Biosearch),
thereby providing an initial lysine residue to serve as the linker
between the two chains to be synthesized. The Fmoc protecting group
was removed with mild base (20% piperidine in DMF). The first
peptide chain was then syntheszed using the resulting free amino
group as a starting point. Peptide synthesis was performed using
the solid phase technique described above. The two cysteine
residues of the first chain were protected with Trt. Following
synthesis of the first peptide chain, the Alloc group was removed
from the support-bound lysine linker with
Pd[P(C.sub.6H.sub.5).sub.3].sub.4, 4-methyl morpholine, and
chloroform. The second peptide chain was then synthesized on this
second free amino group. The two cysteine residues of the second
chain were protected with Acm. An intramolecular disulfide bond was
formed in the first peptide chain by removing the Trt protecting
groups using trifluoroacetic acid, followed by oxidation by
stirring in 20% DMSO overnight. An intramolecular disulfide bond
was then formed in the second peptide chain by simultaneously
removing the Acm protecting groups and oxidizing the deprotected
cysteine residues using iodine, methanol, and thalium
trifluoroacetate.
3. Attachment of Spacers
[0148] Where the spacer was an amino acid (e.g., glycine or lysine
as in AF35462 and AF35464, respectively), the spacer was
incorporated into the peptide during solid phase peptide synthesis.
In this case, the spacer amino acid was coupled to the PAL resin,
and its free amino group served as the basis for the attachment of
another spacer amino acid, or of the lysine linker. Following the
attachment of the lysine linker, dimeric peptides were synthesized
as described above.
4. Synthesis Of Exemplary Peptide Dimers
[0149] Exemplary embodiments of these synthesis techniques are
outlined below. In one example, the synthesis of a peptide dimer
linked via a C-terminal lysine amide is described. In another
example, the synthesis of a peptide dimer linked via a C-terminal
lysine, and containing a spacer molecule attached to the linking
lysine, is described.
Synthesis of a Peptide Dimer Linked Via a C-Terminal Lysine
Amide:
Step 1--Formation of TentaGel-Rink-Lys:
[0150] TentaGel-Rink resin (0.18 mml/g from Rapp Polymere, Germany)
was treated with a activated solution of Fmoc-Lys(Fmoc)-OH
(prepared from 5 eq. of amino acid and 5 eq. of HATU dissolved at
0.5 M in DMF, followed by the addition of 10 eq. of DIEA) and
allowed to gently shake 14 h. The resin was washed (DMF, THF, DCM,
MeOH) and dried to yield the protected resin. Residual amine groups
were capped by treating the resin with a solution of 10% acetic
anhydride, 20% pyridine in DCM for 20 minutes, followed by washing
as above. The Fmoc groups were removed by gently shaking the resin
in 30% piperideine in DMF for 20 minutes, followed by washing (DMF,
THF, DCM, MeOH) and drying.
##STR00154##
[0151] Step 2--Formation of TentaGel-Rink-Lys(Peptide).sub.2:
[0152] The resin from Step 1 was subjected to repeated cycles of
Fmoc-amino acid couplings with HBTU/HOBt activation and Fmoc
removal with piperidine to build both peptide chains
simultaneously. This was convenently carried out on a ABI 433
automated peptide synthesizer available from Applied Biosystems,
Inc. After the final Fmoc removal, the terminal amine groups were
acylated with acetic anhydride (10 eq.) and DIEA (20 eq.) in DMF
for 20 minutes, followed by washing as above.
##STR00155##
[0153] Step 3--Cleavage from Resin:
[0154] The resin from above is suspended in to a solution of TFA
(82.5%), phenol (5%), ethanedithiol (2.5%), water (5%), and
thioanisole (5%) for 3 h at room temperature. Alternative cleavage
cocktails such as TFA (95%), water (2.5%), and triisopropylsilane
(2.5%) can also be used. The TFA solution is cooled to 5.degree. C.
and poured into Et.sub.2O to percipatate the peptide. Filtration
and drying under reduced pressure gave the desired peptide.
Purification via preparative HPLC with a C18 column affords the
pure peptide.
##STR00156##
Synthesis of a peptide dimer linked via a C-terminal lysine amide
and containing a spacer molecule:
[0155] Step 1--Synthesis of Cbz-TAP:
[0156] A solution containing the commercially available diamine
("TAP" from Aldrich Chemical Co.) (10 g, 67.47 mmol) in anhydrous
DCM (100 ml) was cooled to 0.degree. C. A solution of benzyl
chloroformate (4.82 ml, 33.7 mmol) in anhydrous DCM (50 ml) was
added slowly through a dropping funnel over a period of 6-7 h,
maintaining the temperature of the reaction mixture at 0.degree. C.
throughout, then allowed to warm to room temperature (=25.degree.
C.). After a further 16 h, the DCM was removed under vacuum and the
residue partitioned between 3N HCl and ether. The aqueous layers
were collected and neutralized with 50% aq. NaOH to pH8-9 and
extracted with ethyl acetate. The ethyl acetate layer was dried
over anhydrous Na.sub.2SO.sub.4, then concentrated under vacuum to
provide the crude mono-Cbz-TAP (5 g, about 50% yield). This
compound was used for the next reaction without any further
purification.
##STR00157##
[0157] Step 2--Synthesis of Cbz-TAP-Boc:
[0158] To a vigorously stirred suspension of the Cbz-TAP (5 g, 17.7
mmol) in hexane (25 ml) was added Boc.sub.2O (3.86 g, 17.7 mmol)
and stirring continued at RT overnight. The reaction mixture was
diluted with DCM (25 ml) and washed with 10% aq. citric acid
(2.times.), water (2.times.) and brine. The organic layer was dried
over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The
crude product (yield 5 g) was used directly in the next
reaction.
##STR00158##
[0159] Step 3--Synthesis of Boc-TAP:
[0160] The crude product from the previous reaction was dissolved
in methanol (25 ml) and hydrogenated in presence of 5% Pd on Carbon
(5% w/W) under balloon pressure for 16 hrs. The mixture was
filtered, washed with methanol and the filtrate concentrated in
vacuo to provide the crude H-TAP-Boc product (yield 3.7 g). The
overall approximate yield of Boc-TAP after Steps 1-3 was 44%
(calculated based on the amount of Cbz-Cl used.)
##STR00159##
[0161] Step 4--Synthesis of TentaGel-Linker:
[0162] TentaGel bromide (2.5 g, 0.48 mmol/g, from Rapp Polymere,
Germany), phenolic linker (5 equivalent, and K.sub.2CO.sub.3 (5
equivalent) were heated in 20 mL of DMFto 70.degree. C. for 14 h.
After cooling to room temperature, the resin was washed (0.1 N HCl,
water, ACN, DMF, MeOH) and dried to give an amber-colored
resin.
##STR00160##
[0163] Step 5--Synthesis of TentaGel-Linker-TAP(Boc):
[0164] 2.5 gms of the resin from above and H-TAP-Boc (1.5 gms,
Seq.) and glacial AcOH (34 .mu.l, 5 eq.) was taken in a mixture of
1:1 MeOH-THF and shaken overnight. A 1M solution of sodium
cyanoborohydride (5 eq) in THF was added to this and shaken for
another 7 hrs. The resin was filtered washed (DMF, THF, 0.1 N HCl,
water, MeOH) and dried. A small amount of the resin was benzoylated
with Bz-Cl and DIEA in DCM and cleaved with 70% TFA-DCM and checked
by LCMS and HPLC.
##STR00161##
[0165] Step 6--Synthesis of TentaGel-linker-TAP-Lys:
[0166] The resin from above was treated with a activated solution
of Fmoc-Lys(Fmoc)-OH (prepared from 5 eq. of amino acid and 5 eq.
of HATU dissolved at 0.5 M in DMF, followed by the addition of 10
eq. of DIEA) and allowed to gently shake 14 h. The resin was washed
(DMF, THF, DCM, MeOH) and dried to yield the protected resin.
Residual amine groups were capped by treating the resin with a
solution of 10% acetic anhydride, 20% pyridine in DCM for 20
minutes, followed by washing as above. The Fmoc groups are removed
by gently shaking the resin in 30% piperideine in DMF for 20
minutes, followed by washing (DMF, THF, DCM, MeOH) and drying.
##STR00162##
[0167] Step 7--Synthesis of
TentaGel-Linker-TAP-Lys(Peptide).sub.2:
[0168] The resin from above was subjected to repeated cycles of
Fmoc-amino acid couplings with HBTU/HOBt activation and Fmoc
removal with piperidine to build both peptide chains
simultaneously. This was convenently carried out on a ABI 433
automated peptide synthesizer available from Applied Biosystems,
Inc. After the final Fmoc removal, the terminal amine groups were
acylated with acetic anhydride (10 eq.) and DIEA (20 eq.) in DMF
for 20 minutes, follwed by washing as above.
##STR00163##
[0169] Step 8--Cleavage from Resin:
[0170] The resin from above was suspended in a solution of TFA
(82.5%), phenol (5%), ethanedithiol (2.5%), water (5%), and
thioanisole (5%) for 3 h at room temperature. Alternative cleavage
cocktails such as TFA (95%), water (2.5%), and triisopropylsilane
(2.5%) can also be used. The TFA solution was cooled to 5.degree.
C. and poured into Et.sub.2O to percipatate the peptide. Filtration
and drying under reduced pressure gave the desired peptide.
Purification via preparative HPLC with a C18 column afforded the
pure peptide.
##STR00164##
5. Oxidation of Peptides to Form Intramolecular Disulfide Bonds
[0171] The peptide dimer was dissolved in 20% DMSO/water (1 mg dry
weight peptide/mL) and allowed to stand at room temperature for 36
h. The peptide was purified by loading the reaction mixture onto a
C18 HPLC column (Waters Delta-Pak C18, 15 micron particle size, 300
angstrom pore size, 40 mm.times.200 mm length), followed by a
linear ACN/water/0.01% TFA gradiant from 5 to 95% ACN over 40
minutes. Lypholization of the fractions containing the desired
peptide affords the product as a fluffy white solid. For example,
in the case of AF35525, this reaction may be illustrated
schematically as follows:
##STR00165##
6. PEGylation of Peptides
[0172] PEGylation of the peptides of the invention was carried out
using several different techniques.
[0173] PEGylation of a Terminal --NH.sub.2 Group:
[0174] The peptide dimer was mixed with 1.5 eq. (mole basis) of
activated PEG species (mPEG-NPC from NOF Corp. Japan) in dry DMF to
afford a clear solution. After 5 minutes 4 eq of DIEA was added to
above solution. The mixture was stirred at ambient temperature 14
h, followed by purification with C18 reverse phase HPLC. The
structure of PEGylated peptide was confirmed by MALDI mass. The
purified peptide was also subjected to purification via cation ion
exchange chromatography as outlined below. For example, in the case
of AF35593, the monoPEGylation of the terminal --NH.sub.2 group of
the spacer moiety may be illustrated schematically as follows:
##STR00166##
[0175] DiPEGylation of the N-Termini of a Peptide Dimer:
[0176] The peptide dimer is mixed with 2.5 eq. (mole basis) of
activated PEG species (mPEG-NPC from NOF Corp. Japan) in dry DMF to
afford a clear solution. After 5 minutes 4 eq of DIEA is added to
above solution. The mixture is stirred at ambient temperature 14 h,
followed by purification with C18 reverse phase HPLC. The purified
peptide is also subjected to purification via cation ion exchange
chromatography as outlined below. For example, in the case of
AF35083, this reaction may be illustrated schematically as
follows:
##STR00167##
[0177] Peptide Dimerization via PEGylation of N-Termini:
[0178] The peptide (2.5 eq.) and PEG-(SPA-NHS).sub.2 (1 eq. from
Shearwater Corp, USA.) were dissolved in dry DMF at 0.25M to afford
a clear solution. After 5 minutes 10 eq of DIEA is added to above
solution. The mixture is stirred at ambient temperature 2 h,
followed by purification with C18 reverse phase HPLC. The purified
peptide is also subjected to purification via cation ion exchange
chromatography as outlined below. For example, in the case of
AF33131, this reaction may be illustrated schematically as
follows:
##STR00168##
[0179] Peptide Dimerization via PEGylation of C-Termini:
[0180] The peptide (2.5 eq.) and PEG-(SPA-NHS).sub.2 (1 eq. from
Shearwater Corp, USA.) were dissolved in dry DMF at 0.25M to afford
a clear solution. After 5 minutes 10 eq of DMA is added to above
solution. The mixture is stirred at ambient temperature 2 h,
followed by purification with C18 reverse phase HPLC. The purified
peptide is also subjected to purification via cation ion exchange
chromatography as outlined below. For example, this reaction may be
summarized as follows:
##STR00169##
7. Ion Exchange Purification of Peptides.
[0181] Several exchange supports were surveyed for their ability to
separate the above peptide-PEG conjugate from unreacted (or
hydrolyzed) PEG, in addition to their ability to retain the
starting dimeric peptides. The ion exchange resin (2-3 g) was
loaded into a 1 cm column, followed by conversion to the sodium
form (0.2 N NaOH loaded onto column until elutant was pH 14, ca. 5
column volumes), and than to the hydrogen form (eluted with either
0.1 N HCl or 0.1 M HOAc until elutant matched load pH, ca. 5 column
volumes), followed by washing with 25% ACN/water until pH 6. Either
the peptide prior to conjugation or the peptide-PEG conjugate was
dissolved in 25% ACN/water (10 mg/mL) and the pH adjusted to <3
with TFA, then loaded on the column. After washing with 2-3 column
volumes of 25% ACN/water and collecting 5 mL fractions, the peptide
was released from the column by elution with 0.1 M NH.sub.4OAc in
25% ACN/water, again collecting 5 mL fractions. Analysis via HPLC
revealed which fractions contained the desired peptide. Analysis
with an Evaporative Light-Scattering Detector (ELSD) indicated that
when the peptide was retained on the column and was eluted with the
NH.sub.4OAc solution (generally between fractions 4 and 10), no
non-conjugated PEG was observed as a contaminant. When the peptide
eluted in the initial wash buffer (generally the first 2
fractions), no separation of desired PEG-conjugate and excess PEG
was observed.
[0182] The following columns successfully retained both the peptide
and the peptide-PEG conjugate, and successfully purified the
peptide-PEG conjugate from the unconjugates peptide:
TABLE-US-00004 TABLE 1 Ion Exhange Resins Support Source Mono S HR
5/5 strong cation Amersham Biosciences exchange pre-loaded column
SE53 Cellulose, microgranular Whatman strong cation exchange
support SP Sepharose Fast Flow strong Amersham Biosciences cation
exchange support
Example 2
In Vitro Activity Assays
[0183] This example describes various in vitro assays that are
useful in evaluating the activity and potency of EPO-R agonist
peptides of the invention. The results for these assays demonstrate
that the novel peptides of this invention bind to EPO-R and
activate EPO-R signaling. Moreover, the results for these assays
show that the novel peptide compositions exhibit a surprising
increase in EPO-R binding affinity and biological activity compared
to EPO mimetic peptides that have been previously described.
[0184] EPO-R agonist peptide monomers and dimers are prepared
according to the methods provided in Example 1. The potency of
these peptide monomers and dimers is evaluated using a series of in
vitro activity assays, including: a reporter assay, a proliferation
assay, a competitive binding assay, and a C/BFU-e assay. These four
assays are described in further detail below.
[0185] The results of these in vitro activity assays are summarized
in Table 2 (for peptide monomers) and Table 3 (for peptide dimers).
These tables provide the compound designation and structure for
each tested peptide, as well as the experimental results for each
of these four assays. These results demonstrate the dramatically
enhanced potency of the novel peptides of the invention.
1. Reporter Assay
[0186] This assay is based upon a on a murine pre-B-cell line
derived reporter cell, Baf3/EpoR/GCSFR fos/lux. This reporter cell
line expresses a chimeric receptor comprising the extra-cellular
portion of the human EPO receptor to the intra-cellular portion of
the human GCSF receptor. This cell line is further transfected with
a fos promoter-driven luciferase reporter gene construct.
Activation of this chimeric receptor through addition of
erythropoietic agent results in the expression of the luciferase
reporter gene, and therefore the production of light upon addition
of the luciferase substrate luciferin. Thus, the level of EPO-R
activation in such cells may be quantitated via measurement of
luciferase activity.
[0187] The Baf3/EpoR/GCSFR fos/lux cells are cultured in DMEM/F12
medium (Gibco) supplemented with 10% fetal bovine serum (FBS;
Hyclone), 10% WEHI-3 supernatant (the supernatant from a culture of
WEHI-3 cells, ATCC # TIB-68), and penicillin/streptomycin.
Approximately 18 h before the assay, cells are starved by
transferring them to DMEM/F12 medium supplemented with 10% FBS and
0.1% WEHI-3 supernatant. On the day of assay, cells are washed once
with DMEM/F12 medium supplemented with 10% FBS (no WEHI-3
supernatant), then 1.times.10.sup.6 cells/mL are cultured in the
presence of a known concentration of test peptide, or with EPO(R
& D Systems Inc., Minneapolis, Minn.) as a positive control, in
DMEM/F12 medium supplemented with 10% FBS (no WEHI-3 supernatant).
Serial dilutions of the test peptide are concurrently tested in
this assay. Assay plates are incubated for 4 h at 37.degree. C. in
a 5% CO.sub.2 atmosphere, after which luciferin (Steady-Glo;
Promega, Madison, Wi) is added to each well. Following a 5-minute
incubation, light emission is measured on a Packard Topcount
Luminometer (Packard Instrument Co., Downers Grove, Ill.). Light
counts are plotted relative to test peptide concentration and
analysed using Graph Pad software. The concentration of test
peptide that results in a half-maximal emission of light is
recorded as the EC50 [See Tables 2 and 3: Reporter EC50].
2. Proliferation Assay
[0188] This assay is based upon a murine pre-B-cell line, Baf3,
transfected to express human EPO-R. Proliferation of the resulting
cell line, BaF3/Gal4/Elk/EPOR, is dependent on EPO-R activation.
The degree of cell proliferation is quantitated using MTT, where
the signal in the MTT assay is proportional to the number of viable
cells.
[0189] The BaF3/Gal4/Elk/EPOR cells are cultured in spinner flasks
in DMEM/F12 medium (Gibco) supplemented with 10% FBS (Hyclone) and
2% WEHI-3 supernatant (ATCC # TIB-68). Cultured cells are starved
overnight, in a spinner flask at a cell density of 1.times.10.sup.6
cells/ml, in DMEM/F12 medium supplemented with 10% FBS and 0.1%
WEHI-3 supernatant. The starved cells are then washed twice with
Dulbecco's PBS (Gibco), and resuspended to a density of
1.times.10.sup.6 cells/ml in DMEM/F12 supplemented with 10% FBS (no
WEHI-3 supernatant). 500, aliquots (50,000 cells) of the cell
suspension are then plated, in triplicate, in 96 well assay plates.
504 aliquots of dilution series of test EPO mimetic peptides, or 50
.mu.L EPO (R & D Systems Inc., Minneapolis, Minn.) or
Aranesp.TM. (darbepoeitin alpha, an ERO-R agonist commerically
available from Amgen) in DMEM/F12 media supplemented with 10% FBS
(no WEHI-3 supernatant I) are added to the 96 well assay plates
(final well volume of 1004). For example, 12 different dilutions
may be tested where the final concentration of test peptide (or
control EPO peptide) ranges from 810 pM to 0.0045 pM. The plated
cells are then incubated for 48 h at 37.degree. C. Next, 14 .mu.L
of MIT (Roche Diagnostics) is added to each culture dish well, and
then allowed to incubate for 4 h. The reaction is then stopped by
adding 10% SDS+0.01N HCl. The plates are then incubated overnight
at 37.degree. C. Absorbance of each well at a wavelength of 595 nm
is then measured by spectrophotometry. Plots of the absorbance
readings versus test peptide concentration are constructed and the
EC50 calculated using Graph Pad software. The concentration of test
peptide that results in a half-maximal absorbance is recorded as
the EC50 [See Table 3: Proliferation EC50].
3. Competitive Binding Assay
[0190] Competitive binding calculations are made using an assay in
which a light signal is generated as a function of the proximity of
two beads: a streptavidin donor bead bearing a biotinylated
EPO-R-binding peptide tracer and an acceptor bead to which is bound
EPO-R. Light is generated by non-radiative energy transfer, during
which a singlet oxygen is released from a first bead upon
illumination, and contact with the released singlet oxygen causes
the second bead to emit light. These bead sets are commercially
available (Packard). Bead proximity is generated by the binding of
the EPO-R-binding peptide tracer to the EPO-R. A test peptide that
competes with the EPO-R-binding peptide tracer for binding to EPO-R
will prevent this binding, causing a decrease in light
emission.
[0191] In more detail the method is as follows: Add 4 .mu.L of
serial dilutions of the test EPO-R agonist peptide, or positive or
negative controls, to wells of a 384 well plate. Thereafter, add 2
.mu.L/well of receptor/bead cocktail. Receptor bead cocktail
consists of: 15 .mu.L of 5 mg/ml streptavidin donor beads
(Packard), 15 .mu.L of 5 mg/ml monoclonal antibody ab179 (this
antibody recognizes the portion of the human placental alkaline
phosphatase protein contained in the recombinant EPO-R), protein
A-coated acceptor beads (protein A will bind to the ab179 antibody;
Packard), 112.5 .mu.L of a 1:6.6 dilution of recombinant EPO-R
(produced in Chinese Hamster Ovary cells as a fusion protein to a
portion of the human placental alkaline phosphatase protein which
contains the ab179 target epitope) and 607.54 of Alphaquest buffer
(40 mM HEPES, pH 7.4; 1 mM MgCl.sub.2; 0.1% BSA, 0.05% Tween 20).
Tap to mix. Add 2 .mu.L/well of the biotinylated EPO-R-binding
peptide tracer, AF33068 (30 nM final concentration). AF33068, an
EPO-R binding peptide (see Table 3 "Reporter EC50 (pM)"), is made
according to the methods described in Example 1.
##STR00170##
[0192] Centrifuge 1 min to mix. Seal plate with Packard Top Seal
and wrap in foil. Incubate overnight at room temperature. After 18
hours read light emission using an AlphaQuest reader (Packard).
Plot light emission vs concentration of peptide and analyse with
Graph Pad or Excel.
[0193] The concentration of test peptide that results in a 50%
decrease in light emission, relative to that observed without test
peptide, is recorded as the IC.sub.50 [See Tables 2 and 3: AQ
IC.sub.50].
4. C/BFU-e Assay
[0194] EPO-R signaling stimulates the differentiation of bone
marrow stem cells into proliferating red blood cell presursors.
This assay measures the ability of test peptides to stimulate the
proliferation and differentiation of red blood cell precursors from
primary human bone marrow pluripotent stem cells.
[0195] For this assay, serial dilutions of test peptide are made in
IMDM medium (Gibco) supplemented with 10% FBS (Hyclone). These
serial dilutions, or positive control EPO peptide, are then added
to methylcellulose to give a final volume of 1.5 mL. The
methylcellulose and peptide mixture is then vortexed thoroughly.
Aliquots (100,000 cells/mL) of human, bone marrow derived CD34+
cells (Poietics/Cambrex) are thawed. The thawed cells are gently
added to 0.1 mL of 1 mg/ml DNAse (Stem Cells) in a 50 mL tube.
Next, 40-50 mL IMDM medium is added gently to cells: the medium is
added drop by drop along the side of the 50 mL tube for the first
10 mL, and then the remaining volume of medium is slowly dispensed
along the side of the tube. The cells are then spun at 900 rpm for
20 min, and the media removed carefully by gentle aspiration. The
cells are resuspended in 1 ml of IMDM medium and the cell density
per mL is counted on hemacytometer slide (10 .mu.L aliquot of cell
suspension on slide, and cell density is the average
count.times.10,000 cells/ml). The cells are then diluted in IMDM
medium to a cell density of 15,000 cells/mL. A 100 .mu.L of diluted
cells is then added to each 1.5 mL methyl cellulose plus peptide
sample (final cell concentration in assay media is 1000 cells/mL),
and the mixture is vortexed. Allow the bubbles in the mixture to
disappear, and then aspirate 1 mL using blunt-end needle. Add 0.25
mL aspirated mixture from each sample into each of 4 wells of a
24-well plate (Falcon brand). Incubate the plated mixtures at
37.degree. C. under 5% CO.sub.2 in a humid incubator for 14 days.
Score for the presence of erythroid colonies using a phase
microscope (5.times.-10.times. objective, final magnification of
100.times.). The concentration of test peptide at which the numer
of formed colonies is 90% of maximum, relative to that observed
with the EPO positive control, is recorded as the EC90 [See Table
3: C/BFU-e EC90].
5. Radioligand Competitive Binding Assay
[0196] An alternative radioligand competition binding assay can
also be used to measure IC50 values for peptides of the present
invention. This assay measures binding of .sup.125I-EPO to EPOr.
The assay may be performed according to the following exemplary
protocol:
[0197] A. Materials
TABLE-US-00005 Recombinant Identification: Recombinant Human EPO
R/Fc Chimera Human Supplier: R&D Systems (Minneapolis, MN, US)
EPO R/Fc Catalog number: 963-ER Chimera Lot number: EOK033071
Storage: 4.degree. C. Iodinated Identification:
(3[.sup.125I]iodotyrosyl)Erythropoietin, human recombinant
recombinant, high specific activity, 370 kBq, 10 .mu.Ci human
Supplier: Amersham Biosciences (Piscataway, NJ, US) Erythropoietin
Catalog number: IM219-10 .mu.Ci Lot number: Storage: 4.degree. C.
Protein-G Identification: Protein-G Sepharose 4 Fast Flow Sepharose
Supplier: Amersham Biosciences (Piscataway, NJ, US) Catalog number
17-0618-01 Lot number: Storage: 4.degree. C. Assay Buffer Phosphate
Buffered Saline (PBS), pH 7.4, containing 0.1% Bovine Serum Albumin
and 0.1% Sodium Azide Storage: 4.degree. C.
[0198] B. Determination of Appropriate Receptor Concentration.
[0199] One 50 .mu.g vial of lyophilized recombinant EPOr
extracellular domain fused to the Fc portion of human IgG1 is
reconstituted in 1 mL of assay buffer. To determine the correct
amount of receptor to use in the assay, 100 .mu.L serial dilutions
of this receptor preparation are combined with approximately 20,000
cpm in 200 .mu.L of iodinated recombinant human Erythropoietin
(.sup.125I-EPO) in 12.times.75 mm polypropylene test tubes. Tubes
are capped and mixed gently at 4.degree. C. overnight on a LabQuake
rotating shaker.
[0200] The next day, 50 .mu.L of a 50% slurry of Protein-G
Sepharose is added to each tube. Tubes are then incubated for 2
hours at 4.degree. C., mixing gently. The tubes are then
centrifuged for 15 min at 4000 RPM (3297.times.G) to pellet the
protein-G sepharose. The supernatants are carefully removed and
discarded. After washing 3 times with 1 mL of 4.degree. C. assay
buffer, the pellets are counted in a Wallac Wizard gamma counter.
Results were then analyzed and the dilution required to reach 50%
of the maximum binding value was calculated.
[0201] C. IC.sub.50 Determination for Peptide
[0202] To determine the IC.sub.50 of a peptide of the present
invention, 100 .mu.L serial dilutions of the peptide are combined
with 100 .mu.L of recombinant erythropoietin receptor (100 pg/tube)
in 12.times.75 mm polypropylene test tubes. Then 100 .mu.L of
iodinated recombinant human Erythropoietin (.sup.125I-EPO) is added
to each tube and the tubes were capped and mixed gently at
4.degree. C. overnight.
[0203] The next day, bound .sup.125I-EPO is quantitated as
described above. The results are analyzed and the IC.sub.50 value
calculated using Graphpad Prism version 4.0, from GraphPad
Software, Inc. (San Diego, Calif.) The assay is preferably repeated
2 or more times for each peptide whose IC.sub.50 value is measured
by this procedure, for a total of 3 replicate IC.sub.50
determinations.
6. Discussion
[0204] The in vitro reporter assay results for peptide monomers of
the present invention were directly compared with those for related
peptide sequences previously disclosed (see AF31552 and AF31748 in
Table 2): namely,
TABLE-US-00006 SEQ ID NO: 32 GGLYACHMGPMTVCQPLRG and SEQ ID NO: 33
GGLYACHMGPMT(1-nal)VCQPLRG.
[0205] These results demonstrate the dramatically improved potency
of the novel peptide monomers of the invention, as the novel
peptide dimers were 3 to 7.5 times as potent as the previously
disclosed peptide monomers in the reporter assay. These novel
peptide monomers were then used to prepare novel peptide dimers of
even greater potency and activity.
TABLE-US-00007 TABLE 2 In vitro reporter assay for peptide monomers
Reporter Compound EC50 designation Peptide monomer (nM) AF31552
##STR00171## 100 AF31748 ##STR00172## 40 AF33128 ##STR00173## 13
AF36729 ##STR00174## 13.3
TABLE-US-00008 TABLE 3 In vitro activity assays for peptide dimers
Com- Re- Prolif- C/ pound porter eration AQ BFU-e desig- EC50 EC50
IC50 EC90 nation Peptide dimer (pM) (pM) (nM) (nM) AF33065
##STR00175## 300 -- -- -- AF34602 ##STR00176## 727 -- -- -- AF34395
##STR00177## 100 -- -- -- AF34601 ##STR00178## 100 -- -- -- AF32579
##STR00179## 47 142 1.7 3 AF33068 ##STR00180## 170 -- -- -- AF33131
##STR00181## 158 -- 18 -- AF34351 ##STR00182## 50 -- -- -- AF34350
##STR00183## 267 -- 16 -- AF34753 ##STR00184## 73 -- -- 1.2 AF34757
##STR00185## 110 -- -- 2.7 AF35062 ##STR00186## 91 -- -- 1.6
AF35218 ##STR00187## 27 -- -- -- AF35462 ##STR00188## 194 -- -- --
AF35464 ##STR00189## 181 -- -- -- AF33197 ##STR00190## 80 -- -- --
AF34994 ##STR00191## 27 170 -- 3.7 AF35083 ##STR00192## 92 -- -- --
AF35525 ##STR00193## 57 57 -- 3 AF35526 ##STR00194## 900 800 -- 13
AF35563 ##STR00195## 135 47 -- 3 AF35575 ##STR00196## 127 57,000 --
1.1 AF35592 ##STR00197## 67 40 -- 1.3 AF35593 ##STR00198## 76 32 --
1.5 AF35594 ##STR00199## 54 40 -- 2.2 AF35219 ##STR00200## 403 --
-- -- AF32876 ##STR00201## 100 -- -- -- AF32881 ##STR00202## 100 --
-- 11 AF35179 ##STR00203## 57 -- -- 3.7 AF35180 ##STR00204## 303 --
-- -- AF35463 ##STR00205## 125 -- -- -- AF35090 ##STR00206## 35 190
-- 3.7 AF35148 ##STR00207## 3600 -- -- -- AF35149 ##STR00208## 43
57 -- 2 AF35168 ##STR00209## 1800 -- -- -- AF35361 ##STR00210## 77
-- -- -- AF35595 ##STR00211## 65 42 -- 1.5 AF35564 ##STR00212## 887
270 -- 4.5
Example 3
In Vivo Activity Assays
[0206] This example describes various in vivo assays that are
useful in evaluating the activity and potency of EPO-R agonist
peptides of the invention. EPO-R agonist peptide monomers and
dimers are prepared according to the methods provided in Example 1.
The in vivo activity of these peptide monomers and dimers is
evaluated using a series assays, including a polycythemic exhypoxic
mouse bioassay and a reticulocyte assay. These two assays are
described in further detail below.
1. Polycythemic Exhypoxic Mouse Bioassay
[0207] Test peptides are assayed for in vivo activity in the
polycythemic exhypoxic mouse bioassay adapted from the method
described by Cotes and Bangham (1961), Nature 191: 1065-1067. This
assay examines the ability of a test peptide to function as an EPO
mimetic: i.e., to activate EPO-R and induce new red blood cell
synthesis. Red blood cell synthesis is quantitated based upon
incorporation of radiolabeled iron into hemoglobin of the
synthesized red blood cells.
[0208] BDF1 mice are allowed to acclimate to ambient conditions for
7-10 days. Body weights are determined for all animals, and low
weight animals (<15 grams) are not used. Mice are subjected to
successive conditioning cycles in a hypobaric chamber for a total
of 14 days. Each 24 hour cycle consista of 18 hr at 0.40.+-.0.02%
atmospheric pressure and 6 hr at ambient pressure. After
conditioning the mice are maintained at ambient pressure for an
additional 72 hr prior to dosing.
[0209] Test peptides, or recombinant human EPO standards, are
diluted in PBS+0.1% BSA vehicle (PBS/BSA). Peptide monomer stock
solutions are first solubilized in dimethyl sulfoxide (DMSO).
Negative control groups include one group of mice injected with
PBS/BSA alone, and one group injected with 1% DMSO. Each dose group
containa 10 mice. Mice are injected subcutaneously (scruff of neck)
with 0.5 mL of the appropriate sample.
[0210] Forty eight hours following sample injection, the mice are
administered an intraperitoneal injection of 0.2 ml of Fe.sup.59
(Dupont, NEN), for a dose of approximately 0.75 .mu.Curies/mouse.
Mouse body weights are determined 24 hr after Fe.sup.59
administration, and the mice are sacrificed 48 hr after Fe.sup.59
administration. Blood is collected from each animal by cardiac
puncture and hematocrits are determined (heparin was used as the
anticoagulant). Each blood sample (0.2 ml) is analyzed for
Fe.sup.59 incorporation using a Packard gamma counter.
Non-responder mice (i.e., those mice with radioactive incorporation
less than the negative control group) are eliminated from the
appropriate data set. Mice that have hematocrit values less than
53% of the negative control group are also eliminated.
[0211] Results are derived from sets of 10 animals for each
experimental dose. The average amount of radioactivity incorporated
[counts per minute (CPM)] into blood samples from each group is
calculated.
2. Reticulocyte Assay
[0212] Normal BDF1 mice are dosed (0.5 mL, injected subcutaneously)
on three consecutive days with either EPO control or test peptide.
At day three, mice are also dosed (0.1 mL, injected
intraperitoneally) with iron dextran (100 mg/ml). At day five, mice
are anesthetized with CO.sub.2 and bled by cardiac puncture. The
percent (%) reticulocytes for each blood sample is determined by
thiazole orange staining and flow cytometer analysis (retic-count
program). Hematocrits are manually determined. The corrected
percent of reticulocytes is determined using the following
formula:
RETIC.sub.CORRECTED=%
RETIC.sub.OBSERVED.times.(Hematocrit.sub.INDIVIDUAL/Hematocrit.sub.NORMAL-
)
3. Hematological Assay
[0213] Normal CD1 mice are dosed with four weekly bolus intravenous
injections of either EPO positive control, test peptide, or
vehicle. A range of positive control and test peptide doses,
expressed as mg/kg, are tested by varying the active compound
concentration in the formulation. Volumes injected are 5 ml/kg. The
vehicle control group is comprised twelve animals, while 8 animals
are in each of the remaining dose groups. Daily viability and
weekly body weights are recorded.
[0214] The dosed mice are mice are fasted and then anesthetized
with inhaled isoflurane and terminal blood samples are collected
via cardiac or abdominal aorta puncture on Day 1 (for vehicle
control mice) and on Days 15 and 29 (4 mice/group/day). The blood
is transferred to Vacutainer.RTM. brand tubes. Preferred
anticoagulant is ethylenediaminetetraacetic acid (EDTA).
[0215] Blood samples are evaluated for endpoints measuring red
blood synthesis and physiology such as hematocrit (Hct), hemoglobin
(Hgb) and total erythrocyte count (RBC) using automated clinical
analysers well known in the art (e.g., those made by Coulter,
Inc.).
[0216] Data for representative EPO-R agonist peptides in this assay
are given in Table 4. Results are given as increase in percent (%)
hematocrit (Ht), relative to vehicle injected control mice, at day
15 and at day 29. The indicated peptide compounds were administered
to test mice at a dose of 1 mg/kg.
TABLE-US-00009 TABLE 4 In vivo hematological assay for peptide
dimers Increase Increase Compound in Ht (%) in Ht (%) designation
Peptide dimer at day 15 at day 29 AF35526 ##STR00213## 22.1 28.6
AF35527 ##STR00214## 6.8 15.9 AF35563 ##STR00215## 18.3 23.8
AF35594 ##STR00216## 7.9 12.8
[0217] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0218] It is further to be understood that all values are
approximate, and are provided for description.
[0219] Numerous references, including patents, patent applications,
and various publications are cited and discussed in the description
of this invention. The citation and/or discussion of such
references is provided merely to clarify the description of the
present invention and is not an admission that any such reference
is "prior art" to the present invention. All references cited and
discussed in this specification are incorporated herein by
reference in their entirety and to the same extent as if each
reference was individually incorporated by reference.
Sequence CWU 1
1
142117PRTartificialsynthetic peptide 1Leu Tyr Ala Cys His Xaa Gly
Pro Ile Thr Xaa Val Cys Gln Pro Leu 1 5 10 15 Arg
217PRTartificialsynthetic peptide 2Leu Tyr Ala Cys His Met Gly Pro
Ile Thr Trp Val Cys Gln Pro Leu 1 5 10 15 Arg
317PRTartificialsynthetic peptide 3Leu Tyr Ala Cys His Met Gly Pro
Ile Thr Xaa Val Cys Gln Pro Leu 1 5 10 15 Arg
417PRTartificialsynthetic peptide 4Leu Tyr Ala Cys His Met Gly Pro
Ile Thr Xaa Val Cys Gln Pro Leu 1 5 10 15 Arg
520PRTartificialaynthetic peptide 5Gly Gly Leu Tyr Ala Cys His Met
Gly Pro Ile Thr Trp Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
620PRTartificialsynthetic peptide 6Gly Gly Leu Tyr Ala Cys His Met
Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
720PRTartificialsynthetic peptide 7Gly Gly Leu Tyr Ala Cys His Met
Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
820PRTartificialsynthetic peptide 8Xaa Gly Leu Tyr Ala Cys His Met
Gly Pro Ile Thr Trp Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
920PRTartificialsynthetic peptide 9Xaa Gly Leu Tyr Ala Cys His Met
Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
1020PRTartificialsynthetic peptide 10Xaa Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
1120PRTartificialsynthetic peptide 11Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Trp Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa 20
1220PRTartificialsynthetic peptide 12Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa 20
1320PRTartificialsynthetic peptide 13Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa 20
1421PRTartificialsynthetic peptide 14Xaa Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Trp Val Cys Gln 1 5 10 15 Pro Leu Arg Gly Xaa
20 1521PRTartificialsynthetic peptide 15Xaa Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly Xaa
20 1621PRTartificialsynthetic peptide 16Xaa Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly Xaa
20 1717PRTartificialsynthetic peptide 17Leu Tyr Ala Cys His Xaa Gly
Pro Ile Thr Trp Val Cys Gln Pro Leu 1 5 10 15 Arg
1817PRTartificialsynthetic peptide 18Leu Tyr Ala Cys His Xaa Gly
Pro Ile Thr Xaa Val Cys Gln Pro Leu 1 5 10 15 Arg
1917PRTartificialsynthetic peptide 19Leu Tyr Ala Cys His Xaa Gly
Pro Ile Thr Xaa Val Cys Gln Pro Leu 1 5 10 15 Arg
2020PRTartificialsynthetic peptide 20Gly Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Trp Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
2120PRTartificialsynthetic peptide 21Gly Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
2220PRTartificialsynthetic peptide 22Gly Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
2320PRTartificialsynthetic peptide 23Xaa Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Trp Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
2420PRTartificialsynthetic peptide 24Xaa Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
2520PRTartificialsyntheric peptide 25Xaa Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
2620PRTartificialsynthetic peptide 26Gly Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Trp Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa 20
2720PRTartificialsynthetic peptide 27Gly Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa 20
2820PRTartificialsynthetic peptide 28Gly Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa 20
2921PRTartificialsynthetic peptide 29Xaa Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Trp Val Cys Gln 1 5 10 15 Pro Leu Arg Gly Xaa
20 3021PRTartificialsynthetic peptide 30Xaa Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly Xaa
20 3121PRTartificialsynthetic peptide 31Xaa Gly Leu Tyr Ala Cys His
Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly Xaa
20 3219PRTartificialsynthetic peptide 32Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Met Thr Val Cys Gln Pro 1 5 10 15 Leu Arg Gly
3320PRTartificialsynthetic peptide 33Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Met Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Gly 20
3421PRTartificialsynthetic peptide 34Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa Lys
20 3520PRTartificialsynthetic peptide 35Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa 20
3621PRTartificialsynthetic peptide 36Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa Lys
20 3720PRTartificialsynthetic peptide 37Ser Arg Thr Arg Tyr Arg Cys
Glu Met Gly Pro Leu Thr Trp Val Cys 1 5 10 15 Arg Arg Trp Lys 20
3822PRTartificialsythetic peptide 38Leu Thr Arg Leu Tyr Ser Cys His
Met Gly Pro Ser Thr Trp Val Cys 1 5 10 15 Ser Thr Ala Leu Arg Lys
20 3923PRTartificialsynthetic peptide 39Arg Gly Gln Leu Tyr Ala Cys
His Phe Gly Pro Val Thr Trp Val Cys 1 5 10 15 Arg Arg Arg Arg Arg
Val Lys 20 4023PRTartificialsynthetic peptide 40Ser Gly Ile Leu Tyr
Glu Cys His Met Gly Pro Leu Thr Trp Val Cys 1 5 10 15 Thr Pro Ser
Arg Arg Arg Lys 20 4123PRTartificialsynthetic peptide 41Leu Gly Arg
Arg Tyr Ser Cys His Phe Gly Ala Leu Thr Trp Val Cys 1 5 10 15 Gln
Pro Ala Arg Arg Asp Lys 20 4221PRTartificialsynthetic peptide 42Gly
Ser Arg Thr Tyr Ser Cys Gln Leu Gly Pro Val Asp Trp Val Cys 1 5 10
15 Gly Arg Arg Arg Lys 20 4323PRTartificialsynthetic peptide 43Ala
Arg Gly Arg Tyr Gln Cys Gln Phe Gly Pro Leu Thr Trp Glu Cys 1 5 10
15 Ala Pro Ile Arg Pro Arg Lys 20 4419PRTartificialsynthetic
peptide 44Val Thr Arg Met Tyr Arg Cys Arg Met Gly Pro Leu Thr Trp
Val Cys 1 5 10 15 Glu Arg Lys 4523PRTartificialsynthetic peptide
45Arg Pro Ser Leu Tyr Glu Cys His Leu Gly Pro Leu Thr Trp Glu Cys 1
5 10 15 Arg Pro Arg Arg Arg Glu Lys 20 4623PRTartificialsynthetic
peptide 46Arg Gly His Met Tyr Ser Cys Gln Leu Gly Pro Val Thr Trp
Val Cys 1 5 10 15 Arg Pro Leu Ser Gly Arg Lys 20
4725PRTartificialsynthetic peptide 47Ile Thr Pro Thr Tyr His Cys
Arg Phe Gly Pro Gln Thr Trp Val Cys 1 5 10 15 Ala Pro Arg Arg Ser
Ala Leu Thr Lys 20 25 4823PRTartificialsynthetic peptide 48Gly Asn
Arg Met Tyr Gln Cys His Met Gly Pro Leu Thr Trp Val Cys 1 5 10 15
Gln Pro Thr Arg Ile His Lys 20 4923PRTartificialsynthetic peptide
49Ala Arg Gly Arg Tyr Gln Cys Gln Phe Gly Pro Leu Thr Trp Glu Cys 1
5 10 15 Leu Pro Ile Arg Pro Arg Lys 20 5019PRTartificialsynthetic
peptide 50Val Thr Arg Met Tyr Arg Cys Arg Met Gly Pro Leu Thr Trp
Val Cys 1 5 10 15 Glu Arg Lys 5123PRTartificialsynthetic peptide
51Arg Pro Ser Leu Tyr Glu Cys His Leu Gly Pro Leu Thr Trp Glu Cys 1
5 10 15 Arg Pro Arg Arg Arg Glu Lys 20 5223PRTartificialsynthetic
peptide 52Arg Gly His Met Tyr Ser Cys Gln Leu Gly Pro Val Thr Trp
Val Cys 1 5 10 15 Arg Pro Leu Ser Gly Arg Lys 20
5325PRTartificialsynthetic peptide 53Ile Thr Pro Thr Tyr His Cys
Arg Phe Gly Pro Gln Thr Trp Val Cys 1 5 10 15 Ala Pro Arg Arg Ser
Ala Leu Thr Lys 20 25 5423PRTartificialsynthetic peptide 54Gly Asn
Arg Met Tyr Gln Cys His Met Gly Pro Leu Thr Trp Val Cys 1 5 10 15
Gln Pro Thr Arg Ile His Lys 20 5523PRTartificialsynthetic peptide
55Arg Asn His Leu Tyr Gly Cys Arg Met Gly Pro Leu Thr Trp Val Cys 1
5 10 15 Ser Ser Arg Gly Thr Gln Lys 20 5621PRTartificialsynthetic
peptide 56Pro Asp Leu Ala Tyr Ser Cys Arg Met Gly Pro Leu Thr Trp
Val Cys 1 5 10 15 Ala Pro Asn Arg Lys 20 5723PRTartificialsynthetic
peptide 57Leu Gly Arg Arg Tyr Ser Cys His Phe Gly Pro Leu Thr Trp
Val Cys 1 5 10 15 Gln Pro Ala Arg Arg Asp Lys 20
5823PRTartificialsynthetic peptide 58Leu Leu Arg Gly Tyr Glu Cys
Tyr Met Gly Pro Leu Thr Trp Val Cys 1 5 10 15 Arg Ser Ser Arg Pro
Arg Lys 20 5922PRTartificialsynthetic peptide 59Met Arg Thr Arg Tyr
Arg Cys Tyr Met Gly Pro Leu Thr Trp Val Cys 1 5 10 15 Glu Gly Ser
Arg Leu Lys 20 6023PRTartificialsynthetic peptide 60His Leu Gly Arg
Tyr Asp Cys Ser Phe Gly Pro Gln Thr Trp Val Cys 1 5 10 15 Arg Pro
Arg Arg Ser Leu Lys 20 6123PRTartificialsynthetic peptide 61Ile Arg
Gly Arg Asn Arg Cys Arg Phe Gly Pro Gln Thr Trp Val Cys 1 5 10 15
Pro Asp Ser Tyr Glu Phe Lys 20 6225PRTartificialsynthetic peptide
62Gln Arg Arg His Val Phe Leu Ser Asp Gly Ala Ala Tyr Val Gly Leu 1
5 10 15 Trp Val Glu Cys Asp Asp Ile Ser Lys 20 25
6324PRTartificialsynthetic peptide 63Val Leu Pro Leu Tyr Arg Cys
Arg Met Gly Arg Glu Thr Trp Glu Cys 1 5 10 15 Met Arg Ala Ala Gly
Val Thr Lys 20 6423PRTartificialsynthetic peptide 64Pro Gly Asn Ser
Tyr Arg Cys His Met Gly Pro Leu Thr Trp Val Cys 1 5 10 15 Gly Arg
Asp Arg His Leu Lys 20 6523PRTartificialsynthetic peptide 65Arg Asn
His Leu Tyr Gly Cys Arg Met Gly Pro Leu Thr Trp Val Cys 1 5 10 15
Ser Ser Arg Gly Thr Gln Lys 20 6621PRTartificialsynthetic peptide
66Pro Asp Leu Ala Tyr Ser Cys Arg Met Gly Pro Leu Thr Trp Val Cys 1
5 10 15 Ala Pro Asn Arg Lys 20 6723PRTartificialsynthetic peptide
67Leu Gly Arg Arg Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys 1
5 10 15 Gln Pro Ala Arg Arg Asp Lys 20 6823PRTartificialsynthetic
peptide 68Leu Leu Arg Gly Tyr Glu Cys Tyr Met Gly Pro Leu Thr Trp
Val Cys 1 5 10 15 Arg Ser Ser Arg Pro Arg Lys 20
6922PRTartificialsynthetic peptide 69Met Arg Thr Arg Tyr Arg Cys
Tyr Met Gly Pro Leu Thr Trp Val Cys 1 5 10 15 Glu Gly Ser Arg Leu
Lys 20 7023PRTartificialsynthetic peptide 70His Leu Gly Arg Tyr Asp
Cys Ser Phe Gly Pro Gln Thr Trp Val Cys 1 5 10 15 Arg Pro Arg Arg
Ser Leu Lys 20 7123PRTartificialsynthetic peptide 71Ile Arg Gly Arg
Asn Arg Cys Arg Phe Gly Pro Gln Thr Trp Val Cys 1 5 10 15 Pro Asp
Ser Tyr Glu Phe Lys 20 7223PRTartificialsynthetic peptide 72Arg Pro
Arg Pro Tyr Ser Cys Thr Met Gly Pro Arg Thr Trp Val Cys 1 5 10 15
Gly Gly Val Arg Ala Gly Lys 20 7324PRTartificialsynthetic peptide
73Val Leu Pro Leu Tyr Arg Cys Arg Met Gly Arg Glu Thr Trp Glu Cys 1
5 10 15 Met Arg Ala Ala Gly Val Thr Lys 20
7422PRTartificialsynthetic peptide 74Pro Gly Asn Ser Tyr Arg Cys
Met Gly Pro Leu Thr Trp Val Cys Gly 1 5 10 15 Arg Asp Arg His Leu
Lys 20 7521PRTartificialsynthetic peptide 75Gly Gly Xaa Tyr Ala Cys
His Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa
Lys 20 7621PRTartificialsynthetic peptide 76Gly Gly Leu Tyr Ala Cys
His Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa
Lys 20 7721PRTartificialsynthetic peptide 77Gly Gly Leu Tyr Ala Xaa
His Met Gly Pro Ile Thr Xaa Val Xaa Gln 1 5 10 15 Pro Leu Arg Xaa
Lys 20 7821PRTartificialsynthetic peptide 78Gly Gly Leu Tyr Ala Xaa
His Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa
Lys 20 7921PRTartificialsynthetic peptide 79Gly Gly Leu Tyr Ala Cys
His Met Gly Pro Ile Thr Xaa Val Xaa Gln 1 5 10 15 Pro Leu Arg Xaa
Lys 20 8021PRTartificialsynthetic peptide 80Gly Gly Leu Tyr Ala Cys
His Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Xaa Leu Arg Xaa
Lys 20 8117PRTartificialsynthetic peptide 81Glu Tyr Leu Cys Arg Met
Gly Pro Ile Thr Trp Val Cys Glu Arg Tyr 1 5 10 15 Lys
8217PRTartificialsynthetic peptide 82Thr Tyr Ser Cys His Phe Gly
Pro Leu Thr Trp Val Cys Arg Pro Gln 1 5 10 15 Lys
8317PRTartificialsynthetic peptide 83Asp Tyr His Cys Arg Met Gly
Pro Leu Thr Trp Val Cys Arg Pro Leu 1 5 10 15 Lys
8417PRTartificialsynthetic peptide 84Leu Tyr Glu Cys Arg Met Gly
Pro Met Thr Trp Val Cys Arg Pro Gly 1 5 10 15 Lys
8517PRTartificialsynthetic peptide 85Leu Tyr Leu Cys Arg Met Gly
Pro Val Thr Trp Glu Cys Gln Pro Arg 1 5 10 15 Lys
8617PRTartificialsynthetic peptide 86Asp Tyr Asn Cys Arg Phe Gly
Pro Leu Thr Trp Val Cys Arg Pro Ser 1 5 10 15 Lys
8717PRTartificialsynthetic peptide 87Ser Tyr Leu Cys Arg Arg Gly
Pro Thr Thr Trp Leu Cys Thr Ala Gln 1 5 10 15 Lys
8817PRTartificialsynthetic peptide 88Glu Tyr Ser
Cys Arg Met Gly Pro Met Thr Trp Val Cys Ser Pro Thr 1 5 10 15 Lys
8916PRTartificialsynthetic peptide 89Leu Tyr Leu Cys Arg Phe Gly
Pro Val Thr Trp Asp Cys Gly Tyr Lys 1 5 10 15
9017PRTartificialsynthetic peptide 90Ile Tyr Arg Cys Leu Met Gly
Pro Leu Thr Trp Val Cys Thr Pro Asp 1 5 10 15 Lys
9121PRTartificialsynthetic peptide 91Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Xaa Arg Xaa Lys
20 9221PRTartificialsynthetic peptide 92Gly Gly Leu Tyr Ala Cys His
Met Gly Xaa Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa Lys
20 9321PRTartificialsynthetic peptide 93Gly Gly Leu Tyr Ala Cys Xaa
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa Lys
20 9421PRTartificialsynthetic peptide 94Gly Gly Leu Tyr Xaa Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa Lys
20 9521PRTartificialsynthetic peptide 95Arg Thr Arg Glu Tyr Ser Cys
Gln Met Gly Pro Leu Thr Trp Thr Cys 1 5 10 15 Val Pro Arg Ser Lys
20 9621PRTartificialsynthetic peptide 96Ser Arg Ala Arg Tyr Met Cys
His Met Gly Pro Leu Thr Trp Val Cys 1 5 10 15 Arg Pro Glu Val Lys
20 9723PRTartificialsynthetic peptide 97Gly Gly Arg Ala Tyr Met Cys
Arg Leu Gly Pro Val Thr Trp Val Cys 1 5 10 15 Ser Pro Arg Ile Arg
Ile Lys 20 9823PRTartificialsynthetic peptide 98Thr Ile Ala Gln Tyr
Ile Cys Tyr Met Gly Pro Glu Thr Trp Glu Cys 1 5 10 15 Arg Pro Ser
Pro Arg Ala Lys 20 9923PRTartificialsynthetic peptide 99Asn Gly Arg
Thr Tyr Ser Cys Gln Leu Gly Pro Val Thr Trp Val Cys 1 5 10 15 Ser
Arg Gly Val Arg Arg Lys 20 10022PRTartificialsynthetic peptide
100Met Arg Thr Arg Tyr Arg Cys Tyr Met Gly Pro Leu Thr Trp Val Cys
1 5 10 15 Glu Gly Ser Arg Leu Lys 20 10120PRTartificialsynthetic
peptide 101Ser Arg Thr Arg Tyr Arg Cys Glu Met Gly Pro Leu Thr Trp
Val Cys 1 5 10 15 Glu Arg Trp Lys 20 10221PRTartificialsynthetic
peptide 102Gly Ser Arg Thr Tyr Ser Cys Gln Leu Gly Pro Val Thr Trp
Val Cys 1 5 10 15 Gly Arg Arg Arg Lys 20
10323PRTartificialsynthetic peptide 103Arg Pro Arg Pro Tyr Ser Cys
Thr Met Gly Pro Arg Thr Trp Val Cys 1 5 10 15 Gly Gly Val Arg Ala
Gly Lys 20 10421PRTartificialsynthetic peptide 104Gly Gly Thr Tyr
Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Arg 1 5 10 15 Pro Gln
Gly Gly Lys 20 10521PRTartificialsynthetic peptide 105Gly Gly Asp
Tyr His Cys Arg Met Gly Pro Leu Thr Trp Val Cys Arg 1 5 10 15 Pro
Leu Gly Gly Lys 20 10621PRTartificialsynthetic peptide 106Gly Gly
Val Tyr Ala Cys Arg Met Gly Pro Ile Thr Trp Val Cys Ser 1 5 10 15
Pro Leu Gly Gly Lys 20 10721PRTartificialsynthetic peptide 107Gly
Gly Leu Tyr Ala Cys His Met Gly Pro Ile Tyr Xaa Val Cys Glu 1 5 10
15 Pro Leu Arg Xaa Lys 20 10821PRTartificialsynthetic peptide
108Gly Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln
1 5 10 15 Pro Leu Arg Xaa Xaa 20 10921PRTartificialsynthetic
peptide 109Gly Gly Leu Tyr Ala Cys His Met Gly Pro Ile Xaa Xaa Val
Cys Gln 1 5 10 15 Pro Leu Arg Xaa Lys 20
11021PRTartificialsynthetic peptide 110Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Xaa 1 5 10 15 Pro Leu Arg Xaa Lys
20 11121PRTartificialsynthetic peptide 111Gly Gly Leu Xaa Ala Cys
His Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Xaa
Lys 20 11221PRTartificialsynthetic peptide 112Gly Gly Leu Tyr Ala
Cys His Met Gly Pro Xaa Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg
Xaa Lys 20 11321PRTartificialsynthetic peptide 113Gly Gly Leu Tyr
Ala Cys His Met Gly Pro Ile Thr Xaa Xaa Cys Gln 1 5 10 15 Pro Leu
Arg Xaa Lys 20 11421PRTartificialsynthetic peptide 114Gly Gly Leu
Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro
Leu Arg Xaa Lys 20 11522PRTartificialsynthetic peptide 115Gly Gly
Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys 1 5 10 15
Gln Pro Leu Arg Xaa Lys 20 11622PRTartificialsynthetic peptide
116Gly Gly Leu Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys
1 5 10 15 Gln Pro Leu Arg Xaa Lys 20 11722PRTartificialsynthetic
peptide 117Gly Gly Leu Tyr Tyr Ala Cys His Met Gly Pro Ile Thr Xaa
Val Cys 1 5 10 15 Gln Pro Leu Arg Xaa Lys 20
11822PRTartificialsynthetic peptide 118Gly Gly Leu Tyr Ala Ala Cys
His Met Gly Pro Ile Thr Xaa Val Cys 1 5 10 15 Gln Pro Leu Arg Xaa
Lys 20 11922PRTartificialsynthetic peptide 119Gly Gly Leu Tyr Ala
Cys His His Met Gly Pro Ile Thr Xaa Val Cys 1 5 10 15 Gln Pro Leu
Arg Xaa Lys 20 12022PRTartificialsynthetic peptide 120Gly Gly Leu
Tyr Ala Cys His Met Met Gly Pro Ile Thr Xaa Val Cys 1 5 10 15 Gln
Pro Leu Arg Xaa Lys 20 12117PRTartificialsynthetic peptide 121Asn
Tyr Thr Cys Arg Phe Gly Pro Leu Thr Trp Glu Cys Thr Pro Gln 1 5 10
15 Lys 12217PRTartificialsynthetic peptide 122Ser Trp Asp Cys Arg
Ile Gly Pro Ile Thr Trp Val Cys Arg Trp Ser 1 5 10 15 Lys
12317PRTartificialsynthetic peptide 123Asn Tyr Met Cys His Phe Gly
Pro Ile Thr Trp Val Cys Arg Pro Gly 1 5 10 15 Lys
12417PRTartificialsynthetic peptide 124Leu Tyr Leu Cys Arg Met Gly
Pro Gln Thr Trp Met Cys Gln Pro Gly 1 5 10 15 Lys
12517PRTartificialsynthetic peptide 125Trp Tyr Ser Cys Leu Met Gly
Pro Met Thr Trp Val Cys Arg Ala His 1 5 10 15 Lys
12617PRTartificialsynthetic peptide 126Glu Tyr Phe Cys Arg Met Gly
Pro Ile Thr Trp Val Cys Gln Arg Ser 1 5 10 15 Lys
12722PRTartificialsynthetic peptide 127Gly Gly Leu Tyr Ala Cys His
Met Gly Gly Pro Ile Thr Xaa Val Cys 1 5 10 15 Gln Pro Leu Arg Xaa
Lys 20 12822PRTartificialsynthetic sequence 128Gly Gly Leu Tyr Ala
Cys His Met Gly Pro Ile Ile Thr Xaa Val Cys 1 5 10 15 Gln Pro Leu
Arg Xaa Lys 20 12922PRTartificialsynthetic peptide 129Gly Gly Leu
Tyr Ala Cys His Met Gly Pro Ile Thr Thr Xaa Val Cys 1 5 10 15 Gln
Pro Leu Arg Xaa Lys 20 13022PRTartificialsynthetic peptide 130Gly
Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Val Cys 1 5 10
15 Gln Pro Leu Arg Xaa Lys 20 13122PRTartificialsynthetic 131Gly
Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10
15 Gln Pro Leu Arg Xaa Lys 20 13222PRTartificialsynthetic peptide
132Gly Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln
1 5 10 15 Pro Pro Leu Arg Xaa Lys 20 13322PRTartificialsynthetic
peptide 133Gly Gly Leu Tyr Ala Cys His Met Gly Pro Ile Thr Xaa Val
Cys Gln 1 5 10 15 Pro Leu Leu Arg Xaa Lys 20
13422PRTartificialsynthetic peptide 134Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg Arg Xaa
Lys 20 13522PRTartificialsynthetic peptide 135Gly Gly Leu Tyr Ala
Cys His Met Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro Leu Arg
Xaa Xaa Lys 20 13621PRTartificialsynthetic peptide 136Gly Gly Leu
Tyr Ala Cys His Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15 Pro
Leu Arg Xaa Lys 20 13721PRTartificialsynthetic peptide 137Gly Gly
Leu Tyr Ala Cys His Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10 15
Pro Leu Arg Xaa Lys 20 13821PRTartificialsynthetic peptide 138Gly
Gly Leu Tyr Ala Cys His Xaa Gly Pro Ile Thr Xaa Val Cys Gln 1 5 10
15 Pro Leu Arg Xaa Lys 20 13921PRTartificialsynthetic peptide
139Gly Gly Leu Tyr Ala Xaa His Met Gly Pro Ile Thr Xaa Val Xaa Gln
1 5 10 15 Pro Leu Arg Xaa Lys 20 14021PRTartificialsynthetic
peptide 140Gly Gly Leu Tyr Ala Xaa His Met Gly Pro Ile Thr Xaa Val
Cys Gln 1 5 10 15 Pro Leu Arg Xaa Lys 20
14121PRTartificialsynthetic peptide 141Gly Gly Leu Tyr Ala Xaa His
Met Gly Pro Ile Thr Xaa Val Xaa Gln 1 5 10 15 Pro Leu Arg Xaa Lys
20 14221PRTartificialsynthetic peptide 142Gly Gly Leu Tyr Ala Xaa
His Met Gly Pro Ile Thr Xaa Val Xaa Gln 1 5 10 15 Pro Leu Arg Xaa
Lys 20
* * * * *