U.S. patent application number 11/702156 was filed with the patent office on 2007-08-09 for long-acting veterinary polypeptides and methods of producing and administering same.
Invention is credited to Fuad Fares, Udi Eyal Fima.
Application Number | 20070184530 11/702156 |
Document ID | / |
Family ID | 38345655 |
Filed Date | 2007-08-09 |
United States Patent
Application |
20070184530 |
Kind Code |
A1 |
Fares; Fuad ; et
al. |
August 9, 2007 |
Long-acting veterinary polypeptides and methods of producing and
administering same
Abstract
A polypeptide and polynucleotides comprising at least two
carboxy-terminal peptides (CTP) of chorionic gonadotrophin attached
to a non-human peptide-of-interest are disclosed. Pharmaceutical
compositions comprising the non-human polypeptides and
polynucleotides of the invention and methods of using both human
and non-human polypeptides and polynucleotides are also
disclosed.
Inventors: |
Fares; Fuad; (Hourfish
Village, IL) ; Fima; Udi Eyal; (Beer-Sheva,
IL) |
Correspondence
Address: |
PEARL COHEN ZEDEK LATZER, LLP
1500 BROADWAY 12TH FLOOR
NEW YORK
NY
10036
US
|
Family ID: |
38345655 |
Appl. No.: |
11/702156 |
Filed: |
February 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60764761 |
Feb 3, 2006 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/397; 530/399; 536/23.5 |
Current CPC
Class: |
C07K 2319/31 20130101;
C07K 2319/00 20130101; A61P 5/06 20180101; A61P 35/00 20180101;
A61P 7/00 20180101; A61P 3/00 20180101; C07K 14/59 20130101; A61P
3/04 20180101; C07K 14/61 20130101; C07K 14/555 20130101; A61K
38/00 20130101; A61P 31/12 20180101; A61P 43/00 20180101; C07K
14/505 20130101; A61P 7/06 20180101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325; 530/397; 530/399; 536/023.5 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07H 21/04 20060101 C07H021/04; C07K 14/59 20060101
C07K014/59; C07K 14/51 20060101 C07K014/51 |
Claims
1. A polypeptide comprising a non-human peptide of interest and at
least two chorionic gonadotrophin carboxy terminal peptides,
wherein a first chorionic gonadotrophin carboxy terminal peptide of
said at least two chorionic gonadotrophin carboxy terminal peptides
is attached to an amino terminus of said non-human peptide of
interest, and a second chorionic gonadotrophin carboxy terminal
peptide of said at least two chorionic gonadotrophin carboxy
terminal peptides is attached to a carboxy terminus of said
non-human peptide of interest.
2. The polypeptide of claim 1, wherein the sequence of at least 1
of said at least two chorionic gonadotrophin carboxy terminal
peptides comprises an amino acid sequence selected from sequences
set forth in SEQ ID NO: 17 and SEQ ID NO: 18.
3. The polypeptide of claim 1, wherein at least 1 of said at least
two chorionic gonadotrophin carboxy terminal peptides is
truncated.
4. The polypeptide of claim 1, wherein said non-human peptide of
interest is a GH peptide.
5. The polypeptide of claim 1, wherein said non-human peptide of
interest is an EPO peptide.
6. The polypeptide of claim 1, wherein said non-human peptide of
interest is selected from an interferon peptide and a GLP-1
peptide.
7. The polypeptide of claim 1, wherein said non-human peptide of
interest is glycosylated.
8. The polypeptide of claim 1, wherein said non-human peptide of
interest is non-glycosylated.
9. The polypeptide of claim 1, wherein at least 1 of said at least
two chorionic gonadotrophin carboxy terminal peptides is
glycosylated.
10. The polypeptide of claim 1, wherein at least 1 of said at least
two chorionic gonadotrophin carboxy terminal peptides is attached
to said non-human peptide of interest via a linker.
11. The polypeptide of claim 10, wherein said linker is a peptide
bond.
12. The polypeptide of claim 1, further comprising a signal
peptide.
13. The polypeptide of claim 12, wherein said signal peptide is as
set forth in SEQ ID NO: 19.
14. The polypeptide of claim 1, wherein said polypeptide further
comprises a third chorionic gonadotrophin carboxy terminal peptide
attached in tandem to said second chorionic gonadotrophin carboxy
terminal peptide.
15. A polynucleotide comprising a coding portion encoding a
polypeptide, said polypeptide comprising a non-human peptide of
interest and at least two chorionic gonadotrophin carboxy terminal
peptides, wherein a first chorionic gonadotrophin carboxy terminal
peptide of said at least two chorionic gonadotrophin carboxy
terminal peptides is attached to an amino terminus of said
non-human peptide of interest, and a second chorionic gonadotrophin
carboxy terminal peptide of said at least two chorionic
gonadotrophin carboxy terminal peptides is attached to the carboxy
terminus of said non-human peptide of interest.
16. The polynucleotide of claim 15, wherein said polypeptide
further comprises a third chorionic gonadotrophin carboxy terminal
peptide attached in tandem to said second chorionic gonadotrophin
carboxy terminal peptide.
17. The polynucleotide of claim 15, wherein said non-human peptide
of interest is a GH peptide.
18. The polynucleotide of claim 15, wherein said non-human peptide
of interest is an EPO peptide.
19. The polynucleotide of claim 15, wherein said non-human peptide
of interest is selected from an interferon peptide and a GLP-1
peptide.
20. The polynucleotide of claim 15, wherein said non-human
polypeptide further comprises a signal peptide.
21. The polynucleotide of claim 20, wherein the sequence of said
signal peptide is as set forth in SEQ ID NO: 19.
22. A cell comprising the expression vector of claim 22.
23. A veterinary pharmaceutical composition comprising the
expression vector of claim 22.
24. A method of treating or reducing the incidence associated with
a growth, weight-related, or metabolic condition in a non-human
subject, said method comprising administering to said subject a
therapeutically effective amount of a polypeptide, said polypeptide
comprising: (a) a peptide of interest and (b) at least two
chorionic gonadotrophin carboxy terminal peptides, wherein: a first
chorionic gonadotrophin carboxy terminal peptide of said at least
two chorionic gonadotrophin carboxy terminal peptides is attached
to an amino terminus of said peptide of interest, and a second
chorionic gonadotrophin carboxy terminal peptide of said at least
two chorionic gonadotrophin carboxy terminal peptides is attached
to the carboxy terminus of said peptide of interest, thereby
treating said non-human subject having a growth, weight-related, or
metabolic condition.
25. A method of administering a peptide of interest to a non-human
subject in need thereof, comprising the step of attaching a first
chorionic gonadotrophin carboxy terminal peptide to an amino
terminus of said peptide of interest and a second chorionic
gonadotrophin carboxy terminal peptide to the carboxy terminus of
said peptide of interest, thereby generating an improved
polypeptide for administration to said subject, thereby
administering a peptide of interest to a subject in need
thereof.
26. The method of claim 25, further comprising the step of
attaching a third chorionic gonadotrophin carboxy terminal peptide
in tandem to said second chorionic gonadotrophin carboxy terminal
peptide.
27. The method of claim 25, wherein the sequence of at least one of
said chorionic gonadotrophin carboxy terminal peptides comprises an
amino acid sequence selected from the sequences as set forth in SEQ
ID NO: 17-18.
28. The method of claim 25, wherein said peptide of interest is a
GH peptide.
29. The method of claim 25, wherein said peptide of interest is an
EPO peptide.
30. The method of claim 25, wherein said peptide of interest is
selected from an interferon peptide and a GLP-1 peptide.
31. The method of claim 25, wherein said peptide of interest is
glycosylated.
32. The method of claim 25, wherein said improved polypeptide
further comprises a signal peptide.
33. The method of claim 32, wherein the sequence of said signal
peptide is as set forth in SEQ ID NO: 19.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of U.S. Provisional
Application Ser. No. 60/764,761, filed Feb. 3, 2006, which is
hereby incorporated in its entirety by reference herein.
FIELD OF INVENTION
[0002] A polypeptide and polynucleotides encoding same comprising
at least two carboxy-terminal peptides (CTP) of chorionic
gonadotrophin attached to a peptide-of-interest are disclosed.
Pharmaceutical compositions comprising the polypeptide and
polynucleotides of the invention and methods of using same are also
disclosed.
BACKGROUND OF THE INVENTION
[0003] Polypeptides are susceptible to denaturation or enzymatic
degradation in the blood, liver or kidney. Accordingly,
polypeptides typically have short circulatory half-lives of several
hours. Because of their low stability, peptide drugs are usually
delivered in a sustained frequency so as to maintain an effective
plasma concentration of the active peptide. Moreover, since peptide
drugs are usually administrated by infusion, frequent injection of
peptide drugs cause considerable discomfort to a subject. Thus,
there is a need for technologies that will prolong the half-lives
of therapeutic polypeptides while maintaining a high
pharmacological efficacy thereof. Such desirous peptide drugs
should also meet the requirements of enhanced serum stability, high
activity and a low probability of inducing an undesired immune
response when injected into a subject.
[0004] Unfavorable pharmacokinetics, such as a short serum
half-life, can prevent the pharmaceutical development of many
otherwise promising drug candidates. Serum half-life is an
empirical characteristic of a molecule, and must be determined
experimentally for each new potential drug. For example, with lower
molecular weight polypeptide drugs, physiological clearance
mechanisms such as renal filtration can make the maintenance of
therapeutic levels of a drug unfeasible because of cost or
frequency of the required dosing regimen. Conversely, a long serum
half-life is undesirable where a drug or its metabolites have toxic
side effects.
SUMMARY OF THE INVENTION
[0005] In one embodiment, the present invention provides a
polypeptide comprising at least two chorionic gonadotrophin carboxy
terminal peptide (CTP) attached to a non-human peptide of
interest.
[0006] In another embodiment, the present invention provides a
polypeptide comprising a first chorionic gonadotrophin CTP attached
to an amino terminus of a non-human peptide of interest and a
second chorionic gonadotrophin CTP attached to a carboxy terminus
of a polypeptide sequence of interest.
[0007] In another embodiment, the present invention provides a
polypeptide, comprising two chorionic gonadotrophin CTP attached to
a carboxy terminus of a non-human peptide of interest.
[0008] In another embodiment, the present invention provides a
polypeptide comprising a first chorionic gonadotrophin CTP attached
to an amino terminus of non-human peptide of interest, and a second
and third chorionic gonadotrophin CTP attached to a carboxy
terminus of a polypeptide sequence of interest.
[0009] In another embodiment, the present invention provides a
polypeptide comprising at least three chorionic gonadotrophin CTP
attached to a non-human peptide of interest.
[0010] In another embodiment, the present invention provides a
polynucleotide comprising a sequence encoding a polypeptide,
comprising at least two chorionic gonadotrophin CTP attached to a
non-human peptide of interest.
[0011] In another embodiment, the present invention provides a
polynucleotide comprising a sequence encoding a first chorionic
gonadotrophin CTP attached to an amino terminus of non-human
peptide of interest and a second chorionic gonadotrophin CTP
attached to a carboxy terminus of a polypeptide sequence of
interest.
[0012] In another embodiment, the present invention provides a
polynucleotide comprising a sequence encoding two chorionic
gonadotrophin CTP attached to a carboxy terminus of non-human
peptide of interest.
[0013] In another embodiment, the present invention provides a
polynucleotide comprising a sequence encoding a first chorionic
gonadotrophin CTP attached to an amino terminus of non-human
peptide of interest, and a second and third chorionic gonadotrophin
CTP attached to a carboxy terminus of a polypeptide sequence of
interest.
[0014] In another embodiment, the present invention provides a
polynucleotide comprising a sequence encoding at least three
chorionic gonadotrophin CTP attached to a non-human peptide of
interest.
[0015] In another embodiment, the present invention provides a
method of treating or reducing the incidence associated with a
growth, weight-related, or metabolic condition in a non-human
subject, comprising administering to a subject a therapeutically
effective amount of CTP-GH, thereby treating a non-human subject
having a growth, weight-related, or metabolic condition. In another
embodiment, the GH peptide is a non-human GH peptide. In another
embodiment, the GH peptide is a human peptide. Each possibility
represents a separate embodiment of the present invention.
[0016] In another embodiment, the present invention provides a
method of improving a biological half life of a non-human peptide
of interest, comprising the step of attaching at least two
chorionic gonadotrophin CTP sequences to the non-human peptide of
interest, thereby improving a biological half life of a non-human
peptide of interest.
[0017] In another embodiment, the present invention provides a
method of administering a peptide of interest to a non-human
subject in need thereof, comprising the step of attaching at least
two chorionic gonadotrophin CTP sequences to the peptide of
interest, thereby administering a peptide of interest to a
non-human subject in need thereof. In another embodiment, the
peptide of interest is a non-human peptide of interest. In another
embodiment, the peptide of interest is a human peptide of interest.
Each possibility represents a separate embodiment of the present
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-1F are diagrams illustrating six EPO-CTP
constructs.
[0019] FIG. 1A--is a diagram of the polypeptide of SEQ ID NO: 1
[0020] FIG. 1B is a diagram of the polypeptide of SEQ ID NO: 2
[0021] FIG. 1C is a diagram of the polypeptide of SEQ ID NO: 3
[0022] FIG. 1D is a diagram of the polypeptide of SEQ ID NO: 4.
[0023] FIG. 1E is a diagram of the polypeptide of SEQ ID NO: 5.
[0024] FIG. 1F is a diagram of the polypeptide of SEQ ID NO: 6.
[0025] FIG. 2 is a photograph illustrating the expression of the
EPO-CTP variants from transfected DG44 cells. Final test samples
from transfected cells were prepared as described under "sample
preparation" and run on SDS/PAGE. Proteins were detected by western
blot.
[0026] FIG. 3 is a graph illustrating the in vivo bioactivity of
recombinant hEPO derivatives and EPO-3 (SEQ ID NO: 3). ICR mice
(n=7/group) received a single IV injection/week (15 .mu.g/kg) for
three weeks of EPO-3, rhEPO-WT (SEQ ID NO: 16), Recormon
(Commercial EPO) or Recormon (5 .mu.g/kg) 3 times a week. Control
animals were injected IV with PBS. Blood samples were collected
three times a week and haematocrit levels were detected. Each point
represents the group average of haematocrit (%).+-.SE.
[0027] FIG. 4 is a graph illustrating the in vivo bioactivity of
recombinant hEPO derivatives and EPO-1 (SEQ ID NO: 1). ICR mice
(n=7/group) received a single IV injection/week (15 .mu.g/kg) for
three weeks of EPO-1, rhEPO-WT (SEQ ID NO: 16), Recormon or
Recormon (5 .mu.g/kg) 3 times a week. Control animals were injected
IV with PBS. Blood samples were collected three times a week and
haematocrit levels were detected. Each point represents the group
average of haematocrit (%).+-.SE.
[0028] FIG. 5 is a graph illustrating the in vivo bioactivity of
recombinant hEPO derivatives and EPO-2 (SEQ ID NO: 2). ICR mice
(n=7/group) received a single IV injection/week (15 .mu.g/kg) for
three weeks of EPO-2 (SEQ ID NO: 2), rhEPO-WT (SEQ ID NO: 16),
Recormon or Recormon (5 .mu.g/kg) 3 times a week. Control animals
were injected IV with PBS. Blood samples were collected three times
a week and haematocrit levels were detected. Each point represents
the group average of haematocrit (%).+-.SE.
[0029] FIG. 6 is a time graph illustrating the change in
reticulocyte level following a single bolus dose of EPO-0 (SEQ ID
NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp.
[0030] FIG. 7 is a time graph illustrating the change in hemoglobin
level (presented as change from baseline) following a single bolus
dose of EPO-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and
Aranesp.
[0031] FIG. 8 is a time graph illustrating the change in hematocrit
level following a single bolus dose of EPO-0 (SEQ ID NO: 16), EPO-3
(SEQ ID NO: 3) and Aranesp.
[0032] FIG. 9 is a graph illustrating the change in serum
concentration of EPO-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and
Aranesp post i.v. injection.
[0033] FIG. 10 is a Western blot illustrating the molecular weight
& identity of MOD-4020 (SEQ ID NO: 36), MOD-4021 (SEQ ID NO:
37), MOD-4022 (SEQ ID NO: 38), MOD-4023 (SEQ ID NO: 39) and
MOD-4024 (SEQ ID NO: 40). PAGE SDS gel was blotted and stained
using monoclonal anti-hGH antibodies. The photograph indicates that
like commercial and wild type hGH, MOD-7020-4 variants are
recognized by anti-hGH antibodies.
[0034] FIG. 11 is a bar graph illustrating the weight gain of
hypophysectomized rats following administration of the GH-CTP
polypeptides of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0035] In one embodiment, the present invention describes
long-acting polypeptides and methods of producing and using same.
In another embodiment, long-acting polypeptides comprise carboxy
terminal peptide (CTP) of human Chorionic Gonadotropin (hCG). In
another embodiment, CTP acts as a protectant against degradation of
proteins or peptides derived therefrom. In another embodiment, CTP
extends circulatory half-lives of proteins or peptides derived
therefrom. In some embodiments, CTP enhances the potency of
proteins or peptides derived therefrom.
[0036] In another embodiment, "CTP peptide," "carboxy terminal
peptide," and "CTP sequence" are used interchangeably herein. In
another embodiment, the carboxy terminal peptide is a full-length
CTP. In another embodiment, the carboxy terminal peptide is a
truncated CTP. Each possibility represents a separate embodiment of
the present invention.
[0037] In another embodiment, "signal sequence" and "signal
peptide" are used interchangeably herein. In another embodiment,
"sequence" when in reference to a polynucleotide can refer to a
coding portion. Each possibility represents a separate embodiment
of the present invention.
[0038] In another embodiment, "peptide of interest" and
"polypeptide sequence-of-interest" are used interchangeably herein.
In another embodiment, the peptide of interest is a full-length
protein. In another embodiment, the peptide of interest is a
protein fragment. Each possibility represents a separate embodiment
of the present invention.
[0039] In another embodiment, a polypeptide comprising at least two
carboxy-terminal peptide (CTP) sequences of chorionic gonadotrophin
attached to a polypeptide sequence-of-interest, wherein a first CTP
sequence of the at least two CTP sequences is attached to an amino
terminus of the polypeptide sequence of interest and a second CTP
sequence of the at least two CTP sequences is attached to the
carboxy terminus of the polypeptide sequence of interest is
provided. In another embodiment, the carboxy-terminal peptide (CTP)
sequence is of human chorionic gonadotrophin.
[0040] In another embodiment, the carboxy-terminal peptide (CTP) is
attached to the polypeptide sequence of interest via a linker. In
another embodiment, the linker which connects the CTP sequence to
the polypeptide sequence of interest is a covalent bond. In another
embodiment, the linker which connects the CTP sequence to the
polypeptide sequence of interest is a peptide bond. In another
embodiment, the linker which connects the CTP sequence to the
polypeptide sequence of interest is a substituted peptide bond.
[0041] The phrase "polypeptide sequence of interest" refers, in
another embodiment, to any polypeptide sequence, such as one
comprising a biological activity. In another embodiment, the
peptide is glycosylated. In another embodiment, the peptide is
non-glycosylated. Examples of polypeptides which benefit from an
extension in their circulatory half-life include, but are not
limited to erythropoietin (EPO), interferons, human growth hormone
(hGH) and glucagon-like peptide-1 (GLP-1).
[0042] In another embodiment, the carboxy terminal peptide (CTP) of
human Chorionic Gonadotropin (hCG) is fused to a protein. In
another embodiment, the carboxy terminal peptide (CTP) of human hCG
is fused to a glycoprotein. In another embodiment, the carboxy
terminal peptide (CTP) of hCG is fused to a glycoprotein hormone.
In another embodiment, the CTP of hCG is fused to a peptide derived
from a glycoprotein hormone. In some embodiments, glycoprotein
hormones comprise EPO, FSH, or TSH and peptides derived
therefrom.
[0043] In some embodiments, a CTP sequences at both the amino
terminal end of a polypeptide and at the carboxy terminal end of
the polypeptide provide enhanced protection against degradation of
a protein. In some embodiments, CTP sequences at both the amino
terminal end of a polypeptide and at the carboxy terminal end of
the polypeptide provide extended half-life of the attached
protein.
[0044] In some embodiments, a CTP sequence at the amino terminal
end of a polypeptide, a CTP sequence at the carboxy terminal end of
the polypeptide, and at least one additional CTP sequence attached
in tandem to the CTP sequence at the carboxy terminus provide
enhanced protection against degradation of a protein. In some
embodiments, a CTP sequence at the amino terminal end of a
polypeptide, a CTP sequence at the carboxy terminal end of the
polypeptide, and at least one additional CTP sequence attached in
tandem to the CTP sequence at the carboxy terminus provide extended
half-life of the attached protein. In some embodiments, a CTP
sequence at the amino terminal end of a polypeptide, a CTP sequence
at the carboxy terminal end of the polypeptide, and at least one
additional CTP sequence attached in tandem to the CTP sequence at
the carboxy terminus provide enhanced activity of the attached
protein.
[0045] In some embodiments, a CTP sequence at the amino terminal
end of a polypeptide, a CTP sequence at the carboxy terminal end of
the polypeptide, and at least one additional CTP sequence attached
in tandem to the CTP sequence at the amino terminus provide
enhanced protection against degradation of the attached protein. In
some embodiments, a CTP sequence at the amino terminal end of a
polypeptide, a CTP sequence at the carboxy terminal end of the
polypeptide, and at least one additional CTP sequence attached in
tandem to the CTP sequence at the amino terminus provide extended
half-life of the attached protein. In some embodiments, a CTP
sequence at the amino terminal end of a polypeptide, a CTP sequence
at the carboxy terminal end of the polypeptide, and at least one
additional CTP sequence attached in tandem to the CTP sequence at
the amino terminus provide enhanced activity the attached
protein.
[0046] In another embodiment, the carboxy terminal peptide (CTP)
peptide of the present invention comprises the amino acid sequence
from amino acid 112 to position 145 of human chorionic
gonadotrophin, as set forth in SEQ ID NO: 17. In another
embodiment, the CTP sequence of the present invention comprises the
amino acid sequence from amino acid 118 to position 145 of human
chorionic gonadotropin, as set forth in SEQ ID NO: 18. In another
embodiment, the CTP sequence also commences from any position
between positions 112-118 and terminates at position 145 of human
chorionic gonadotrophin. In some embodiments, the CTP sequence
peptide is 28, 29, 30, 31, 32, 33 or 34 amino acids long and
commences at position 112, 113, 114, 115, 116, 117 or 118 of the
CTP amino acid sequence.
[0047] In another embodiment, the CTP peptide is a variant of
chorionic gonadotrophin CTP which differs from the native CTP by
1-5 conservative amino acid substitutions as described in U.S. Pat.
No. 5,712,122. In another embodiment, the CTP peptide is a variant
of chorionic gonadotrophin CTP which differs from the native CTP by
1 conservative amino acid substitution. In another embodiment, the
CTP peptide is a valiant of chorionic gonadotrophin CTP which
differs from the native CTP by 2 conservative amino acid
substitutions. In another embodiment, the CTP peptide is a variant
of chorionic gonadotrophin CTP which differs from the native CTP by
3 conservative amino acid substitutions. In another embodiment, the
CTP peptide is a variant of chorionic gonadotrophin CTP which
differs from the native CTP by 4 conservative amino acid
substitutions. In another embodiment, the CTP peptide is a variant
of chorionic gonadotrophin CTP which differs from the native CTP by
5 conservative amino acid substitutions. In another embodiment, the
CTP peptide amino acid sequence of the present invention is at
least 70% homologous to the native CTP amino acid sequence or a
peptide thereof. In another embodiment, the CTP peptide amino acid
sequence of the present invention is at least 80% homologous to the
native CTP amino acid sequence or a peptide thereof. In another
embodiment, the CTP peptide amino acid sequence of the present
invention is at least 90% homologous to the native CTP amino acid
sequence or a peptide thereof. In another embodiment, the CTP
peptide amino acid sequence of the present invention is at least
95% homologous to the native CTP amino acid sequence or a peptide
thereof.
[0048] In another embodiment, the CTP peptide DNA sequence of the
present invention is at least 70% homologous to the native CTP DNA
sequence or a peptide thereof. In another embodiment, the CTP
peptide DNA sequence of the present invention is at least 80%
homologous to the native CTP DNA sequence or a peptide thereof. In
another embodiment, the CTP peptide DNA sequence of the present
invention is at least 90% homologous to the native CTP DNA sequence
or a peptide thereof. In another embodiment, the CTP peptide DNA
sequence of the present invention is at least 95% homologous to the
native CTP DNA sequence or a peptide thereof.
[0049] In one embodiment, at least one of the chorionic
gonadotrophin CTP amino acid sequences is truncated. In another
embodiment, both of the chorionic gonadotrophin CTP amino acid
sequences are truncated. In another embodiment, 2 of the chorionic
gonadotrophin CTP amino acid sequences are truncated. In another
embodiment, 2 or more of the chorionic gonadotrophin CTP amino acid
sequences are truncated. In another embodiment, all of the
chorionic gonadotrophin CTP amino acid sequences are truncated. In
one embodiment, the truncated CTP comprises the first 10 amino
acids of SEQ ID NO:43. In one embodiment, the truncated CTP
comprises the first 11 amino acids of SEQ ID NO:43. In one
embodiment, the truncated CTP comprises the first 12 amino acids of
SEQ ID NO:43. In one embodiment, the truncated CTP comprises the
first 13 amino acids of SEQ ID NO:43. In one embodiment, the
truncated CTP comprises the first 14 amino acids of SEQ ID NO:43.
In one embodiment, the truncated CTP comprises the first 15 amino
acids of SEQ ID NO:43. In one embodiment, the truncated CTP
comprises the first 16 amino acids of SEQ ID NO:43. In one
embodiment, the truncated CTP comprises the last 14 amino acids of
SEQ ID NO:43.
[0050] In one embodiment, at least one of the chorionic
gonadotrophin CTP amino acid sequences is glycosylated. In another
embodiment, both of the chorionic gonadotrophin CTP amino acid
sequences are glycosylated. In another embodiment, 2 of the
chorionic gonadotrophin CTP amino acid sequences are glycosylated.
In another embodiment, 2 or more of the chorionic gonadotrophin CTP
amino acid sequences are glycosylated. In another embodiment, all
of the chorionic gonadotrophin CTP amino acid sequences are
glycosylated. In one embodiment, the CTP sequence of the present
invention comprises at least one glycosylation site. In one
embodiment, the CTP sequence of the present invention comprises 2
glycosylation sites. In one embodiment, the CTP sequence of the
present invention comprises 3 glycosylation sites. In one
embodiment, the CTP sequence of the present invention comprises 4
glycosylation sites. Each possibility represents a separate
embodiment of the present invention.
[0051] As provided herein, attachment of CTP sequence to both the
amino and carboxy termini of the EPO protein results in increased
potency at stimulating erythropoiesis (FIGS. 3-5) and (Table 6 of
Example 4), as compared to recombinant EPO and other combinations
of EPO and CTP. In some embodiments, an EPO attached to three CTP
sequences does not impair binding to its receptor as evidenced in
Table 4 of Example 3, which demonstrates that EPO attached to three
CTP sequences is equally effective at stimulating proliferation of
TF-1 cells as wild-type EPO.
[0052] In some embodiments, "homology" according to the present
invention also encompasses deletions, insertions, or substitution
variants, including an amino acid substitution, thereof and
biologically active polypeptide fragments thereof. In one
embodiment the substitution variant comprises a glycine in position
104 of erythropoietin amino acid sequence is substituted by a
serine (SEQ ID NO: 22).
[0053] In another embodiment, the methods of the present invention
provide an EPO peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for the treatment of anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
the treatment of anemia. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 1
having additionally at least one CTP amino acid peptide on the
N-terminus for the treatment of anemia. In another embodiment, the
methods of the present invention provide an EPO peptide set forth
in SEQ ID NO: 1 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one additional CTP amino
acid peptide on the C-terminus for the treatment of anemia. In
another embodiment, the methods of the present invention provide an
EPO peptide set forth in SEQ ID NO: 2 having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for the treatment of anemia.
In another embodiment, the methods of the present invention provide
an EPO peptide set forth in SEQ ID NO: 3 having additionally at
least one CTP amino acid peptide on the N-terminus and at least one
CTP amino acid peptide on the C-terminus for the treatment of
anemia. In another embodiment, the methods of the present invention
provide an EPO peptide set forth in SEQ ID NO: 4 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of anemia. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 5
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of anemia. In another embodiment, the
methods of the present invention provide an EPO peptide set forth
in SEQ ID NO: 6 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for the treatment of anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 16 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of anemia. In
another embodiment, the methods of the present invention provide an
EPO peptide set forth in SEQ ID NO: 22 having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for the treatment of
anemia.
[0054] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding an EPO peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of anemia. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding an EPO
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
the treatment of anemia. In another embodiment, the methods of the
present invention provide a nucleic acid set forth in SEQ ID NO: 20
encoding an EPO peptide and one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of anemia. In another embodiment, the
methods of the present invention provide a nucleic acid set forth
in SEQ ID NO: 21 encoding an EPO peptide and one CTP amino acid
peptide on the N-terminus and two CTP amino acid peptides on the
C-terminus for the treatment of anemia.
[0055] In another embodiment, the methods of the present invention
provide an EPO peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for inhibiting anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
inhibiting anemia. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 1
having additionally at least one CTP amino acid peptide on the
N-terminus for inhibiting anemia. In another embodiment, the
methods of the present invention provide an EPO peptide set forth
in SEQ ID NO: 1 having additionally at least one CTP amino acid
peptide on the N-terminus and at least additional one CTP amino
acid peptide on the C-terminus for inhibiting anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 2 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 3 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 4 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 5 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 6 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 16 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 22 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting anemia.
[0056] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding an EPO peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
inhibiting anemia. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding an EPO
peptide having one CTP amino acid peptide on the N-terminus and two
CTP amino acid peptides on the C-terminus for inhibiting anemia. In
another embodiment, the methods of the present invention provide a
nucleic acid set forth in SEQ ID NO: 20 encoding an EPO peptide and
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for inhibiting anemia. In
another embodiment, the methods of the present invention provide a
nucleic acid set forth in SEQ ID NO: 21 encoding an EPO peptide and
one CTP amino acid peptide on the N-terminus and two CTP amino acid
peptides on the C-terminus for inhibiting anemia.
[0057] In another embodiment, the methods of the present invention
provide an EPO peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for the treatment of tumor-associated
anemia. In another embodiment, the methods of the present invention
provide an EPO peptide having additionally one CTP amino acid
peptide on the N-terminus and two CTP amino acid peptides on the
C-terminus for the treatment of tumor-associated anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 1 having additionally at least one
CTP amino acid peptide on the N-terminus for the treatment of
tumor-associated anemia. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 1
having additionally at least one CTP amino acid peptide on the
N-terminus and at least additional one CTP amino acid peptide on
the C-terminus for the treatment of tumor-associated anemia. In
another embodiment, the methods of the present invention provide an
EPO peptide set forth in SEQ ID NO: 2 having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for the treatment of
tumor-associated anemia. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 3
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of tumor-associated anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 4 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of
tumor-associated anemia. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 5
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of tumor-associated anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 6 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of
tumor-associated anemia. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 16
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of tumor-associated anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 22 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of
tumor-associated anemia.
[0058] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding an EPO peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of tumor-associated anemia. In another embodiment, the
methods of the present invention provide a nucleic acid sequence
encoding an EPO peptide having additionally one CTP amino acid
peptide on the N-terminus and two CTP amino acid peptides on the
C-terminus for the treatment of tumor-associated anemia. In another
embodiment, the methods of the present invention provide a nucleic
acid set forth in SEQ ID NO: 20 encoding an EPO peptide having
additionally one CTP amino acid peptide on the N-terminus and at
least one CTP amino acid peptide on the C-terminus for the
treatment of tumor-associated anemia. In another embodiment, the
methods of the present invention provide a nucleic acid set forth
in SEQ ID NO: 21 encoding an EPO peptide having additionally one
CTP amino acid peptide on the N-terminus and two CTP amino acid
peptides on the C-terminus for the treatment of tumor-associated
anemia.
[0059] In another embodiment, the methods of the present invention
provide an EPO peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for inhibiting tumor-associated anemia.
In another embodiment, the methods of the present invention provide
an EPO peptide having additionally one CTP amino acid peptide on
the N-terminus and two CTP amino acid peptides on the C-terminus
for inhibiting tumor-associated anemia. In another embodiment, the
methods of the present invention provide an EPO peptide set forth
in SEQ ID NO: 1 having additionally at least one CTP amino acid
peptide on the N-terminus for inhibiting tumor-associated anemia.
In another embodiment, the methods of the present invention provide
an EPO peptide set forth in SEQ ID NO: 1 having additionally at
least one CTP amino acid peptide on the N-terminus and at least
additional one CTP amino acid peptide on the C-terminus for
inhibiting tumor-associated anemia. In another embodiment, the
methods of the present invention provide an EPO peptide set forth
in SEQ ID NO: 2 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for inhibiting tumor-associated anemia. In
another embodiment, the methods of the present invention provide an
EPO peptide set forth in SEQ ID NO: 3 having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for inhibiting
tumor-associated anemia. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 4
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for inhibiting tumor-associated anemia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 5 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting tumor-associated
anemia. In another embodiment, the methods of the present invention
provide an EPO peptide set forth in SEQ ID NO: 6 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
inhibiting tumor-associated anemia. In another embodiment, the
methods of the present invention provide an EPO peptide set forth
in SEQ ID NO: 16 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for inhibiting tumor-associated anemia. In
another embodiment, the methods of the present invention provide an
EPO peptide set forth in SEQ ID NO: 22 having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for inhibiting
tumor-associated anemia.
[0060] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding an EPO peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
inhibiting tumor-associated anemia. In another embodiment, the
methods of the present invention provide a nucleic acid sequence
encoding an EPO peptide having additionally one CTP amino acid
peptide on the N-terminus and two CTP amino acid peptides on the
C-terminus for inhibiting tumor-associated anemia. In another
embodiment, the methods of the present invention provide a nucleic
acid set forth in SEQ ID NO: 20 encoding an EPO peptide and one CTP
amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting tumor-associated
anemia. In another embodiment, the methods of the present invention
provide a nucleic acid set forth in SEQ ID NO: 21 encoding an EPO
peptide and one CTP amino acid peptide on the N-terminus and two
CTP amino acid peptides on the C-terminus for inhibiting
tumor-associated anemia.
[0061] In another embodiment, the methods of the present invention
provide an EPO peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for the treatment of tumor hypoxia. In
another embodiment, the methods of the present invention provide an
EPO peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
the treatment of tumor hypoxia. In another embodiment, the methods
of the present invention provide an EPO peptide set forth in SEQ ID
NO: 1 having additionally at least one CTP amino acid peptide on
the N-terminus for the treatment of tumor hypoxia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 1 having additionally at least one
CTP amino acid peptide on the N-terminus and at least additional
one CTP amino acid peptide on the C-terminus for the treatment of
tumor hypoxia. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 2 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of tumor hypoxia. In another embodiment, the methods of
the present invention provide an EPO peptide set forth in SEQ ID
NO: 3 having additionally at least one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of tumor hypoxia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 4 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of tumor hypoxia.
In another embodiment, the methods of the present invention provide
an EPO peptide set forth in SEQ ID NO: 5 having additionally at
least one CTP amino acid peptide on the N-terminus and at least one
CTP amino acid peptide on the C-terminus for the treatment of tumor
hypoxia. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 6 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of tumor hypoxia. In another embodiment, the methods of
the present invention provide an EPO peptide set forth in SEQ ID
NO: 16 having additionally at least one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of tumor hypoxia. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 22 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of tumor
hypoxia.
[0062] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding an EPO peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of tumor hypoxia. In another embodiment, the methods of
the present invention provide a nucleic acid sequence encoding an
EPO peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
the treatment of tumor hypoxia. In another embodiment, the methods
of the present invention provide a nucleic acid set forth in SEQ ID
NO: 20 encoding an EPO peptide and one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of tumor hypoxia. In another
embodiment, the methods of the present invention provide a nucleic
acid set forth in SEQ ID NO: 21 encoding an EPO peptide having
additionally one CTP amino acid peptide on the N-terminus and two
CTP amino acid peptides on the C-terminus for the treatment of
tumor hypoxia.
[0063] In another embodiment, the methods of the present invention
provide an EPO peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for the treatment of chronic infections
such as HIV, inflammatory bowel disease, or septic episodes. In
another embodiment, the methods of the present invention provide an
EPO peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
the treatment of chronic infections such as HIV, inflammatory bowel
disease, or septic episodes. In another embodiment, the methods of
the present invention provide an EPO peptide set forth in SEQ ID
NO: 1 having additionally at least one CTP amino acid peptide on
the N-terminus for the treatment of chronic infections such as HIV,
inflammatory bowel disease, or septic episodes. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 1 having additionally at least one
CTP amino acid peptide on the N-terminus and at least additional
one CTP amino acid peptide on the C-terminus for the treatment of
chronic infections such as HIV, inflammatory bowel disease, or
septic episodes. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 2 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of chronic infections such as HIV, inflammatory bowel
disease, or septic episodes. In another embodiment, the methods of
the present invention provide an EPO peptide set forth in SEQ ID
NO: 3 having additionally at least one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of chronic infections such as HIV,
inflammatory bowel disease, or septic episodes. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 4 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of chronic
infections such as HIV, inflammatory bowel disease, or septic
episodes. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 5 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of chronic infections such as HIV, inflammatory bowel
disease, or septic episodes. In another embodiment, the methods of
the present invention provide an EPO peptide set forth in SEQ ID
NO: 6 having additionally at least one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of chronic infections such as HIV,
inflammatory bowel disease, or septic episodes. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 16 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of chronic
infections such as HIV, inflammatory bowel disease, or septic
episodes. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 22 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of chronic infections such as HIV, inflammatory bowel
disease, or septic episodes.
[0064] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding an EPO peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of chronic infections such as HIV, inflammatory bowel
disease, or septic episodes. In another embodiment, the methods of
the present invention provide a nucleic acid sequence encoding an
EPO peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
the treatment of chronic infections such as HIV, inflammatory bowel
disease, or septic episodes. In another embodiment, the methods of
the present invention provide a nucleic acid set forth in SEQ ID
NO: 20 encoding an EPO peptide and one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of chronic infections such as HIV,
inflammatory bowel disease, or septic episodes. In another
embodiment, the methods of the present invention provide a nucleic
acid set forth in SEQ ID NO: 21 encoding an EPO peptide and one CTP
amino acid peptide on the N-terminus and two CTP amino acid
peptides on the C-terminus for the treatment of chronic infections
such as HIV, inflammatory bowel disease, or septic episodes.
[0065] In another embodiment, the methods of the present invention
provide an EPO peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for inhibiting chronic infections such as
HIV, inflammatory bowel disease, or septic episodes. In another
embodiment, the methods of the present invention provide an EPO
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
inhibiting chronic infections such as HIV, inflammatory bowel
disease, or septic episodes. In another embodiment, the methods of
the present invention provide an EPO peptide set forth in SEQ ID
NO: 1 having additionally at least one CTP amino acid peptide on
the N-terminus for inhibiting chronic infections such as HIV,
inflammatory bowel disease, or septic episodes. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 1 having additionally at least one
CTP amino acid peptide on the N-terminus and at least additional
one CTP amino acid peptide on the C-terminus for inhibiting chronic
infections such as HIV, inflammatory bowel disease, or septic
episodes. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 2 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
inhibiting chronic infections such as HIV, inflammatory bowel
disease, or septic episodes. In another embodiment, the methods of
the present invention provide an EPO peptide set forth in SEQ ID
NO: 3 having additionally at least one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for inhibiting chronic infections such as HIV,
inflammatory bowel disease, or septic episodes. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 4 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting chronic infections
such as HIV, inflammatory bowel disease, or septic episodes. In
another embodiment, the methods of the present invention provide an
EPO peptide set forth in SEQ ID NO: 5 having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for inhibiting chronic
infections such as HIV, inflammatory bowel disease, or septic
episodes. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 6 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
inhibiting chronic infections such as HIV, inflammatory bowel
disease, or septic episodes. In another embodiment, the methods of
the present invention provide an EPO peptide set forth in SEQ ID
NO: 16 having additionally at least one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for inhibiting chronic infections such as HIV,
inflammatory bowel disease, or septic episodes. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 22 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for inhibiting chronic infections
such as HIV, inflammatory bowel disease, or septic episodes.
[0066] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding an EPO peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
inhibiting chronic infections such as HIV, inflammatory bowel
disease, or septic episodes. In another embodiment, the methods of
the present invention provide a nucleic acid sequence encoding an
EPO peptide having one CTP amino acid peptide on the N-terminus and
two CTP amino acid peptides on the C-terminus for inhibiting
chronic infections such as HIV, inflammatory bowel disease, or
septic episodes. In another embodiment, the methods of the present
invention provide a nucleic acid set forth in SEQ ID NO: 20
encoding an EPO peptide and one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for inhibiting chronic infections such as HIV,
inflammatory bowel disease, or septic episodes. In another
embodiment, the methods of the present invention provide a nucleic
acid set forth in SEQ ID NO: 21 encoding an EPO peptide and one CTP
amino acid peptide on the N-terminus and two CTP amino acid
peptides on the C-terminus for inhibiting chronic infections such
as HIV, inflammatory bowel disease, or septic episodes.
[0067] In another embodiment, the methods of the present invention
provide an EPO peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for the treatment of fatigue syndrome
following cancer chemotherapy. In another embodiment, the methods
of the present invention provide an EPO peptide having additionally
one CTP amino acid peptide on the N-terminus and two CTP amino acid
peptides on the C-terminus for the treatment of fatigue syndrome
following cancer chemotherapy. In another embodiment, the methods
of the present invention provide an EPO peptide set forth in SEQ ID
NO: 1 having additionally at least one CTP amino acid peptide on
the N-terminus for the treatment of fatigue syndrome following
cancer chemotherapy. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 1
having additionally at least one CTP amino acid peptide on the
N-terminus and at least additional one CTP amino acid peptide on
the C-terminus for the treatment of fatigue syndrome following
cancer chemotherapy. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 2
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of fatigue syndrome following cancer
chemotherapy. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 3 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of fatigue syndrome following cancer chemotherapy. In
another embodiment, the methods of the present invention provide an
EPO peptide set forth in SEQ ID NO: 4 having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for the treatment of fatigue
syndrome following cancer chemotherapy. In another embodiment, the
methods of the present invention provide an EPO peptide set forth
in SEQ ID NO: 5 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for the treatment of fatigue syndrome following
cancer chemotherapy. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 6
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of fatigue syndrome following cancer
chemotherapy. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 16 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of fatigue syndrome following cancer chemotherapy. In
another embodiment, the methods of the present invention provide an
EPO peptide set forth in SEQ ID NO: 22 having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for the treatment of fatigue
syndrome following cancer chemotherapy.
[0068] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding an EPO peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for the
treatment of fatigue syndrome following cancer chemotherapy. In
another embodiment, the methods of the present invention provide a
nucleic acid sequence encoding an EPO peptide having additionally
one CTP amino acid peptide on the N-terminus and two CTP amino acid
peptides on the C-terminus for the treatment of fatigue syndrome
following cancer chemotherapy. In another embodiment, the methods
of the present invention provide a nucleic acid set forth in SEQ ID
NO: 20 encoding an EPO peptide and one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of fatigue syndrome following cancer
chemotherapy. In another embodiment, the methods of the present
invention provide a nucleic acid set forth in SEQ ID NO: 21
encoding an EPO peptide and one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
the treatment of fatigue syndrome following cancer
chemotherapy.
[0069] In another embodiment, the methods of the present invention
provide an EPO peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for improving stem cell engraftment. In
another embodiment, the methods of the present invention provide an
EPO peptide having additionally one to CTP amino acid peptide on
the N-terminus and two CTP amino acid peptides on the C-terminus
for improving stem cell engraftment. In another embodiment, the
methods of the present invention provide an EPO peptide set forth
in SEQ ID NO: 1 having additionally at least one CTP amino acid
peptide on the N-terminus for improving stem cell engraftment. In
another embodiment, the methods of the present invention provide an
EPO peptide set forth in SEQ ID NO: 1 having additionally at least
one CTP amino acid peptide on the N-terminus and at least
additional one CTP amino acid peptide on the C-terminus for
improving stem cell engraftment. In another embodiment, the methods
of the present invention provide an EPO peptide set forth in SEQ ID
NO: 2 having additionally at least one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for improving stem cell engraftment. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 3 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for improving stem cell engraftment.
In another embodiment, the methods of the present invention provide
an EPO peptide set forth in SEQ ID NO: 4 having additionally at
least one CTP amino acid peptide on the N-terminus and at least one
CTP amino acid peptide on the C-terminus for improving stem cell
engraftment. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 5 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
improving stem cell engraftment. In another embodiment, the methods
of the present invention provide an EPO peptide set forth in SEQ ID
NO: 6 having additionally at least one CTP amino acid peptide on
the N-terminus and at least one CTP amino acid peptide on the
C-terminus for improving stem cell engraftment. In another
embodiment, the methods of the present invention provide an EPO
peptide set forth in SEQ ID NO: 16 having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for improving stem cell engraftment.
In another embodiment, the methods of the present invention provide
an EPO peptide set forth in SEQ ID NO: 22 having additionally at
least one CTP amino acid peptide on the N-terminus and at least one
CTP amino acid peptide on the C-terminus for improving stem cell
engraftment.
[0070] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding an EPO peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
improving stem cell engraftment. In another embodiment, the methods
of the present invention provide a nucleic acid sequence encoding
an EPO peptide having additionally one CTP amino acid peptide on
the N-terminus and two CTP amino acid peptides on the C-terminus
for improving stem cell to engraftment. In another embodiment, the
methods of the present invention provide a nucleic acid set forth
in SEQ ID NO: 20 encoding an EPO peptide and one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for improving stem cell engraftment. In another
embodiment, the methods of the present invention provide a nucleic
acid set forth in SEQ ID NO: 21 encoding an EPO peptide and one CTP
amino acid peptide on the N-terminus and two CTP amino acid
peptides on the C-terminus for improving stem cell engraftment.
[0071] In another embodiment, the methods of the present invention
provide an EPO peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for increasing the survival rate of a
patient with aplastic anemia or myelodysplastic syndrome. In
another embodiment, the methods of the present invention provide an
EPO peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
increasing the survival rate of a patient with aplastic anemia or
myelodysplastic syndrome. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 1
having additionally at least one CTP amino acid peptide on the
N-terminus for increasing the survival rate of a patient with
aplastic anemia or myelodysplastic syndrome. In another embodiment,
the methods of the present invention provide an EPO peptide set
forth in SEQ ID NO: 1 having additionally at least one CTP amino
acid peptide on the N-terminus and at least additional one CTP
amino acid peptide on the C-terminus for increasing the survival
rate of a patient with aplastic anemia or myelodysplastic syndrome.
In another embodiment, the methods of the present invention provide
an EPO peptide set forth in SEQ ID NO: 2 having additionally at
least one CTP amino acid peptide on the N-terminus and at least one
CTP amino acid peptide on the C-terminus for increasing the
survival rate of a patient with aplastic anemia or myelodysplastic
syndrome. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 3 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
increasing the survival rate of a patient with aplastic anemia or
myelodysplastic syndrome. In another embodiment, the methods of the
present invention provide an EPO peptide set forth in SEQ ID NO: 4
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for increasing the survival rate of a patient with
aplastic anemia or myelodysplastic syndrome. In another embodiment,
the methods of the present invention provide an EPO peptide set
forth in SEQ ID NO: 5 having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for increasing the survival rate of a
patient with aplastic anemia or myelodysplastic syndrome. In
another embodiment, the methods of the present invention provide an
EPO peptide set forth in SEQ ID NO: 6 having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for increasing the survival
rate of a patient with aplastic anemia or myelodysplastic syndrome.
In another embodiment, the methods of the present invention provide
an EPO peptide set forth in SEQ ID NO: 16 having additionally at
least one CTP amino acid peptide on the N-terminus and at least one
CTP amino acid peptide on the C-terminus for increasing the
survival rate of a patient with aplastic anemia or myelodysplastic
syndrome. In another embodiment, the methods of the present
invention provide an EPO peptide set forth in SEQ ID NO: 22 having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
increasing the survival rate of a patient with aplastic anemia or
myelodysplastic syndrome.
[0072] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding an EPO peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
increasing the survival rate of a patient with aplastic anemia or
myelodysplastic syndrome. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding an EPO
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
increasing the survival rate of a patient with aplastic anemia or
myelodysplastic syndrome. In another embodiment, the methods of the
present invention provide a nucleic acid set forth in SEQ ID NO: 20
encoding an EPO peptide and one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for increasing the survival rate of a patient with
aplastic anemia or myelodysplastic syndrome. In another embodiment,
the methods of the present invention provide a nucleic acid set
forth in SEQ ID NO: 21 encoding an EPO peptide and one CTP amino
acid peptide on the N-terminus and two CTP amino acid peptides on
the C-terminus for increasing the survival rate of a patient with
aplastic anemia or myelodysplastic syndrome.
[0073] In some embodiments, human growth hormone (hGH) is utilized
according to the teachings of the present invention. In some
embodiments, the attachment of CTP sequence to both the amino and
carboxy termini of the hGH protein results in increased potency
(FIG. 11). In some embodiments, the attachment of CTP sequence to
both the amino and carboxy termini of the hGH protein results in
prolonged in-vivo activity. In one embodiment, CTP-hGH polypeptides
of the present invention are set forth in SEQ ID NO: 39-41.
[0074] As provided herein, growth gain was demonstrated in
hipophysectomized rats (which have no growth hormone secretion)
following injections of CTP-hGH.
[0075] In one embodiment, the phrase "human growth hormone" (hGH)
refers to a polypeptide, such as set forth in Genbank Accession No.
P01241 (SEQ ID NO: 47), exhibiting hGH activity (i.e. stimulation
of growth).
[0076] In another embodiment, "human growth hormone" (hGH) refers
to a polypeptide, such as set forth in Genbank Accession No.
P01241, exhibiting hGH activity (i.e. stimulation of growth). In
another embodiment, "GH" of the present invention also refers to
homologues. In another embodiment, a GH amino acid sequence of
methods and compositions the present invention is at least 50%
homologous to a GH sequence set forth herein as determined using
BlastP software of the National Center of Biotechnology Information
(NCBI) using default parameters. In another embodiment, the percent
homology is 60%. In another embodiment, the percent homology is
70%. In another embodiment, the percent homology is 80%. In another
embodiment, the percent homology is 90%. In another embodiment, the
percent homology is at least 95%. In another embodiment, the
percent homology is greater than 95%. Each possibility represents a
separate embodiment of the present invention.
[0077] Exemplary CTP-GH polypeptides and CTP-hGH polypeptides of
the present invention are set forth in SEQ ID NO: 39, SEQ ID NO: 40
and SEQ ID NO: 41.
[0078] In another embodiment, the methods of the present invention
provide a growth hormone (GH) peptide having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for stimulating muscle growth.
In another embodiment, the methods of the present invention provide
a GH peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
stimulating muscle growth. In another embodiment, the methods of
the present invention provide a GH peptide set forth in SEQ ID NO:
23 having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for stimulating muscle growth. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for stimulating muscle growth. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 37 having additionally at least one
CTP amino acid peptide on the N-terminus for stimulating muscle
growth. In another embodiment, the methods of the present invention
provide a GH peptide set forth in SEQ ID NO: 38, having
additionally at least one CTP amino acid peptide on the N-terminus
for stimulating muscle growth. In another embodiment, the methods
of the present invention provide a GH peptide set forth in SEQ ID
NO: 39 for stimulating muscle growth. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 40 for stimulating muscle growth. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 41 for stimulating muscle growth. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 42 having additionally at least one
CTP amino acid peptide on the N-terminus for stimulating muscle
growth. In another embodiment, the methods of the present invention
provide a GH peptide set forth in SEQ ID NO: 44 for stimulating
muscle growth.
[0079] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
stimulating muscle growth. In another embodiment, the methods of
the present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
stimulating muscle growth. In another embodiment, the methods of
the present invention provide a nucleic acid of SEQ ID NO: 45
encoding a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
stimulating muscle growth. In another embodiment, the methods of
the present invention provide a nucleic acid of SEQ ID NO: 46
encoding a GH peptide and one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
stimulating muscle growth.
[0080] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for stimulating bone growth. In another
embodiment, the methods of the present invention provide a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
stimulating bone growth. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 23
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for stimulating bone growth. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: to 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for stimulating bone growth. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 37 having additionally at least one
CTP amino acid peptide on the N-terminus for stimulating bone
growth. In another embodiment, the methods of the present invention
provide a GH peptide set forth in SEQ ID NO: 38 having additionally
at least one CTP amino acid peptide on the N-terminus for
stimulating bone growth. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 39
for stimulating bone growth. In another embodiment, the methods of
the present invention provide a GH peptide set forth in SEQ ID NO:
40 for stimulating bone growth. In another embodiment, the methods
of the present invention provide a GH peptide set forth in SEQ ID
NO: 41 for stimulating bone growth. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 42 having additionally at least one CTP amino acid
peptide on the N-terminus for stimulating bone growth. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 44 for stimulating bone growth.
[0081] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
stimulating bone growth. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
stimulating bone growth. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 45 encoding
a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
stimulating bone growth. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 46 encoding
a GH peptide and one CTP amino acid peptide on the N-terminus and
two CTP amino acid peptides on the C-terminus for stimulating bone
growth.
[0082] In another embodiment, the methods of the present invention
provide a GH peptide of the present invention for maintaining
muscle quality.
[0083] In another embodiment, the methods of the present invention
provide a GH of the present invention for maintaining bone
quality.
[0084] In another embodiment, the methods of the present invention
provide a GH-CTP nucleic acid sequence of the present invention for
maintaining bone quality.
[0085] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for treating a wasting disease. In
another embodiment, the methods of the present invention provide a
GH peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
treating a wasting disease. In another embodiment, the methods of
the present invention provide a GH peptide set forth in SEQ ID NO:
23 having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for treating a wasting disease. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for treating a wasting disease. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 37 having additionally at least one
CTP amino acid peptide on the N-terminus for treating a wasting
disease. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 38 having
additionally at least one CTP amino acid peptide on the N-terminus
for treating a wasting disease. In another embodiment, the methods
of the present invention provide a GH peptide set forth in SEQ ID
NO: 39 for treating a wasting disease. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 40 for treating a wasting disease. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 41 for treating a wasting disease.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 42 having additionally at
least one CTP amino acid peptide on the N-terminus for treating a
wasting disease. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 44 for
treating a wasting disease.
[0086] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
treating a wasting disease. In another embodiment, the methods of
the present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
treating a wasting disease. In another embodiment, the methods of
the present invention provide a nucleic acid of SEQ ID NO: 45
encoding a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
treating a wasting disease. In another embodiment, to the methods
of the present invention provide a nucleic acid of SEQ ID NO: 46
encoding a GH peptide and one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
treating a wasting disease.
[0087] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for increasing cardiac function. In
another embodiment, the methods of the present invention provide a
GH peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
increasing cardiac function. In another embodiment, the methods of
the present invention provide a GH peptide set forth in SEQ ID NO:
23 having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for increasing cardiac function. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for increasing cardiac function. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 37 having additionally at least one
CTP amino acid peptide on the N-terminus for increasing cardiac
function. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 38 having
additionally at least one CTP amino acid peptide on the N-terminus
for increasing cardiac function. In another embodiment, the methods
of the present invention provide a GH peptide set forth in SEQ ID
NO: 39 for increasing cardiac function. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 40 for increasing cardiac function. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 41 for increasing cardiac function.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 42 having additionally at
least one CTP amino acid peptide on the N-terminus for increasing
cardiac function. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 44 for
increasing cardiac function.
[0088] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
increasing cardiac function. In another embodiment, the methods of
the present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
increasing cardiac function. In another embodiment, the methods of
the present invention provide a nucleic acid of SEQ ID NO: 45
encoding a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
increasing cardiac function. In another embodiment, the methods of
the present invention provide a nucleic acid of SEQ ID NO: 46
encoding a GH peptide and one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
increasing cardiac function.
[0089] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for increasing lipolysis. In another
embodiment, the methods of the present invention provide a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
increasing lipolysis. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 23
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for increasing lipolysis. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for increasing lipolysis. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 37 having additionally at least one CTP amino acid
peptide on the N-terminus for increasing lipolysis. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 38 having additionally at least one
CTP amino acid peptide on the N-terminus for increasing lipolysis.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 39 for increasing lipolysis.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 40 for increasing lipolysis.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 41 for increasing lipolysis.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 42 having additionally at
least one CTP amino acid peptide on the N-terminus for increasing
lipolysis. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 44 for
increasing lipolysis.
[0090] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
increasing lipolysis. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
increasing lipolysis. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 45 encoding
a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
increasing lipolysis. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 46 encoding
a GH peptide and one CTP amino acid peptide on the N-terminus and
two CTP amino acid peptides on the C-terminus for increasing
lipolysis.
[0091] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for improving fluid balance. In another
embodiment, the methods of the present invention provide a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
improving fluid balance. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 23
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for improving fluid balance. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for improving fluid balance. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 37 having additionally at least one
CTP amino acid peptide on the N-terminus for improving fluid
balance. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 38 having
additionally at least one CTP amino acid peptide on the N-terminus
for improving fluid balance. In another embodiment, the methods of
the present invention provide a GH peptide set forth in SEQ ID NO:
39 for improving fluid balance. In another embodiment, the methods
of the present invention provide a GH peptide set forth in SEQ ID
NO: 40 for improving fluid balance. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 41 for improving fluid balance. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 42 having additionally at least one CTP amino acid
peptide on the N-terminus for improving fluid balance. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 44 for improving fluid balance.
[0092] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
improving fluid balance. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
improving fluid balance. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 45 encoding
a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
improving fluid balance. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 46 encoding
a GH peptide and one CTP amino acid peptide on the N-terminus and
two CTP amino acid peptides on the C-terminus for improving fluid
balance.
[0093] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for treating osteoporosis. In another
embodiment, the methods of the present invention provide a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
treating osteoporosis. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 23
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for treating osteoporosis. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for treating osteoporosis. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 37 having additionally at least one CTP amino acid
peptide on the N-terminus for treating osteoporosis. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 38 having additionally at least one
CTP amino acid peptide on the N-terminus for treating osteoporosis.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 39 for treating osteoporosis.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 40 for treating osteoporosis.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 41 for treating osteoporosis.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 42 having additionally at
least one CTP amino acid peptide on the N-terminus for treating
osteoporosis. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 44 for
treating osteoporosis.
[0094] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
treating osteoporosis. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
treating osteoporosis. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 45 encoding
a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
treating osteoporosis. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 46 encoding
a GH peptide and one CTP amino acid peptide on the N-terminus and
two CTP amino acid peptides on the C-terminus for treating
osteoporosis.
[0095] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for inhibiting osteoporosis. In another
embodiment, the methods of the present invention provide a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
inhibiting osteoporosis. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 23
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for inhibiting osteoporosis. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for inhibiting osteoporosis. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 37 having additionally at least one
CTP amino acid peptide on the N-terminus for inhibiting
osteoporosis. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 38 having
additionally at least one CTP amino acid peptide on the N-terminus
for inhibiting osteoporosis. In another embodiment, the methods of
the present invention provide a GH peptide set forth in SEQ ID NO:
39 for inhibiting osteoporosis. In another embodiment, the methods
of the present invention provide a GH peptide set forth in SEQ ID
NO: 40 for inhibiting osteoporosis. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 41 for inhibiting osteoporosis. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 42 having additionally at least one CTP amino acid
peptide on the N-terminus for inhibiting osteoporosis. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 44 for inhibiting osteoporosis.
[0096] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
inhibiting osteoporosis. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
inhibiting osteoporosis. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 45 encoding
a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
inhibiting osteoporosis. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 46 encoding
a GH peptide and one CTP amino acid peptide on the N-terminus and
two CTP amino acid peptides on the C-terminus for inhibiting
osteoporosis.
[0097] In another embodiment, the methods of the present invention
provide a GH peptide of the present invention for improving
exercise capacity.
[0098] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for improving lung function. In another
embodiment, the methods of the present invention provide a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
improving lung function. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 23
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for improving lung function. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for improving lung function. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 37 having additionally at least one
CTP amino acid peptide on the N-terminus for improving lung
function. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 38 having
additionally at least one CTP amino acid peptide on the N-terminus
for improving lung function. In another embodiment, the methods of
the present invention provide a GH peptide set forth in SEQ ID NO:
39 for improving lung function. In another embodiment, the methods
of the present invention provide a GH peptide set forth in SEQ ID
NO: 40 for improving lung function. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 41 for improving lung function. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 42 having additionally at least one CTP amino acid
peptide on the N-terminus for improving lung function. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 44 for improving lung function.
[0099] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
improving lung function. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
improving lung function. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 45 encoding
a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
improving lung function. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 46 encoding
a GH peptide and one CTP amino acid peptide on the N-terminus and
two CTP amino acid peptides on the C-terminus for improving lung
function.
[0100] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for improving immunity. In another
embodiment, the methods of the present invention provide a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
improving immunity. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 23
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for improving immunity. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for improving immunity. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 37 having additionally at least one CTP amino acid
peptide on the N-terminus for improving immunity. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 38 having additionally at least one
CTP amino acid peptide on the N-terminus for improving immunity. In
another embodiment, the methods of the present invention provide a
GH peptide set forth in SEQ ID NO: 39 for improving immunity. In
another embodiment, the methods of the present invention provide a
GH peptide set forth in SEQ ID NO: 40 for improving immunity. In
another embodiment, the methods of the present invention provide a
GH peptide set forth in SEQ ID NO: 41 for improving immunity. In
another embodiment, the methods of the present invention provide a
GH peptide set forth in SEQ ID NO: 42 having additionally at least
one CTP amino acid peptide on the N-terminus for improving
immunity. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 44 for
improving immunity.
[0101] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
improving immunity. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
improving immunity. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 45 encoding
a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
improving immunity. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 46 encoding
a GH peptide and one CTP amino acid peptide on the N-terminus and
two CTP amino acid peptides on the C-terminus for improving
immunity.
[0102] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for regrowing vital organs. In another
embodiment, the methods of the present invention provide a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
regrowing vital organs. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 23
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for regrowing vital organs. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for regrowing vital organs. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 37 having additionally at least one
CTP amino acid peptide on the N-terminus for regrowing vital
organs. In another embodiment, the methods of the present invention
provide a GH peptide set forth in SEQ ID NO: 38 having additionally
at least one CTP amino acid peptide on the N-terminus for regrowing
vital organs. In another embodiment, the methods of the present
invention provide a GH peptide set forth in SEQ ID NO: 39 for
regrowing vital organs. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 40
for regrowing vital organs. In another embodiment, the methods of
the present invention provide a GH peptide set forth in SEQ ID NO:
41 for regrowing vital organs. In another embodiment, the methods
of the present invention provide a GH peptide set forth in SEQ ID
NO: 42 having additionally at least one CTP amino acid peptide on
the N-terminus for regrowing vital organs. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 44 for regrowing vital organs.
[0103] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
regrowing vital organs. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
regrowing vital organs. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 45 encoding
a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
regrowing vital organs. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 46 encoding
a GH peptide and one CTP amino acid peptide on the N-terminus and
two CTP amino acid peptides on the C-terminus for regrowing vital
organs.
[0104] In another embodiment, the methods of the present invention
provide a GH peptide of the present invention for increasing sense
of well-being.
[0105] In another embodiment, the methods of the present invention
provide a GH peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for restoring REM sleep. In another
embodiment, the methods of the present invention provide a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
restoring REM sleep. In another embodiment, the methods of the
present invention provide a GH peptide set forth in SEQ ID NO: 23
having additionally at least one CTP amino acid peptide on the
N-terminus and at least one CTP amino acid peptide on the
C-terminus for restoring REM sleep. In another embodiment, the
methods of the present invention provide a GH peptide set forth in
SEQ ID NO: 36 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for restoring REM sleep. In another embodiment,
the methods of the present invention provide a GH peptide set forth
in SEQ ID NO: 37 having additionally at least one CTP amino acid
peptide on the N-terminus for restoring REM sleep. In another
embodiment, the methods of the present invention provide a GH
peptide set forth in SEQ ID NO: 38 having additionally at least one
CTP amino acid peptide on the N-terminus for restoring REM sleep.
In another embodiment, the methods of the present invention provide
a GH peptide set forth in SEQ ID NO: 39 for restoring REM sleep. In
another embodiment, the methods of the present invention provide a
GH peptide set forth in SEQ ID NO: 40 for restoring REM sleep. In
another embodiment, the methods of the present invention provide a
GH peptide set forth in SEQ ID NO: 41 for restoring REM sleep. In
another embodiment, the methods of the present invention provide a
GH peptide set forth in SEQ ID NO: 42 having additionally at least
one CTP amino acid peptide on the N-terminus for restoring REM
sleep. In another embodiment, the methods of the present invention
provide a GH peptide set forth in SEQ ID NO: 44 for restoring REM
sleep.
[0106] In another embodiment, the methods of the present invention
provide a nucleic acid sequence encoding a GH peptide having
additionally at least one CTP amino acid peptide on the N-terminus
and at least one CTP amino acid peptide on the C-terminus for
restoring REM sleep. In another embodiment, the methods of the
present invention provide a nucleic acid sequence encoding a GH
peptide having additionally one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
restoring REM sleep. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 45 encoding
a GH peptide comprising one CTP amino acid peptide on the
N-terminus and two CTP amino acid peptides on the C-terminus for
restoring REM sleep. In another embodiment, the methods of the
present invention provide a nucleic acid of SEQ ID NO: 46 encoding
a GH peptide and one CTP amino acid peptide on the N-terminus and
two CTP amino acid peptides on the C-terminus for restoring REM
sleep.
[0107] In some embodiments, homology according to the present
invention also encompasses deletions, insertions, or substitution
variants, including an amino acid substitution, thereof and
biologically active polypeptide fragments thereof. In one
embodiment the substitution variant is one, in which the glutamine
in position 65 of hGH is substituted by a valine (SEQ ID NO: 23)
[Gellerfors et al., J Pharm Biomed Anal 1989, 7:173-83].
[0108] In some embodiments, interferon is utilized according to the
teachings of the present invention. In some embodiments, the
attachment of CTP sequence to both the amino and carboxy termini of
the interferon protein results in increased potency. In some
embodiments, the attachment of CTP sequence to both the amino and
carboxy termini of the interferon protein results in prolonged
in-vivo activity.
[0109] In one embodiment, "interferon" refers to the mammalian
interferon polypeptide Type I. In one embodiment, "interferon"
refers to the mammalian interferon polypeptide Type II. In some
embodiments, additional suitable interferon polypeptides as known
to those of ordinary skill in the art are utilized. In some
embodiments, the interferon is alpha-interferon. In some
embodiments, the interferon is beta-interferon. In some
embodiments, the interferon is gamma-interferon. In some
embodiments, the interferon is omega-interferon. In some
embodiments, the interferon is a subspecies interferon. In one
embodiment, the subspecies interferon (IFN) is IFN-.alpha.2a. In
one embodiment, the subspecies interferon (IFN) is IFN-.alpha.2b.
In one embodiment, the subspecies interferon (IFN) is IFN-.beta.1a.
In one embodiment, the interferon (IFN) subspecies is
IFN-.beta.1b.
[0110] In one embodiment, interferon of the present invention
exhibits interferon activity, such as antiviral or
antiproliferative activity. In some embodiments, GenBank accession
nos. of non-limiting examples of interferons are listed in Table 1
below.
[0111] In one embodiment, an interferon of the present invention
also refers to homologues. In one embodiment, interferon amino acid
sequence of the present invention is at least 50% homologous to an
interferon sequence disclosed herein). In one embodiment,
interferon amino acid sequence of the present invention is at least
60% homologous an interferon sequence disclosed herein). In one
embodiment, interferon amino acid sequence of the present invention
is at least 70% homologous an interferon sequence disclosed
herein). In one embodiment, interferon amino acid sequence of the
present invention is at least 80% homologous to an interferon
sequence disclosed herein). In one embodiment, interferon amino
acid sequence of the present invention is at least 90% homologous
to an interferon sequence disclosed herein). In one embodiment,
interferon amino acid sequence of the present invention is at least
95% homologous an interferon sequence disclosed herein). In some
embodiments, homology according to the present invention also
encompasses deletions, insertions, or substitution variants,
including an amino acid substitution, thereof and biologically
active polypeptide fragments thereof. In one embodiment the
cysteine in position 17 of interferon .beta. is substituted by a
Serine (SEQ ID NO: 24).
[0112] Table 1 below lists examples of interferons with their
respective NCBI sequence numbers TABLE-US-00001 TABLE 1 Interferon
name NCBI sequence number interferon, .alpha.1 NP_076918.1
interferon, .alpha.10 NP_002162.1 interferon, .alpha.13 NP_008831.2
interferon, .alpha.14 NP_002163.1 interferon, .alpha.16 NP_002164.1
interferon, .alpha.17 NP_067091.1 interferon, .alpha.2 NP_000596.2
interferon, .alpha.21 NP_002166.1 interferon, .alpha.4 NP_066546.1
interferon, .alpha.5 NP_002160.1 interferon, .alpha.6 NP_066282.1
interferon, .alpha.7 NP_066401.2 interferon, .alpha.8 NP_002161.2
interferon, .beta.1 NP_002167.1 interferon, .epsilon.1 NP_795372.1
interferon, .gamma. NP_000610.2 interferon, .epsilon. NP_064509.1
interferon, .OMEGA.1 NP_002168.1
[0113] In another embodiment, the methods of the present invention
provide an interferon beta 1 peptide having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for treating or inhibiting
multiple sclerosis. In another embodiment, the methods of the
present invention provide an interferon beta 1 peptide having
additionally one CTP amino acid peptide on the N-terminus and two
CTP amino acid peptides on the C-terminus for treating or
inhibiting multiple sclerosis. In another embodiment, the methods
of the present invention provide an interferon beta 1 peptide set
forth in SEQ ID NO: 24 having additionally at least one CTP amino
acid peptide on the N-terminus and one CTP amino acid peptide on
the C-terminus for treating or inhibiting multiple sclerosis. In
another embodiment, the methods of the present invention provide an
interferon beta 1 peptide set forth in SEQ ID NO: 24 having
additionally on the N-terminus the signal peptide of SEQ ID NO: 26
and at least one CTP amino acid peptide on the N-terminus of SEQ ID
NO: 26 and at least one CTP amino acid peptide on the C-terminus of
SEQ ID NO: 24 for treating or inhibiting multiple sclerosis.
[0114] In some embodiments, glucagon-like peptide-1 is utilized
according to the teachings of the present invention. In some
embodiments, the attachment of CTP sequences to both the amino and
carboxy termini of a "glucagon-like peptide-1" results in increased
potency. In some embodiments, the attachment of CTP to both the
amino and carboxy termini of a peptide results in prolonged in-vivo
activity. In some embodiments, the attachment of CTP to both the
amino and carboxy termini of the glucagon-like peptide-results in
prolonged in-vivo activity.
[0115] In one embodiment, "glucagon-like peptide-1" (GLP-1) refers
to a mammalian polypeptide. In one embodiment, "glucagon-like
peptide-1" (GLP-1) refers to a human polypeptide. In some
embodiments, GLP-1 is cleaved from the glucagon preproprotein
(Genbank ID No. NP002045) that has the ability to bind to the GLP-1
receptor and initiate a signal transduction pathway resulting in
insulinotropic activity. In one embodiment, "insulinotropic
activity" refers to the ability to stimulate insulin secretion in
response to elevated glucose levels, thereby causing glucose uptake
by cells and decreased plasma glucose levels. In some embodiments,
GLP-1 polypeptides include, but are not limited to those described
in U.S. Pat. No. 5,118,666; which is incorporated by reference
herein.
[0116] In one embodiment, "GLP-1" refers to a polypeptide, such as
set forth in sequences set forth in SEQ ID NO: 25 as determined
using BlastP software of the National Center of Biotechnology
Information (NCBI) using default parameters). In one embodiment, a
GLP-1 of the to present invention also refers to a GLP-1 homologue.
In one embodiment, GLP-1 amino acid sequence of the present
invention is at least 50% homologous to GLP-1 sequences set forth
in SEQ ID NO: 25 as determined using BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters). In one embodiment, GLP-1 amino acid sequence of the
present invention is at least 60% homologous to GLP-1 sequences set
forth in SEQ ID NO: 25 as determined using BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters). In one embodiment, GLP-1 amino acid sequence of the
present invention is at least 70% homologous to GLP-1 sequences set
forth in SEQ ID NO: 25 as determined using BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters). In one embodiment, GLP-1 amino acid sequence of the
present invention is at least 80% homologous to GLP-1 sequences set
forth in SEQ ID NO: 25 as determined using BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters). In one embodiment, GLP-1 amino acid sequence of the
present invention is at least 90% homologous to GLP-1 sequences set
forth in SEQ ID NO: 25 as determined using BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters). In one embodiment, GLP-1 amino acid sequence of the
present invention is at least 95% homologous to GLP-1 sequences set
forth in SEQ ID NO: 25 as determined using BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters).
[0117] In another embodiment, the methods of the present invention
provide a GLP-1 peptide having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for treating or inhibiting type II
diabetes. In another embodiment, the methods of the present
invention provide a GLP-1 peptide having additionally one CTP amino
acid peptide on the N-terminus and two CTP amino acid peptides on
the C-terminus for treating or inhibiting type II diabetes. In
another embodiment, the methods of the present invention provide a
GLP-1 peptide set forth in SEQ ID NO: 25 having additionally at
least one CTP amino acid peptide on the N-terminus and at least one
CTP amino acid peptide on the C-terminus for treating or inhibiting
type II diabetes.
[0118] In one embodiment, the homologue also refers to a deletion,
insertion, or substitution variant, including an amino acid
substitution, thereof and biologically active polypeptide fragments
thereof.
[0119] In one embodiment the polypeptide sequence-of-interest is an
EPO. In one embodiment the polypeptide sequence-of-interest is an
interferon. In another embodiment the polypeptide
sequence-of-interest is an hGH. In another embodiment the
polypeptide sequence-of-interest is a GLP-1. In another embodiment
the polypeptide sequence-of-interest is an insulin. In another
embodiment the polypeptide sequence-of-interest is enkephalin. In
another embodiment the polypeptide sequence-of-interest is an ACTH.
In another embodiment the polypeptide sequence-of-interest is a
glucagon. In another embodiment the polypeptide
sequence-of-interest is an insulin-like growth factor. In another
embodiment the polypeptide sequence-of-interest is an epidermal
growth factor. In another embodiment the polypeptide
sequence-of-interest is an acidic or basic fibroblast growth
factor. In another embodiment the polypeptide sequence-of-interest
is a platelet-derived growth factor. In another embodiment the
polypeptide sequence-of-interest is a granulocyte-CSF. In another
embodiment the polypeptide sequence-of-interest is a
macrophage-CSF. In another embodiment the polypeptide
sequence-of-interest is an IL-2. In another embodiment the
polypeptide sequence-of-interest is an IL-3. In another embodiment
the polypeptide sequence-of-interest is a tumor necrosis factor. In
another embodiment the polypeptide sequence-of-interest is an LHRH.
In another embodiment the polypeptide sequence-of-interest is an
LHRH analog. In another embodiment the polypeptide
sequence-of-interest is a somatostatin. In another embodiment the
polypeptide sequence-of-interest is a growth hormone releasing
factor. In another embodiment the polypeptide sequence-of-interest
is an endorphin. In another embodiment the polypeptide
sequence-of-interest is an alveolar surfactant protein. In another
embodiment the polypeptide sequence-of-interest is a natriuretic
factor. In another embodiment the polypeptide sequence-of-interest
is an adhesion. In another embodiment the polypeptide
sequence-of-interest is an angiostatin. In another embodiment the
polypeptide sequence-of-interest is an endostatin. In another
embodiment the polypeptide sequence-of-interest is a receptor
peptide. In another embodiment the polypeptide sequence-of-interest
is a receptor binding ligand. In another embodiment the polypeptide
sequence-of-interest is an antibody. In another embodiment the
polypeptide sequence-of-interest is an antibody fragment. In
another embodiment the polypeptide sequence-of-interest is a
peptide or a protein including any modified form.
[0120] In another embodiment, the peptide of the invention
comprises a peptide of interest having additionally at least one
CTP amino acid peptide on the N-terminus and one CTP amino acid
peptide on the C-terminus. In another embodiment, the peptide of
interest having additionally at least one CTP amino acid peptide on
the N-terminus and one CTP amino acid peptide on the C-terminus
comprises a protein selected from the following list: insulin,
Albutein/albumin, Activase altiplase/tPA, adenosine deaminase,
immune globulin, glucocerebrosidase, Leukine-sargramostim/GM-CSF,
G-CSF, Venoglobulin-S/IgG, Proleukin aldesleukin, DNase, factor
VIII, Helixate, L-asparaginase, WinRho SDF Rh I, Retavase
retaplase/tPA, Factor IX, FSH, globulin, fibrin, interleukin-11,
becaplermin/PDGF, lepirudin/herudin, TNF, Thymoglobulin, factor
VIIa, interferon alpha-2a, interferon alfa n-1, interferon alfa-N3,
interferon beta-1b, interferon gamma-1b, Interleukin-2, HGH, or
monoclonal antibodies.
[0121] In another embodiment, the methods of the present invention
provide insulin having additionally at least one CTP amino acid
peptide on the N-terminus and one CTP amino acid peptide on the
C-terminus for the treatment of diabetes.
[0122] In another embodiment, the methods of the present invention
provide albumin having additionally at least one CTP amino acid
peptide on the N-terminus and one CTP amino acid peptide on the
C-terminus for the treatment of hypovolemic shock, hemodialysis or
cardiopulmonary bypass.
[0123] In another embodiment, the methods of the present invention
provide Activase-altiplase/tPA having additionally at least one CTP
amino acid peptide on the N-terminus and one CTP amino acid peptide
on the C-terminus for the treatment of acute myocardial infarction,
acute massive pulmonary embolism, or (change throughout) ischemic
stroke.
[0124] In another embodiment, the methods of the present invention
provide adenosine deaminase having additionally at least one CTP
amino acid peptide on the N-terminus and one CTP amino acid peptide
on the C-terminus for the treatment of severe combined
immunodeficiency disease.
[0125] In another embodiment, the methods of the present invention
provide immune globulin having additionally at least one CTP amino
acid peptide on the N-terminus and one CTP amino acid peptide on
the C-terminus for the treatment of transplant recipients.
[0126] In another embodiment, the methods of the present invention
provide immune globulin is a CMV immune globulin. In another
embodiment, the methods of the present invention provide
glucocerebrosidase having additionally at least one CTP amino acid
peptide on the N-terminus and one CTP amino acid peptide on the
C-terminus for the treatment of Gaucher disease.
[0127] In another embodiment, the methods of the present invention
provide Leukine-sargramostim/GM-CSF having additionally at least
one CTP amino acid peptide on the N-terminus and one CTP amino acid
peptide on the C-terminus for the Stimulation of hematopoietic
progenitor cells.
[0128] In another embodiment, the methods of the present invention
provide G-CSF having additionally at least one CTP amino acid
peptide on the N-terminus and one CTP amino acid peptide on the
C-terminus for the treatment of Neutropenia. In another embodiment,
the methods of the present invention provide Venoglobulin-S/IgG
having additionally at least one CTP amino acid peptide on the
N-terminus and one CTP amino acid peptide on the C-terminus for the
treatment of Immunodeficiency diseases.
[0129] In another embodiment, the methods of the present invention
provide Proleukin-aldesleukin having additionally at least one CTP
amino acid peptide on the N-terminus and one CTP amino acid peptide
on the C-terminus for the treatment of renal carcinoma or
metastatic melanoma.
[0130] In another embodiment, the methods of the present invention
provide DNase having additionally at least one CTP amino acid
peptide on the N-terminus and one CTP amino acid peptide on the
C-terminus for the treatment of Cystic fibrosis.
[0131] In another embodiment, the methods of the present invention
provide factor VIII having additionally at least one CTP amino acid
peptide on the N-terminus and one CTP amino acid peptide on the
C-terminus for the treatment of Hemophilia A.
[0132] In another embodiment, the methods of the present invention
provide Helixate having additionally at least one CTP amino acid
peptide on the N-terminus and one CTP amino acid peptide on the
C-terminus for the treatment of Hemophilia A.
[0133] In another embodiment, the methods of the present invention
provide L-asparaginase having additionally at least one CTP amino
acid peptide on the N-terminus and one CTP amino acid peptide on
the C-terminus for the treatment of acute lymphoblastic
leukemia.
[0134] In another embodiment, the methods of the present invention
provide WinRho SDF Rh IgG having additionally at least one CTP
amino acid peptide on the N-terminus and one CTP amino acid peptide
on the C-terminus for the treatment of Rh isoimmunization and
immune thrombocytopenic purpura.
[0135] In another embodiment, the methods of the present invention
provide Retavase retaplase/tPA having additionally at least one CTP
amino acid peptide on the N-terminus and one CTP amino acid peptide
on the C-terminus for the treatment of acute myocardial
infarction.
[0136] In another embodiment, the methods of the present invention
provide Factor IX having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for the treatment of Hemophilia B.
[0137] In another embodiment, the methods of the present invention
provide Factor IX having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for the treatment of Hemophilia B.
[0138] In another embodiment, the methods of the present invention
provide FSH having additionally at least one CTP amino acid peptide
on the N-terminus and at least one CTP amino acid peptide on the
C-terminus for stimulation of ovulation during assisted
reproduction.
[0139] In another embodiment, the methods of the present invention
provide globulin having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for the prevention of respiratory syncytial virus
disease.
[0140] In another embodiment, the methods of the present invention
provide fibrin having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for wound management and hemostasis. In another
embodiment, the methods of the present invention provide
interleukin-11 having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for chemotherapy-induced thrombocytopenia.
[0141] In another embodiment, the methods of the present invention
provide becaplermin/PDGF having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for the treatment of diabetic foot
ulcers.
[0142] In another embodiment, the methods of the present invention
provide lepirudin/herudin having additionally at least one CTP
amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for anticoagulation in
heparin-induced thrombocytopenia.
[0143] In another embodiment, the methods of the present invention
provide soluble TNF having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for the treatment of rheumatoid arthritis.
[0144] In another embodiment, the methods of the present invention
provide Thymoglobulin having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for the treatment of organ transplant
rejection disease.
[0145] In another embodiment, the methods of the present invention
provide factor VIIa having additionally at least one CTP amino acid
peptide on the N-terminus and at least one CTP amino acid peptide
on the C-terminus for the treatment of hemophilia.
[0146] In another embodiment, the methods of the present invention
provide interferon alpha-2a having additionally at least one CTP
amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of hairy cell
leukemia and AIDS-related Kaposi's sarcoma.
[0147] In another embodiment, the methods of the present invention
provide interferon alpha-2b having additionally at least one CTP
amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of Hairy cell
leukemia, genital warts, AIDS-related Kaposi's sarcoma, hepatitis
C, hepatitis B, malignant melanoma, and follicular lymphoma.
[0148] In another embodiment, the methods of the present invention
provide interferon alfa-N3 having additionally at least one CTP
amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of genital
warts.
[0149] In another embodiment, the methods of the present invention
provide interferon gamma-1b having additionally at least one CTP
amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of chronic
granulomatous disease.
[0150] In another embodiment, the methods of the present invention
provide interferon alfa n-1 having additionally at least one CTP
amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of hepatitis C
infection.
[0151] In another embodiment, the methods of the present invention
provide Interleukin-2 having additionally at least one CTP amino
acid peptide on the N-terminus and at least one CTP amino acid
peptide on the C-terminus for the treatment of renal carcinoma and
metastatic melanoma.
[0152] In another embodiment, the methods of the present invention
provide interferon beta-1b having additionally at least one CTP
amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for the treatment of multiple
sclerosis.
[0153] In another embodiment, the methods of the present invention
provide hGH having additionally at least one CTP amino acid peptide
on the N-terminus and at least one CTP amino acid peptide on the
C-terminus for the treatment of wasting disease, AIDS, cachexia, or
hGH deficiency.
[0154] In another embodiment, the methods of the present invention
provide an OKT3 monoclonal antibody having additionally at least
one CTP amino acid peptide on the N-terminus and at least one CTP
amino acid peptide on the C-terminus for organ transplant.
[0155] In another embodiment, the methods of the present invention
provide a Reo monoclonal antibody having additionally at least one
CTP amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for prevention of complications from
coronary intervention and angioplasty.
[0156] In another embodiment, the methods of the present invention
provide a monoclonal antibody having additionally at least one CTP
amino acid peptide on the N-terminus and at least one CTP amino
acid peptide on the C-terminus for treating colorectal cancer,
Non-Hodgkin's lymphoma, kidney transplant rejection, metastatic
breast cancer, or the prevention of respiratory syncytial virus
disease.
[0157] In one embodiment, the invention is employed in veterinary
medicine. In one embodiment, the present invention provides
treatment of domesticated mammals which are maintained as human
companions (e.g., dogs, cats, horses), which have significant
commercial value (e.g., dairy cows. beef cattle, sporting animals),
which have significant scientific value (e.g., captive or free
specimens of endangered species), or which otherwise have
value.
[0158] In one embodiment, polypeptides, antibodies, or
polynucleotides of the present invention are administered to an
animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs,
micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat,
non-human primate, and human. In one embodiment, the recited
applications have uses in a wide variety of hosts. In some
embodiments, such hosts include, but are not limited to, human,
murine, rabbit, goat, guinea pig, camel, horse, mouse, rat,
hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, or
non-human primate.
[0159] In one embodiment, farm animals are treated by the methods
of the present invention. In one embodiment, farm animals include
pigs, cattle, dairy cows, horses, goats, sheep, chickens, turkeys,
geese, ducks and related species. In one embodiment, laboratory
animals are treated by the methods of the present invention. In one
embodiment, laboratory animals include rats, mice, guinea pigs,
rabbits, goats, monkeys, dogs, cats and others. In one embodiment,
zoo animals are treated by the methods of the present invention. In
one embodiment, zoo animals include all vertebrate animals kept in
zoos. In one embodiment, aquatic animals are treated by the methods
of the present invention. In one embodiment, aquatic animals
include fish, eels, turtles, seals, penguins, sharks, whales, and
related species. In one embodiment, domesticated animals are
treated by the methods of the present invention. In one embodiment,
domesticated animals include any pet, such as cats and dogs, or
animal that is kept by humans, e.g., horses, cattle, pigs, goats,
rabbits, chickens, turkeys, geese, ducks and the like.
[0160] According to the present invention the term pigs includes
pigs, piglets, hogs, gilts, barrows, boars and sows. In another
embodiment, "cattle" refers to calves, cows, dairy cows, heifers,
steers and bulls.
[0161] In one embodiment, bovine growth hormone is utilized by the
methods of the present invention. In one embodiment, artificial
bovine growth hormone is utilized by the methods of the present
invention. In one embodiment, the artificial bovine growth hormone
has a sequence set forth in NCBI sequence ID number AAA72262. In
another embodiment, the artificial bovine growth hormone is any
other artificial bovine growth hormone known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0162] In one embodiment, sheep growth hormone is utilized by the
methods of the present invention. In one embodiment, sheep growth
hormone has a sequence set forth in NCBI sequence ID number
NP.sub.--001009315. In another embodiment, the sheep growth hormone
is any other sheep growth hormone known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0163] In one embodiment, horse growth hormone is utilized by the
methods of the present invention. In one embodiment, horse growth
hormone has a sequence set forth in NCBI sequence ID number
AAA21027. In another embodiment, the horse growth hormone is any
other horse growth hormone known in the art. Each possibility
represents a separate embodiment of the present invention.
[0164] In one embodiment, chicken growth hormone is utilized by the
methods of the present invention. In one embodiment, chicken growth
hormone has a sequence set forth in NCBI sequence ID number
CAA3561. In another embodiment, the chicken growth hormone is any
other chicken growth hormone known in the art. Each possibility
represents a separate embodiment of the present invention.
[0165] In one embodiment, murine growth hormone is utilized by the
methods of the present invention. In one embodiment, the murine
growth hormone has a sequence set forth in NCBI sequence ID number
NP.sub.--032143. In another embodiment, the murine growth hormone
is any other murine growth hormone known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0166] In one embodiment, tilapia growth hormone is utilized by the
methods of the present invention. In one embodiment, the tilapia
growth hormone has a sequence set forth in NCBI sequence ID number
CAA00818. In another embodiment, the tilapia growth hormone is any
other tilapia growth hormone known in the art. Each possibility
represents a separate embodiment of the present invention.
[0167] In one embodiment, bovine EPO is utilized by the methods of
the present invention. In one embodiment, artificial bovine growth
hormone is utilized by the methods of the present invention. In one
embodiment, artificial bovine growth hormone has a sequence set
forth in NCBI sequence ID number NP.sub.--776334. In another
embodiment, the bovine EPO is any other bovine EPO known in the
art. Each possibility represents a separate embodiment of the
present invention.
[0168] In one embodiment, pig EPO is utilized by the methods of the
present invention. In one embodiment, pig EPO has a sequence set
forth in NCBI sequence ID number NP.sub.--999299. In another
embodiment, the pig EPO is any other pig EPO known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0169] In one embodiment, sheep EPO is utilized by the methods of
the present invention. In one embodiment, sheep growth hormone has
a sequence set forth in NCBI sequence ID number NP.sub.--001019908.
In another embodiment, the sheep growth hormone is any other sheep
growth hormone known in the art. Each possibility represents a
separate embodiment of the present invention.
[0170] In one embodiment, murine EPO is utilized by the methods of
the present invention. In one embodiment, the murine growth hormone
has a sequence set forth in NCBI sequence ID number CAA72707. In
another embodiment, the murine growth hormone is any other murine
growth hormone known in the art. Each possibility represents a
separate embodiment of the present invention.
[0171] In one embodiment, bovine GLP-1 is utilized by the methods
of the present invention. In one embodiment, bovine GLP-1 has a
sequence set forth in NCBI sequence ID number P01272. In another
embodiment, the bovine GLP-1 is any other bovine GLP-1 known in the
art. Each possibility represents a separate embodiment of the
present invention.
[0172] In one embodiment, sheep GLP-1 is utilized by the methods of
the present invention. In one embodiment, sheep GLP-1 has a
sequence set forth in NCBI sequence ID number Q8MJ25. In another
embodiment, the sheep GLP-1 is any other sheep GLP-1 known in the
art. Each possibility represents a separate embodiment of the
present invention.
[0173] In one embodiment, pig GLP-1 is utilized by the methods of
the present invention. In one embodiment, chicken GLP-1 has a
sequence set forth in NCBI sequence ID number P01274. In another
embodiment, the chicken GLP-1 is any other chicken GLP-1 known in
the art. Each possibility represents a separate embodiment of the
present invention.
[0174] In one embodiment, murine GLP-1 is utilized by the methods
of the present invention. In one embodiment, the murine GLP-1 has a
sequence set forth in NCBI sequence ID number NP.sub.--032127. In
another embodiment, the murine GLP-1 is any other murine GLP-1
known in the art. Each possibility represents a separate embodiment
of the present invention.
[0175] In one embodiment, bovine interferon alpha is utilized by
the methods of the present invention. In one embodiment, bovine
interferon alpha has a sequence set forth in NCBI sequence ID
number ABD57311. In another embodiment, the bovine interferon alpha
is any other bovine interferon alpha known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0176] In one embodiment, sheep interferon alpha is utilized by the
methods of the present invention. In one embodiment, sheep
interferon alpha has a sequence set forth in NCBI sequence ID
number CAA41790. In another embodiment, the sheep interferon alpha
is any other sheep interferon alpha known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0177] In one embodiment, pig interferon alpha is utilized by the
methods of the present invention. In one embodiment, chicken
interferon alpha has a sequence set forth in NCBI sequence ID
number AAP92118. In another embodiment, the pig interferon alpha is
any other pig interferon alpha known in the art. Each possibility
represents a separate embodiment of the present invention.
[0178] In one embodiment, murine interferon alpha is utilized by
the methods of the present invention. In one embodiment, the murine
interferon alpha has a sequence set forth in NCBI sequence ID
number AAA37886. In another embodiment, the murine interferon alpha
is any other murine interferon alpha known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0179] In some embodiments, the CTP sequences modification is
advantageous in permitting lower dosages to be used.
[0180] In some embodiments, "polypeptide" as used herein
encompasses native polypeptides (either degradation products,
synthetically synthesized polypeptides or recombinant polypeptides)
and peptidomimetics (typically, synthetically synthesized
polypeptides), as well as peptoids and semipeptoids which are
polypeptide analogs, which have, in some embodiments, modifications
rendering the polypeptides even more stable while in a body or more
capable of penetrating into cells.
[0181] In some embodiments, modifications include, but are not
limited to N terminus modification, C terminus modification,
polypeptide bond modification, including, but not limited to,
CH2--NH, CH2--S, CH2--S.dbd.O, O.dbd.C--NH, CH2--O, CH2--CH2,
S.dbd.C--NH, CH.dbd.CH or CF.dbd.CH, backbone modifications, and
residue modification. Methods for preparing peptidomimetic
compounds are well known in the art and are specified, for example,
in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F.
Choplin Pergamon Press (1992), which is incorporated by reference
as if fully set forth herein. Further details in this respect are
provided hereinunder.
[0182] In some embodiments, polypeptide bonds (--CO--NH--) within
the polypeptide are substituted. In some embodiments, the
polypeptide bonds are substituted by N-methylated bonds
(--N(CH3)--CO--). In some embodiments, the polypeptide bonds are
substituted by ester bonds (--C(R)H--C--O--O--C(R)--N--). In some
embodiments, the polypeptide bonds are substituted by ketomethylen
bonds (--CO--CH2--). In some embodiments, the polypeptide bonds are
substituted by .alpha.-aza bonds (--NH--N(R)--CO--), wherein R is
any alkyl, e.g., methyl, carba bonds (--CH2--NH--). In some
embodiments, the polypeptide bonds are substituted by
hydroxyethylene bonds (--CH(OH)--CH2--). In some embodiments, the
polypeptide bonds are substituted by thioamide bonds (--CS--NH--).
In some embodiments, the polypeptide bonds are substituted by
olefinic double bonds (--CH.dbd.CH--). In some embodiments, the
polypeptide bonds are substituted by retro amide bonds
(--NH--CO--). In some embodiments, the polypeptide bonds are
substituted by polypeptide derivatives (--N(R)--CH2--CO--), wherein
R is the "normal" side chain, naturally presented on the carbon
atom. In some embodiments, these modifications occur at any of the
bonds along the polypeptide chain and even at several (2-3 bonds)
at the same time.
[0183] In some embodiments, natural aromatic amino acids of the
polypeptide such as Trp, Tyr and Phe, be substituted for synthetic
non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol),
ring-methylated derivatives of Phe, halogenated derivatives of Phe
or o-methyl-Tyr. In some embodiments, the polypeptides of the
present invention include one or more modified amino acid or one or
more non-amino acid monomers (e.g. fatty acid, complex
carbohydrates etc).
[0184] In one embodiment, "amino acid" or "amino acid" is
understood to include the 20 naturally occurring amino acid; those
amino acid often modified post-translationally in vivo, including,
for example, hydroxyproline, phosphoserine and phosphothreonine;
and other unusual amino acid including, but not limited to,
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine. In one embodiment, "amino acid" includes
both D- and L-amino acid.
[0185] In some embodiments, the polypeptides of the present
invention are utilized in therapeutics which requires the
polypeptides to be in a soluble form. In some embodiments, the
polypeptides of the present invention include one or more
non-natural or natural polar amino acid, including but not limited
to serine and threonine which are capable of increasing polypeptide
solubility due to their hydroxyl-containing side chain.
[0186] In some embodiments, the polypeptides of the present
invention are utilized in a linear form, although it will be
appreciated by one skilled in the art that in cases where
cyclicization does not severely interfere with polypeptide
characteristics, cyclic forms of the polypeptide can also be
utilized.
[0187] In some embodiments, the polypeptides of present invention
are biochemically synthesized such as by using standard solid phase
techniques. In some embodiments, these biochemical methods include
exclusive solid phase synthesis, partial solid phase synthesis,
fragment condensation, or classical solution synthesis. In some
embodiments, these methods are used when the polypeptide is
relatively short (about 5-15 kDa) and/or when it cannot be produced
by recombinant techniques (i.e., not encoded by a nucleic acid
sequence) and therefore involves different chemistry.
[0188] In some embodiments, solid phase polypeptide synthesis
procedures are well known to one skilled in the art and further
described by John Morrow Stewart and Janis Dillaha Young, Solid
Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company,
1984). In some embodiments, synthetic polypeptides are purified by
preparative high performance liquid chromatography [Creighton T.
(1983) Proteins, structures and molecular principles. WH Freeman
and Co. N.Y.] and the composition of which can be confirmed via
amino acid sequencing by methods known to one skilled in the
art.
[0189] In some embodiments, recombinant protein techniques are used
to generate the polypeptides of the present invention. In some
embodiments, recombinant protein techniques are used for generation
of relatively long polypeptides (e.g., longer than 18-25 amino
acid). In some embodiments, recombinant protein techniques are used
for the generation of large amounts of the polypeptide of the
present invention. In some embodiments, recombinant techniques are
described by Bitter et al., (1987) Methods in Enzymol. 153:516-544,
Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al.
(1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J.
6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et
al, (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell.
Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for
Plant Molecular Biology, Academic Press, NY, Section VIII, pp
421-463.
[0190] In one embodiment, a polypeptide of the present invention is
synthesized using a polynucleotide encoding a polypeptide of the
present invention. In some embodiments, the polynucleotide encoding
a polypeptide of the present invention is ligated into an
expression vector, comprising a transcriptional control of a
cis-regulatory sequence (e.g., promoter sequence). In some
embodiments, the cis-regulatory sequence is suitable for directing
constitutive expression of the polypeptide of the present
invention. In some embodiments, the cis-regulatory sequence is
suitable for directing tissue specific expression of the
polypeptide of the present invention. In some embodiments, the
cis-regulatory sequence is suitable for directing inducible
expression of the polypeptide of the present invention.
[0191] In some embodiments, polynucleotides which express the
polypeptides of the present invention are as set forth in SEQ ID
NOs: 20, 21, 44, 45 and 46.
[0192] In some embodiment, tissue-specific promoters suitable for
use with the present invention include sequences which are
functional in specific cell population, example include, but are
not limited to promoters such as albumin that is liver specific
[Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific
promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in
particular promoters of T-cell receptors [Winoto et al., (1989)
EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell
33729-740], neuron-specific promoters such as the neurofilament
promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA
86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985)
Science 230:912-916] or mammary gland-specific promoters such as
the milk whey promoter (U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). Inducible promoters suitable
for use with the present invention include for example the
tetracycline-inducible promoter (Srour, M. A., et al., 2003.
Thromb. Haemost. 90: 398-405).
[0193] In one embodiment, the phrase "a polynucleotide" refers to a
single or double stranded nucleic acid sequence which be isolated
and provided in the form of an RNA sequence, a complementary
polynucleotide sequence (cDNA), a genomic polynucleotide sequence
and/or a composite polynucleotide sequences (e.g., a combination of
the above).
[0194] In one embodiment, "complementary polynucleotide sequence"
refers to a sequence, which results from reverse transcription of
messenger RNA using a reverse transcriptase or any other RNA
dependent DNA polymerase. In one embodiment, the sequence can be
subsequently amplified in vivo or in vitro using a DNA
polymerase.
[0195] In one embodiment, "genomic polynucleotide sequence" refers
to a sequence derived (isolated) from a chromosome and thus it
represents a contiguous portion of a chromosome.
[0196] In one embodiment, "composite polynucleotide sequence"
refers to a sequence, which is at least partially complementary and
at least partially genomic. In one embodiment, a composite sequence
can include some exonal sequences required to encode the
polypeptide of the present invention, as well as some intronic
sequences interposing there between. In one embodiment, the
intronic sequences can be of any source, including of other genes,
and typically will include conserved splicing signal sequences. In
one embodiment, intronic sequences include cis acting expression
regulatory elements.
[0197] In one embodiment, the polynucleotides of the present
invention further comprise a signal sequence encoding a signal
peptide for the secretion of the polypeptides of the present
invention. In some embodiments, signal sequences include, but are
not limited to the endogenous signal sequence for EPO as set forth
in SEQ ID NO: 19 or the endogenous signal sequence for IFN-.beta.1
as set forth in SEQ ID NO: 26. In another embodiment, the signal
sequence is N-terminal to the CTP sequence that is in turn
N-terminal to the polypeptide sequence of interest; e.g. the
sequence is (a) signal sequence- (b) CTP- (c) sequence-of-interest-
(d) optionally 1 or more additional CTP sequences. In another
embodiment, 1 or more CTP sequences is inserted between the signal
sequence of a polypeptide sequence of interest and the polypeptide
sequence of interest itself, thus interrupting the wild-type
sequence of interest. Each possibility represents a separate
embodiment of the present invention.
[0198] In one embodiment, following expression and secretion, the
signal peptides are cleaved from the precursor proteins resulting
in the mature proteins.
[0199] In some embodiments, polynucleotides of the present
invention are prepared using PCR techniques as described in Example
1, or any other method or procedure known to one skilled in the
art. In some embodiments, the procedure involves the legation of
two different DNA sequences (See, for example, "Current Protocols
in Molecular Biology", eds. Ausubel et al., John Wiley & Sons,
1992).
[0200] In one embodiment, polynucleotides of the present invention
are inserted into expression vectors (i.e., a nucleic acid
construct) to enable expression of the recombinant polypeptide. In
one embodiment, the expression vector of the present invention
includes additional sequences which render this vector suitable for
replication and integration in prokaryotes. In one embodiment, the
expression vector of the present invention includes additional
sequences which render this vector suitable for replication and
integration in eukaryotes. In one embodiment, the expression vector
of the present invention includes a shuttle vector which renders
this vector suitable for replication and integration in both
prokaryotes and eukaryotes. In some embodiments, cloning vectors
comprise transcription and translation initiation sequences (e.g.,
promoters, enhances) and transcription and translation terminators
(e.g., polyadenylation signals).
[0201] In one embodiment, a variety of prokaryotic or eukaryotic
cells can be used as host-expression systems to express the
polypeptides of the present invention. In some embodiments, these
include, but are not limited to, microorganisms, such as bacteria
transformed with a recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vector containing the polypeptide coding
sequence; yeast transformed with recombinant yeast expression
vectors containing the polypeptide coding sequence; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors, such as Ti
plasmid, containing the polypeptide coding sequence.
[0202] In some embodiments, non-bacterial expression systems are
used (e.g. mammalian expression systems such as CHO cells) to
express the polypeptide of the present invention. In one
embodiment, the expression vector used to express polynucleotides
of the present invention in mammalian cells is pCI-DHFR vector
comprising a CMV promoter and a neomycin resistance gene.
Construction of the pCI-dhfr vector is described, according to one
embodiment, in Example 1.
[0203] In some embodiments, in bacterial systems of the present
invention, a number of expression vectors can be advantageously
selected depending upon the use intended for the polypeptide
expressed. In one embodiment, large quantities of polypeptide are
desired. In one embodiment, vectors that direct the expression of
high levels of the protein product, possibly as a fusion with a
hydrophobic signal sequence, which directs the expressed product
into the periplasm of the bacteria or the culture medium where the
protein product is readily purified are desired. In one embodiment,
certain fusion protein engineered with a specific cleavage site to
aid in recovery of the polypeptide. In one embodiment, vectors
adaptable to such manipulation include, but are not limited to, the
pET series of E. coli expression vectors [Studier et al., Methods
in Enzymol. 185:60-89 (1990)].
[0204] In one embodiment, yeast expression systems are used. In one
embodiment, a number of vectors containing constitutive or
inducible promoters can be used in yeast as disclosed in U.S. Pat.
No. 5,932,447. In another embodiment, vectors which promote
integration of foreign DNA sequences into the yeast chromosome are
used.
[0205] In one embodiment, the expression vector of the present
invention can further include additional polynucleotide sequences
that allow, for example, the translation of several proteins from a
single mRNA such as an internal ribosome entry site (IRES) and
sequences for genomic integration of the promoter-chimeric
polypeptide.
[0206] In some embodiments, mammalian expression vectors include,
but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-),
pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5,
DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from
Invitrogen, pCI which is available from Promega, pMbac, pPbac,
pBK-RSV and pBK-CMV which are available from Strategene, pTRES
which is available from Clontech, and their derivatives.
[0207] In some embodiments, expression vectors containing
regulatory elements from eukaryotic viruses such as retroviruses
are used by the present invention. SV40 vectors include pSVT7 and
pMT2. In some embodiments, vectors derived from bovine papilloma
virus include pBV-1MTHA, and vectors derived from Epstein Bar virus
include pHEBO, and p2O5. Other exemplary vectors include pMSG,
pAV009/A.sup.+, pMTO10/A.sup.+, pMAMneo-5, baculovirus pDSVE, and
any other vector allowing expression of proteins under the
direction of the SV-40 early promoter, SV-40 later promoter,
metallothionein promoter, murine mammary tumor virus promoter, Rous
sarcoma virus promoter, polyhedrin promoter, or other promoters
shown effective for expression in eukaryotic cells.
[0208] In some embodiments, recombinant viral vectors are useful
for in vivo expression of the polypeptides of the present invention
since they offer advantages such as lateral infection and targeting
specificity. In one embodiment, lateral infection is inherent in
the life cycle of, for example, retrovirus and is the process by
which a single infected cell produces many progeny virions that bud
off and infect neighboring cells. In one embodiment, the result is
that a large area becomes rapidly infected, most of which was not
initially infected by the original viral particles. In one
embodiment, viral vectors are produced that are unable to spread
laterally. In one embodiment, this characteristic can be useful if
the desired purpose is to introduce a specified gene into only a
localized number of targeted cells.
[0209] In one embodiment, various methods can be used to introduce
the expression vector of the present invention into cells. Such
methods are generally described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New
York (1989, 1992), in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989),
Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.
(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich.
(1995), Vectors: A Survey of Molecular Cloning Vectors and Their
Uses, Butterworths, Boston Mass. (1988) and Gilboa et al.
[Biotechniques 4 (6): 504-512, 1986] and include, for example,
stable or transient transfection, lipofection, electroporation and
infection with recombinant viral vectors. In addition, see U.S.
Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection
methods.
[0210] In some embodiments, introduction of nucleic acid by viral
infection offers several advantages over other methods such as
lipofection and electroporation, since higher transfection
efficiency can be obtained due to the infectious nature of
viruses.
[0211] In one embodiment, it will be appreciated that the
polypeptides of the present invention can also be expressed from a
nucleic acid construct administered to the individual employing any
suitable mode of administration, described hereinabove (i.e.,
in-vivo gene therapy). In one embodiment, the nucleic acid
construct is introduced into a suitable cell via an appropriate
gene delivery vehicle/method (transfection, transduction,
homologous recombination, etc.) and an expression system as needed
and then the modified cells are expanded in culture and returned to
the individual (i.e., ex-vivo gene therapy).
[0212] In one embodiment, in vivo gene therapy using EPO has been
attempted in animal models such as rodents [Bohl et al., Blood.
2000; 95:2793-2798], primates [Gao et al., Blood, 2004, Volume 103,
Number 9] and has proven successful in human clinical trials for
patients with chronic renal failure [Lippin et al Blood 2005, 106,
Number 7].
[0213] In one embodiment, plant expression vectors are used. In one
embodiment, the expression of a polypeptide coding sequence is
driven by a number of promoters. In some embodiments, viral
promoters such as the 35S RNA and 19S RNA promoters of CaMV
[Brisson et al., Nature 310:511-514 (1984)], or the coat protein
promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are
used. In another embodiment, plant promoters are used such as, for
example, the small subunit of RUBISCO [Coruzzi et al., EMBO J.
3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)]
or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B
[Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one
embodiment, constructs are introduced into plant cells using Ti
plasmid, Ri plasmid, plant viral vectors, direct DNA
transformation, microinjection, electroporation and other
techniques well known to the skilled artisan. See, for example,
Weissbach & Weissbach [Methods for Plant Molecular Biology,
Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other
expression systems such as insects and mammalian host cell systems,
which are well known in the art, can also be used by the present
invention.
[0214] It will be appreciated that other than containing the
necessary elements for the transcription and translation of the
inserted coding sequence (encoding the polypeptide), the expression
construct of the present invention can also include sequences
engineered to optimize stability, production, purification, yield
or activity of the expressed polypeptide.
[0215] Various methods, in some embodiments, can be used to
introduce the expression vector of the present invention into the
host cell system. In some embodiments, such methods are generally
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene
Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene
Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of
Molecular Cloning Vectors and Their Uses, Butterworths, Boston
Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986]
and include, for example, stable or transient transfection,
lipofection, electroporation and infection with recombinant viral
vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992
for positive-negative selection methods.
[0216] In some embodiments, transformed cells are cultured under
effective conditions, which allow for the expression of high
amounts of recombinant polypeptide. In some embodiments, effective
culture conditions include, but are not limited to, effective
media, bioreactor, temperature, pH and oxygen conditions that
permit protein production. In one embodiment, an effective medium
refers to any medium in which a cell is cultured to produce the
recombinant polypeptide of the present invention. In some
embodiments, a medium typically includes an aqueous solution having
assimilable carbon, nitrogen and phosphate sources, and appropriate
salts, minerals, metals and other nutrients, such as vitamins. In
some embodiments, cells of the present invention can be cultured in
conventional fermentation bioreactors, shake flasks, test tubes,
microtiter dishes and petri plates. In some embodiments, culturing
is carried out at a temperature, pH and oxygen content appropriate
for a recombinant cell. In some embodiments, culturing conditions
are within the expertise of one of ordinary skill in the art.
[0217] In some embodiments, depending on the vector and host system
used for production, resultant polypeptides of the present
invention either remain within the recombinant cell, secreted into
the fermentation medium, secreted into a space between two cellular
membranes, such as the periplasmic space in E. coli; or retained on
the outer surface of a cell or viral membrane.
[0218] In one embodiment, following a predetermined time in
culture, recovery of the recombinant polypeptide is effected.
[0219] In one embodiment, the phrase "recovering the recombinant
polypeptide" used herein refers to collecting the whole
fermentation medium containing the polypeptide and need not imply
additional steps of separation or purification.
[0220] In one embodiment, polypeptides of the present invention are
purified using a variety of standard protein purification
techniques, such as, but not limited to, affinity chromatography,
ion exchange chromatography, filtration, electrophoresis,
hydrophobic interaction chromatography, gel filtration
chromatography, reverse phase chromatography, concanavalin A
chromatography, chromatofocusing and differential
solubilization.
[0221] In one embodiment, to facilitate recovery, the expressed
coding sequence can be engineered to encode the polypeptide of the
present invention and fused cleavable moiety. In one embodiment, a
fusion protein can be designed so that the polypeptide can be
readily isolated by affinity chromatography; e.g., by
immobilization on a column specific for the cleavable moiety. In
one embodiment, a cleavage site is engineered between the
polypeptide and the cleavable moiety and the polypeptide can be
released from the chromatographic column by treatment with an
appropriate enzyme or agent that specifically cleaves the fusion
protein at this site [e.g., see Booth et al., Immunol. Lett.
19:65-70 (1988); and Gardella et al., J. Biol. Chem.
265:15854-15859 (1990)].
[0222] In one embodiment, the polypeptide of the present invention
is retrieved in "substantially pure" form.
[0223] In one embodiment, the phrase "substantially pure" refers to
a purity that allows for the effective use of the protein in the
applications described herein.
[0224] In one embodiment, the polypeptide of the present invention
can also be synthesized using in vitro expression systems. In one
embodiment, in vitro synthesis methods are well known in the art
and the components of the system are commercially available.
[0225] In one embodiment, production of CTP-EPO-CTP polypeptides
using recombinant DNA technology is illustrated in Example 1.
[0226] In some embodiments, the recombinant polypeptides are
synthesized and purified; their therapeutic efficacy can be assayed
either in vivo or in vitro. In one embodiment, the binding
activities of the recombinant EPO polypeptides of the present
invention can be ascertained using various assays as described in
Examples 2-6 and 8-9. In one embodiment, in vitro binding activity
is ascertained by measuring the ability of the polypeptide to
stimulate proliferation of TF-1 cells. In one embodiment, in vivo
activity is deduced by analyzing hematocrit levels (FIGS. 3-5)
and/or as a percentage of reticulocytes.
[0227] In one embodiment, the EPO polypeptides of the present
invention can be used to treat a subject, with a variety of
erythropoietin-associated conditions. In some embodiments, a
subject is a human subject.
[0228] In some embodiment, the phrase "erythropoietin-associated
conditions" refers to any condition associated with below normal,
abnormal, or inappropriate modulation of erythropoietin. In some
embodiment, levels of erythropoietin associated with such
conditions are determined by any measure accepted and utilized by
those of skill in the art. In some embodiment,
erythropoietin-associated conditions typically include anemic
conditions.
[0229] In some embodiment, "anemic conditions" refers to any
condition, disease, or disorder associated with anemia. In some
embodiment, anemic conditions include, but are not limited to,
aplastic anemia, autoimmune hemolytic anemia, bone marrow
transplantation, Churg-Strauss syndrome, Diamond Blackfan anemia,
Fanconi's anemia, Felty syndrome, graft versus host disease,
hematopoietic stem cell transplantation, hemolytic uremic syndrome,
myelodysplasic syndrome, nocturnal paroxysmal hemoglobinuria,
osteomyelofibrosis, pancytopenia, pure red-cell aplasia, purpura
Schoenlein-Henoch, sideroblastic anemia, refractory anemia with
excess of blasts, rheumatoid arthritis, Shwachman syndrome, sickle
cell disease, thalassemia major, thalassemia minor,
thrombocytopenic purpura, etc.
[0230] In one embodiment, the present invention comprises
CTP-hGH-CTP polypeptides. In one embodiment, recombinant DNA
technology methods are used for the production of CTP-hGH-CTP
polypeptides as illustrated in Example 7. In one embodiment, the
therapeutic efficacy of the CTP-hGH-CTP polypeptides of the present
invention is assayed either in vivo. In one embodiment, the
therapeutic efficacy of the CTP-hGH-CTP polypeptides of the present
invention is assayed either in vitro. In one embodiment, the
binding activities of the recombinant hGH polypeptides of the
present invention are measured using Nb2 (a prolactin-dependent rat
lymphoma cell line (ECACC Cell Bank)) or a FCD-P1 murine cell line,
previously transfected with human growth hormone receptor. In one
embodiment, binding of hGH to these receptors induces cell
proliferation which in one embodiment is measured by the levels of
MTT cellular stain as a function of hGH activity. In one
embodiment, in vivo activity is deduced by measuring weight gain
over time in treated growth hormone deficient animals.
[0231] In some embodiment, human growth hormone polypeptides of the
present invention can be used to treat a subject, with conditions
related to growth and weight, such as a growth deficiency disorder,
AIDS wasting, aging, impaired immune function of HIV-infected
subjects, a catabolic illness, surgical recovery, a congestive
cardiomyopathy, liver transplantation, liver regeneration after
hepatectomy, chronic renal failure, renal osteodystrophy,
osteoporosis, achondroplasia/hypochondroplasia, skeletal dysplasia,
a chronic inflammatory or nutritional disorder such as Crohn's
disease, short bowel syndrome, juvenile chronic arthritis, cystic
fibrosis, male infertility, X-linked hypophosphatemic rickets,
Down's syndrome, Spina bifida, Noonan Syndrome, obesity, impaired
muscle strength and fibromyalgia. In some embodiments, interferon
polypeptides of the present invention are used to treat a subject,
with a variety of conditions such as hairy cell leukemia (HCL),
Kaposi's sarcoma (KS), chronic myelogenous leukemia (CML), chronic
hepatitis C (CHC), condylomata acuminata (CA), chronic hepatitis B,
malignant melanoma, follicular non-Hodgkin's lymphoma, multiple
sclerosis, chronic granulomatous disease, Mycobacterium avium
complex (MAC), pulmonary fibrosis and osteoporosis.
[0232] In some embodiments, Glucagon-like peptide-1 (GLP-1)
polypeptides of the present invention are used to treat a subject
with non-insulin dependent diabetes, obesity, stroke, myocardial
infarction, stroke, stress-induced hyperglycemia, or irritable
bowel syndrome.
[0233] In one embodiment, the polypeptides of the present invention
can be provided to the individual per se. In one embodiment, the
polypeptides of the present invention can be provided to the
individual as part of a pharmaceutical composition where it is
mixed with a pharmaceutically acceptable carrier.
[0234] In one embodiment, a "pharmaceutical composition" refers to
a preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0235] In one embodiment, "active ingredient" refers to the
polypeptide sequence of interest, which is accountable for the
biological effect.
[0236] In some embodiments, any of the compositions of this
invention will comprise at least two CTP sequences bound to a
protein of interest, in any form. In one embodiment, the present
invention provides combined preparations. In one embodiment, "a
combined preparation" defines especially a "kit of parts" in the
sense that the combination partners as defined above can be dosed
independently or by use of different fixed combinations with
distinguished amounts of the combination partners i.e.,
simultaneously, concurrently, separately or sequentially. In some
embodiments, the parts of the kit of pairs can then, e.g., be
administered simultaneously or chronologically staggered, that is
at different time points and with equal or different time intervals
for any part of the kit of parts. The ratio of the total amounts of
the combination partners, in some embodiments, can be administered
in the combined preparation. In one embodiment, the combined
preparation can be varied, e.g., in order to cope with the needs of
a patient subpopulation to be treated or the needs of the single
patient which different needs can be due to a particular disease,
severity of a disease, age, sex, or body weight as can be readily
made by a person skilled in the art.
[0237] In one embodiment, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases. In one
embodiment, one of the ingredients included in the pharmaceutically
acceptable carrier can be for example polyethylene glycol (PEG), a
biocompatible polymer with a wide range of solubility in both
organic and aqueous media (Mutter et al. (1979).
[0238] In one embodiment, "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. In one embodiment,
excipients include calcium carbonate, calcium phosphate, various
sugars and types of starch, cellulose derivatives, gelatin,
vegetable oils and polyethylene glycols.
[0239] Techniques for formulation and administration of drugs are
found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0240] In one embodiment, suitable routes of administration, for
example, include oral, rectal, transmucosal, transnasal, intestinal
or parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
[0241] In one embodiment, the preparation is administered in a
local rather than systemic manner, for example, via injection of
the preparation directly into a specific region of a patient's
body.
[0242] Various embodiments of dosage ranges are contemplated by
this invention. The dosage of the polypeptide of the present
invention, in one embodiment, is in the range of 0.05-80 mg/day. In
another embodiment, the dosage is in the range of 0.05-50 mg/day.
In another embodiment, the dosage is in the range of 0.1-20 mg/day.
In another embodiment, the dosage is in the range of 0.1-10 mg/day.
In another embodiment, the dosage is in the range of 0.1-5 mg/day.
In another embodiment, the dosage is in the range of 0.5-5 mg/day.
In another embodiment, the dosage is in the range of 0.5-50 mg/day.
In another embodiment, the dosage is in the range of 5-80 mg/day.
In another embodiment, the dosage is in the range of 35-65 mg/day.
In another embodiment, the dosage is in the range of 35-65 mg/day.
In another embodiment, the dosage is in the range of 20-60 mg/day.
In another embodiment, the dosage is in the range of 40-60 mg/day.
In another embodiment, the dosage is in a range of 45-60 mg/day. In
another embodiment, the dosage is in the range of 40-60 mg/day. In
another embodiment, the dosage is in a range of 60-120 mg/day. In
another embodiment, the dosage is in the range of 120-240 mg/day.
In another embodiment, the dosage is in the range of 40-60 mg/day.
In another embodiment, the dosage is in a range of 240-400 mg/day.
In another embodiment, the dosage is in a range of 45-60 mg/day. In
another embodiment, the dosage is in the range of 15-25 mg/day. In
another embodiment, the dosage is in the range of 5-10 mg/day. In
another embodiment, the dosage is in the range of 55-65 mg/day.
[0243] In one embodiment, the dosage is 20 mg/day. In another
embodiment, the dosage is 30 mg/day. In another embodiment, the
dosage is 40 mg/day. In another embodiment, the dosage is 50
mg/day. In another embodiment, the dosage is 60 mg/day. In another
embodiment, the dosage is 70 mg/day. In another embodiment, the
dosage is 80 mg/day. In another embodiment, the dosage is 90
mg/day. In another embodiment, the dosage is 100 mg/day.
[0244] Oral administration, in one embodiment, comprises a unit
dosage form comprising tablets, capsules, lozenges, chewable
tablets, suspensions, emulsions and the like. Such unit dosage
forms comprise a safe and effective amount of the desired compound,
or compounds, each of which is in one embodiment, from about 0.7 or
3.5 mg to about 280 mg/70 kg, or in another embodiment, about 0.5
or 10 mg to about 210 mg/70 kg. The pharmaceutically-acceptable
carriers suitable for the preparation of unit dosage forms for
peroral administration are well-known in the art. In some
embodiments, tablets typically comprise conventional
pharmaceutically-compatible adjuvants as inert diluents, such as
calcium carbonate, sodium carbonate, mannitol, lactose and
cellulose; binders such as starch, gelatin and sucrose;
disintegrants such as starch, alginic acid and croscarmelose;
lubricants such as magnesium stearate, stearic acid and talc. In
one embodiment, glidants such as silicon dioxide can be used to
improve flow characteristics of the powder-mixture. In one
embodiment, coloring agents, such as the FD&C dyes, can be
added for appearance. Sweeteners and flavoring agents, such as
aspartame, saccharin, menthol, peppermint, and fruit flavors, are
useful adjuvants for chewable tablets. Capsules typically comprise
one or more solid diluents disclosed above. In some embodiments,
the selection of carrier components depends on secondary
considerations like taste, cost, and shelf stability, which are not
critical for the purposes of this invention, and can be readily
made by a person skilled in the art.
[0245] In one embodiment, the oral dosage form comprises predefined
release profile. In one embodiment, the oral dosage form of the
present invention comprises an extended release tablets, capsules,
lozenges or chewable tablets. In one embodiment, the oral dosage
form of the present invention comprises a slow release tablets,
capsules, lozenges or chewable tablets. In one embodiment, the oral
dosage form of the present invention comprises an immediate release
tablets, capsules, lozenges or chewable tablets. In one embodiment,
the oral dosage form is formulated according to the desired release
profile of the pharmaceutical active ingredient as known to one
skilled in the art.
[0246] Peroral compositions, in some embodiments, comprise liquid
solutions, emulsions, suspensions, and the like. In some
embodiments, pharmaceutically-acceptable carriers suitable for
preparation of such compositions are well known in the art. In some
embodiments, liquid oral compositions comprise from about 0.012% to
about 0.933% of the desired compound or compounds, or in another
embodiment, from about 0.033% to about 0.7%.
[0247] In some embodiments, compositions for use in the methods of
this invention comprise solutions or emulsions, which in some
embodiments are aqueous solutions or emulsions comprising a safe
and effective amount of the compounds of the present invention and
optionally, other compounds, intended for topical intranasal
administration. In some embodiments, h compositions comprise from
about 0.01% to about 10.0% w/v of a subject compound, more
preferably from about 0.1% to about 2.0, which is used for systemic
delivery of the compounds by the intranasal route.
[0248] In another embodiment, the pharmaceutical compositions are
administered by intravenous, intra-arterial, or intramuscular
injection of a liquid preparation. In some embodiments, liquid
formulations include solutions, suspensions, dispersions,
emulsions, oils and the like. In one embodiment, the pharmaceutical
compositions are administered intravenously, and are thus
formulated in a form suitable for intravenous administration. In
another embodiment, the pharmaceutical compositions are
administered intra-arterially, and are thus formulated in a form
suitable for intra-arterial administration. In another embodiment,
the pharmaceutical compositions are administered intramuscularly,
and are thus formulated in a form suitable for intramuscular
administration.
[0249] Further, in another embodiment, the pharmaceutical
compositions are administered topically to body surfaces, and are
thus formulated in a form suitable for topical administration.
Suitable topical formulations include gels, ointments, creams,
lotions, drops and the like. For topical administration, the
compounds of the present invention are combined with an additional
appropriate therapeutic agent or agents, prepared and applied as
solutions, suspensions, or emulsions in a physiologically
acceptable diluent with or without a pharmaceutical carrier.
[0250] In one embodiment, pharmaceutical compositions of the
present invention are manufactured by processes well known in the
art, e.g., by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping or lyophilizing processes.
[0251] In one embodiment, pharmaceutical compositions for use in
accordance with the present invention is formulated in conventional
manner using one or more physiologically acceptable carriers
comprising excipients and auxiliaries, which facilitate processing
of the active ingredients into preparations which, can be used
pharmaceutically. In one embodiment, formulation is dependent upon
the route of administration chosen.
[0252] In one embodiment, injectables, of the invention are
formulated in aqueous solutions. In one embodiment, injectables, of
the invention are formulated in physiologically compatible buffers
such as Hank's solution, Ringer's solution, or physiological salt
buffer. In some embodiments, for transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0253] In one embodiment, the preparations described herein are
formulated for parenteral administration, e.g., by bolus injection
or continuous infusion. In some embodiments, formulations for
injection are presented in unit dosage form, e.g., in ampoules or
in multidose containers with optionally, an added preservative. In
some embodiments, compositions are suspensions, solutions or
emulsions in oily or aqueous vehicles, and contain formulatory
agents such as suspending, stabilizing and/or dispersing
agents.
[0254] The compositions also comprise, in some embodiments,
preservatives, such as benzalkonium chloride and thimerosal and the
like; chelating agents, such as edetate sodium and others; buffers
such as phosphate, citrate and acetate; tonicity agents such as
sodium chloride, potassium chloride, glycerin, mannitol and others;
antioxidants such as ascorbic acid, acetylcystine, sodium
metabisulfote and others; aromatic agents; viscosity adjustors,
such as polymers, including cellulose and derivatives thereof; and
polyvinyl alcohol and acid and bases to adjust the pH of these
aqueous compositions as needed. The compositions also comprise, in
some embodiments, local anesthetics or other actives. The
compositions can be used as sprays, mists, drops, and the like.
[0255] In some embodiments, pharmaceutical compositions for
parenteral administration include aqueous solutions of the active
preparation in water-soluble form. Additionally, suspensions of the
active ingredients, in some embodiments, are prepared as
appropriate oily or water based injection suspensions. Suitable
lipophilic solvents or vehicles include, in some embodiments, fatty
oils such as sesame oil, or synthetic fatty acid esters such as
ethyl oleate, triglycerides or liposomes. Aqueous injection
suspensions contain, in some embodiments, substances, which
increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol or dextran. In another
embodiment, the suspension also contain suitable stabilizers or
agents which increase the solubility of the active ingredients to
allow for the preparation of highly concentrated solutions.
[0256] In another embodiment, the active compound can be delivered
in a vesicle, in particular a liposome (see Langer, Science
249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid).
[0257] In another embodiment, the pharmaceutical composition
delivered in a controlled release system is formulated for
intravenous infusion, implantable osmotic pump, transdermal patch,
liposomes, or other modes of administration. In one embodiment, a
pump is used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989). In another embodiment,
polymeric materials can be used. In yet another embodiment, a
controlled release system can be placed in proximity to the
therapeutic target, i.e., the brain, thus requiring only a fraction
of the systemic dose (see, e.g., Goodson, in Medical Applications
of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other
controlled release systems are discussed in the review by Langer
(Science 249:1527-1533 (1990).
[0258] In some embodiments, the active ingredient is in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use. Compositions are
formulated, in some embodiments, for atomization and inhalation
administration. In another embodiment, compositions are contained
in a container with attached atomizing means.
[0259] In one embodiment, the preparation of the present invention
is formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0260] In some embodiments, pharmaceutical compositions suitable
for use in context of the present invention include compositions
wherein the active ingredients are contained in an amount effective
to achieve the intended purpose. In some embodiments, a
therapeutically effective amount means an amount of active
ingredients effective to prevent, alleviate or ameliorate symptoms
of disease or prolong the survival of the subject being
treated.
[0261] In one embodiment, determination of a therapeutically
effective amount is well within the capability of those skilled in
the art.
[0262] The compositions also comprise preservatives, such as
benzalkonium chloride and thimerosal and the like; chelating
agents, such as edetate sodium and others; buffers such as
phosphate, citrate and acetate; tonicity agents such as sodium
chloride, potassium chloride, glycerin, mannitol and others;
antioxidants such as ascorbic acid, acetylcystine, sodium
metabisulfote and others; aromatic agents; viscosity adjustors,
such as polymers, including cellulose and derivatives thereof; and
polyvinyl alcohol and acid and bases to adjust the pH of these
aqueous compositions as needed. The compositions also comprise
local anesthetics or other actives. The compositions can be used as
sprays, mists, drops, and the like.
[0263] Some examples of substances which can serve as
pharmaceutically-acceptable carriers or components thereof are
sugars, such as lactose, glucose and sucrose; starches, such as
corn starch and potato starch; cellulose and its derivatives, such
as sodium carboxymethyl cellulose, ethyl cellulose, and methyl
cellulose; powdered tragacanth; malt; gelatin; talc; solid
lubricants, such as stearic acid and magnesium stearate; calcium
sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame
oil, olive oil, corn oil and oil of theobroma; polyols such as
propylene glycol, glycerine, sorbitol, mannitol, and polyethylene
glycol; alginic acid; emulsifiers, such as the Tween.TM. brand
emulsifiers; wetting agents, such sodium lauryl sulfate; coloring
agents; flavoring agents; tableting agents, stabilizers;
antioxidants; preservatives; pyrogen-free water; isotonic saline;
and phosphate buffer solutions. The choice of a
pharmaceutically-acceptable carrier to be used in conjunction with
the compound is basically determined by the way the compound is to
be administered. If the subject compound is to be injected, in one
embodiment, the pharmaceutically-acceptable carrier is sterile,
physiological saline, with a blood-compatible suspending agent, the
pH of which has been adjusted to about 7.4.
[0264] In addition, the compositions further comprise binders (e.g.
acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,
hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),
disintegrating agents (e.g. cornstarch, potato starch, alginic
acid, silicon dioxide, croscarmelose sodium, crospovidone, guar
gum, sodium starch glycolate), buffers (e.g., Tris-HCL, acetate,
phosphate) of various pH and ionic strength, additives such as
albumin or gelatin to prevent absorption to surfaces, detergents
(e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease
inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation
enhancers, solubilizing agents (e.g., glycerol, polyethylene
glycerol), anti-oxidants (e.g., ascorbic acid, sodium
metabisulfite, butylated hydroxyanisole), stabilizers (e.g.
hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity
increasing agents (e.g. carbomer, colloidal silicon dioxide, ethyl
cellulose, guar gum), sweeteners (e.g. aspartame, citric acid),
preservatives (e.g., Thimerosal, benzyl alcohol, parabens),
lubricants (e.g. stearic acid, magnesium stearate, polyethylene
glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon
dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate),
emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl
sulfate), polymer coatings (e.g., poloxamers or poloxamines),
coating and film forming agents (e.g. ethyl cellulose, acrylates,
polymethacrylates) and/or adjuvants.
[0265] Typical components of carriers for syrups, elixirs,
emulsions and suspensions include ethanol, glycerol, propylene
glycol, polyethylene glycol, liquid sucrose, sorbitol and water.
For a suspension, typical suspending agents include methyl
cellulose, sodium carboxymethyl cellulose, cellulose (e.g.
Avicel.TM., RC-591), tragacanth and sodium alginate; typical
wetting agents include lecithin and polyethylene oxide sorbitan
(e.g. polysorbate 80). Typical preservatives include methyl paraben
and sodium benzoate. In another embodiment, peroral liquid
compositions also contain one or more components such as
sweeteners, flavoring agents and colorants disclosed above.
[0266] The compositions also include incorporation of the active
material into or onto particulate preparations of polymeric
compounds such as polylactic acid, polyglycolic acid, hydrogels,
etc, or onto liposomes, microemulsions, micelles, unilamellar or
multilamellar vesicles, erythrocyte ghosts, or spheroplasts.) Such
compositions will influence the physical state, solubility,
stability, rate of in vivo release, and rate of in vivo
clearance.
[0267] Also comprehended by the invention are particulate
compositions coated with polymers (e.g. poloxamers or poloxamines)
and the compound coupled to antibodies directed against
tissue-specific receptors, ligands or antigens or coupled to
ligands of tissue-specific receptors.
[0268] In some embodiments, compounds modified by the covalent
attachment of water-soluble polymers such as polyethylene glycol,
copolymers of polyethylene glycol and polypropylene glycol,
carboxymethyl cellulose, dextran, polyvinyl alcohol,
polyvinylpyrrolidone or polyproline. In another embodiment, the
modified compounds exhibit substantially longer half-lives in blood
following intravenous injection than do the corresponding
unmodified compounds. In one embodiment, modifications also
increase the compound's solubility in aqueous solution, eliminate
aggregation, enhance the physical and chemical stability of the
compound, and greatly reduce the immunogenicity and reactivity of
the compound. In another embodiment, the desired in vivo biological
activity is achieved by the administration of such polymer-compound
abducts less frequently or in lower doses than with the unmodified
compound.
[0269] In some embodiments, preparation of effective amount or dose
can be estimated initially from in vitro assays. In one embodiment,
a dose can be formulated in animal models and such information can
be used to more accurately determine useful doses in humans.
[0270] In one embodiment, toxicity and therapeutic efficacy of the
active ingredients described herein can be determined by standard
pharmaceutical procedures in vitro, in cell cultures or
experimental animals. In one embodiment, the data obtained from
these in vitro and cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. In one
embodiment, the dosages vary depending upon the dosage form
employed and the route of administration utilized. In one
embodiment, the exact formulation, route of administration and
dosage can be chosen by the individual physician in view of the
patient's condition. [See e.g., Fingl, et al., (1975) "The
Pharmacological Basis of Therapeutics", Ch. 1 p. 1].
[0271] In one embodiment, depending on the severity and
responsiveness of the condition to be treated, dosing can be of a
single or a plurality of administrations, with course of treatment
lasting from several days to several weeks or until cure is
effected or diminution of the disease state is achieved.
[0272] In one embodiment, the amount of a composition to be
administered will, of course, be dependent on the subject being
treated, the severity of the affliction, the manner of
administration, the judgment of the prescribing physician, etc.
[0273] In one embodiment, compositions including the preparation of
the present invention formulated in a compatible pharmaceutical
carrier are also be prepared, placed in an appropriate container,
and labeled for treatment of an indicated condition.
[0274] In one embodiment, compositions of the present invention are
presented in a pack or dispenser device, such as an FDA approved
kit, which contain one or more unit dosage forms containing the
active ingredient. In one embodiment, the pack, for example,
comprise metal or plastic foil, such as a blister pack. In one
embodiment, the pack or dispenser device is accompanied by
instructions for administration. In one embodiment, the pack or
dispenser is accommodated by a notice associated with the container
in a form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice, in
one embodiment, is labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert.
[0275] In one embodiment, it will be appreciated that the
polypeptides of the present invention can be provided to the
individual with additional active agents to achieve an improved
therapeutic effect as compared to treatment with each agent by
itself. In another embodiment, measures (e.g., dosing and selection
of the complementary agent) are taken to adverse side effects which
are associated with combination therapies.
[0276] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0277] Generally, the nomenclature used herein and the laboratory
procedures utilized in the to present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference. Other general
references are provided throughout this document.
Example 1
Generation of EPO Constructs
Materials and Methods:
[0278] Construction of expression vector pCI-dhfr: pCI-neo
mammalian expression vector was purchased from Promega (catalog no.
E1841). The vector contains a CMV IE enhancer/promoter and neomycin
phosphotransferase gene. The pSV2-dhfr clone was purchased from
ATCC (Catalog No. 37146). The plasmid contains the murine dhfr
gene. The construction of pCI-dhfr vector was performed as follows:
[0279] a. The pSV2-dhfr plasmid was digested with restriction
enzyme BglII (3' end of the dhfr gene). DNA polymerase I, Large
(Klenow) Fragment was used to fill-in the 5' overhangs to form
blunt ends. The DNA was then digested with restriction enzyme AvrII
(5' end of the dhfr gene). The dhfr gene (AvrII-blunt end) fragment
was isolated. [0280] b. The pCI-neo vector was digested with
restriction enzyme BstXI (3' end of the neo gene). DNA polymerase
I, Large (Klenow) Fragment was used to remove the 3' overhangs to
form blunt ends. The DNA was then digested with restriction enzyme
AvrII (5' end of the neo gene). The expression vector (AvrII-blunt
end) was isolated. [0281] c. The dhfr gene was ligated into pCI
vector to form an expression vector containing the dhfr gene
(pCI-dhfr).
[0282] Construction of hEPO-CTP variants: A cassette gene
containing the C-Terminal peptide (CTP) of the beta subunit of hCG
was fused to the coding sequence of human EPO (NP.sub.--000790.2)
at different locations. Four EPO-CTP variants were constructed as
illustrated in FIGS. 1A-D. The proEPO signal peptide was used for
the construction of the secreted EPO-CTP variants. XbaI-NotI
fragments containing Epo sequences were ligated into the pCI-dhfr
expression vector of the present invention.
[0283] Table 2 hereinbelow summarizes the primer sequences used for
constructing the CTP-containing polypeptides of the present
invention. TABLE-US-00002 TABLE 2 Restriction SEQ site Primer ID
(underlined number NO sequence in sequence) 1 7 5'
AATCTAGAGGTCATCATGGGGGTG XbaI C 3' 2 8 5' ATTGCGGCCGCGGATCCAGAAGAC
NotI CTTTATTG 3' 17.sup.R 9 5' TAAATATTGGGGTGTCCGAGGGCC SspI C 3'
10 10 5' CCAATATTACCACAAGCCCCACCA SspI CGCCTCAT 3' 11.sup.R 11 5'
TGCGGCCGCGGATCCTTATCTGTC NotI CCCTGTCCTGC 3' 15 12 5'
GCCCTGCTGTCGGAAGC 3' 2.sup.R 13 5' ATTGCGGCCGCGGATCCAGAAGAC NotI
CTTTATTG 23.sup.R 14 5' CTTTGAGGAAGAGGAGCCCAGGAC TGGGAGGC 3' 24 15
5' CCTGGGCTCCTCTTCCTCAAAGG C 3' 38.sup.R 16 5' GCTTCCGACAGCAGGGC
3'
[0284] EPO-1 701-1-p17-6 (Epo-1--SEQ ID NO: 1): The XbaI-NotI 702
bp fragment was constructed by PCR using the above primers (SEQ ID
NOs: 7-16). Then the XbaI-NotI PCR fragment containing Epo-ctp
sequence was ligated into pCI-dhfr expression vector.
[0285] EPO-2 701-2-p24-2 (Epo-2--SEQ ID NO: 2): The XbaI/ApaI
fragment (hGH-ctp) of pCI-dhfr-401-2-p21-2 (hGH-ctpx2) was replaced
by the XbaI/ApaI fragment (EPO-ctp) of 701-1-p17-6 to create an
Epo-ctpx2.
[0286] EPO-4-701-4-p42-1 (Epo-4--SEQ ID NO: 4): Firstly, a fragment
from pCI-dhfr-EPO-ctp (701-1-p17-6) was constructed by PCR using
primers 1 and 17 followed by XbaI/SspI digestion. This resulted in
a fragment containing EPO and partial 5' CTP.
[0287] Secondly, a new fragment was constructed by overlapping PCR,
on pGT123-hEpo as a template, using primer 10 and primer 11.
SspI/NotI digestion resulted in fragment containing 3' partial CTP
and Epo.
[0288] The two fragments were ligated into pCI-dhfr to construct
the p701-4-p42-1 clone.
[0289] EPO-3-p56-6 (Epo-3 SEQ ID NO; 3): Primers were purchased
from Sigma-Genosys. PCR reaction was performed using primer 15 (SEQ
ID NO: 12) and primer 2.sup.R (SEQ ID NO: 13) and plasmid DNA of
pCI-dhfr-EPO-ctp x2 (701-2-p24-2) as a template. As a result of the
PCR amplification, a 486 bp product was formed and ligated into TA
cloning vector (Invitrogen, catalog K2000-01). Stu I-NotI fragment
containing *Epo-ctp x2 sequence was isolated (209 bp).
[0290] Three sequential PCR reactions were performed. The first
reaction was conducted with primer 1 (SEQ ID NO: 7) and primer
23.sup.R (SEQ ID NO: 14) and plasmid DNA of pGT123-epo-ctp as a
template; as a result of the PCR amplification, an 80 bp product
was formed (signal peptide).
[0291] The second reaction was conducted with primer 24 (SEQ ID NO:
15) and primer 11.sup.R (SEQ ID NO: 11) and plasmid DNA of
701-4-p42-1 as a template; as a result of the PCR amplification, a
610 bp product was formed.
[0292] The last reaction was conducted with primers 1 (SEQ ID NO:
7) and 11.sup.R (SEQ ID NO: 11) and a mixture of the products of
the previous two reactions as a template; as a result of the PCR
amplification, a 700 bp product was formed and the XbaI-StuI
fragment was isolated.
[0293] The two fragments (XbaI-StuI and StuI-NotI) were inserted
into the eukaryotic expression vector pCI-dhfr (triple ligation) to
yield the 701-3-p56-6 clone.
[0294] EPO-5-p91-4 (Epo-5 SEQ ID NO; 5--(ctp-Epo): Primers were
ordered from Sigma-Genosys. A PCR reaction was performed using
primer 1 (SEQ ID NO: 7) and primer 11.sup.R (SEQ ID NO: 11) and
plasmid DNA of pCI-dhfr-ctp-EPO-ctp x2 (701-3-p56-6) as a template;
as a result of the PCR amplification, a 670 bp product was formed
and ligated into TA cloning vector (Invitrogen, catalog K2000-01).
XbaI-NotI fragment containing ctp-Epo sequence was ligated into our
eukaryotic expression vector pCI-dhfr to yield the 701-5-p91-4
clone.
[0295] EPO-6-p90-1 (Epo-6 SEQ ID NO: 6--(ctp-Epo-ctp): Three PCR
reactions were performed. The first reaction was conducted with
primer 1 (SEQ ID NO: 7) and primer 38.sup.R (SEQ ID NO: 16) and
plasmid DNA of 701-3-p56-6 as a template; as a result of the PCR
amplification, a 400 bp product was formed.
[0296] The second reaction was conducted with primer 15 (SEQ ID NO:
12) and primer 2.sup.R (SEQ ID NO: 13) and plasmid DNA of
701-1-p17-6 as a template; as a result of the PCR amplification, a
390 bp product was formed.
[0297] The last reaction was conducted with primers 1 (SEQ ID NO:
7) and 2.sup.R (SEQ ID NO: 13) and a mixture of the products of the
previous two reactions as a template; as a result of the PCR
amplification, a 787 bp product was formed and ligated into TA
cloning vector (Invitrogen, catalog K2000-01). The XbaI-NotI
fragment containing ctp-Epo-ctp sequence was ligated into the
eukaryotic expression vector pCI-dhfr to yield the 701-6-p90-1
clone.
Example 2
Expression and Isolation of EPO-CTP Polypeptides
Materials and Methods
[0298] DNA transfection and clone selection: DG44 cells were
transfected with pCI-DHFR expression vectors containing EPO-CTP
variants using FuGENE6 Reagent (FUGENE Transfection Reagent--Roche
Cat. 11 815 091 001). 48 hr following transfection, cells were
diluted and seeded at 50-200 cells per well in a selective medium
(CD DG44 Medium w/o HT (Gibco: Scotland part: #07990111A) Sku num.:
ME060027 supplemented with 8 mM L-Glutamine Biological Industries:
Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68 solution (Gibco:
Cat: 240040-032). Selected clones were screened for highest protein
production using commercial ELISA. 3-5 producing clones per each
variant were frozen for a backup cell bank. A selected clone for
each variant was adapted to growth in larger scale cultures up to 1
L flasks on an orbital shaker platform. Supernatants were collected
and analyzed by ELISA, SDS-PAGE and western blot. Following the
withdrawal of aliquots, the protein-containing supernatants were
kept frozen until further use.
[0299] Cell culture: DG44 cells were maintained in DG44 medium with
HT (cat #12610-010, Gibco) supplemented with 8 mM L-Glutamine
(Biological Industries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic
F-68 solution (Gibco: Cat: 240040-032), at 37.degree. C. in
humidified 8% CO.sub.2 incubator. Transfected clones were
maintained in DG44 basal medium without HT supplement, hypoxanthine
and thymidine, with pluronic acid and L-glutamine.
[0300] Sample preparation: Supernatants were collected, filtrated
and analyzed by ELISA to determine protein concentration. SDS-PAGE
and western blot were used to determine purity and identity.
Following ELISA, sample concentrations were defined and the
solution was dialyzed against PBS. Following the withdrawal of
aliquots, the protein-contained supernatants were kept frozen at
-20.degree. C. until further use.
[0301] Western Blotting: Samples were electrophoresed on
nondenaturing 15% SDS-polyacrylamide gels. Gels were allowed to
equilibrate for 10 min in 25 mM Tris and 192 mM glycine in 20%
(vol/vol) methanol). Proteins were transferred to a 0.2 .mu.m pore
size nitrocellulose membrane (Sigma, Saint Louis, Mo.) at 250 mA
for 3 h, using a Mini Trans-Blot electrophoresis cell (Biorad
Laboratories, Richmond, Calif.). The nitrocellulose membrane was
incubated in 5% non-fat dry milk for 2 h at room temperature. The
membrane was incubated with EPO anti-serum (1:1000 titer) overnight
at 4.degree. C. followed by three consecutive washes in PBS
containing 0.1% Tween (10 min/wash). The membrane was incubated
with secondary antibody conjugated to Horse Radish Peroxidase (HRP)
(Zymed, San Francisco, Calif.) for 2 h at room temperature,
followed by three washes. Finally, the nitrocellulose paper was
reacted with enhanced chemiluminescent substrate (ECL) (Pierce,
Rockford, Ill.) for 5 min, dried with a Whatman sheet, and exposed
to X-ray film.
Results
[0302] Table 3 hereinbelow shows the concentrations of the various
CTP-modified EPO forms obtained from 5 selected clones and their
preparation for further testing. TABLE-US-00003 TABLE 3 Post
dilution in Mock Stock sup. according Titer to Epo3 Post
ultrafiltration #Version # Clone IU/ml.sup.1 titer IU/ml.sup.2
IU/ml.sup.3 Epo0 17 3093 102 335 SEQ ID NO: 16 Epo1 47 1049 104 291
SEQ ID NO: 1 Epo2 67 2160 110 303 SEQ ID NO: 2 Epo3 85 105 119 392
SEQ ID NO: 3 Epo4 112 6100 ND 342 SEQ ID NO: 4 .sup.1EPO variants
stock concentration were determined by ELISA (Quantikine IVD Epo
ELISA, DEP00, R&D Systems) .sup.2Samples EPO-0, 1, 2 and 4 were
diluted to 105 IU/ml in mock sup (Adjusted to Epo3 titer). Epo0 =
wild type EPO expressed in the same system as the CTP modified EPOs
.sup.3All samples were concentrated and dialyzed by ultrafiltration
against PBS to a final concentration of 180 IU/ml.
[0303] All proteins were detected by Western blot as illustrated in
FIG. 2.
Example 3
Biological Activity of the EPO-CTP Polypeptides of the Present
Invention
[0304] The TF-1 bioactivity test represents the ability of the
EPO-CTP variants to bind its receptor and then stimulate activity
which results in cell proliferation. Therefore, this test was used
as a first step in evaluating the biological potency of the EPO-CTP
polypeptides of the present invention.
Materials and Methods
[0305] Cell Proliferation Analysis: Proliferation assay was
performed with the cell line TF-1, measuring levels of MTT cellular
stain as a function of EPO activity (Kitamura et al., Kitamura, T.
et al. (1989) Establishment and characterization of a unique human
cell line that proliferates; Hammerling U. et al. In vitro bioassay
for human erythropoietin based on proliferative stimulation of an
erythroid cell line and analysis of carbohydrate-dependent
microheterogeneity. Journal of Pharm. Biomed. Analysis 14(11):
1455-1469 (1996). Exponentially growing TF-1 cells were washed
twice, plated at about 10.sup.4 cells/well in microtiter plates,
and incubated in basal medium with a titrated dilution series of
EPO (Recormon), EPO standard (NIBSC standard), rhEPO (MOD-7010),
MOD-701 variants (EPO-1, EPO-2, EPO-3 and EPO-4) for 48 hours. 4
hours prior to assaying for cell proliferation, MTT reagent was
added to the wells, and absorbance was measured by ELISA reader. A
calculated protein concentration value for each variant protein was
obtained from Eprex's (Epoetin (EPO)-man-made form of the human
hormone) dose-response standard curve.
Results
[0306] The in vitro biological activity of EPO polypeptides was
determined with an Epo-dependent cell line, human erythroleukemia
TF-1 (DSMZ Cell Bank) [Dong et al., Biochemical and Biophysical
Research Communications, Volume 339, Issue 1, 6 Jan. 2006, Pages
380-385]. The MTT assay was performed [Hammerling U. et al. In
vitro bioassay for human erythropoietin based on proliferative
stimulation of an erythroid cell line and analysis of
carbohydrate-dependent microheterogeneity. Journal of Pharm.
Biomed. Analysis 14(11): 1455-1469 (1996);], and the laboratory
standard of EPO used to generate the standard curve was calibrated
against the International Standard (Epo ampoule code 87/684 of
NIBSC).
[0307] The results are summarized in Table 4 hereinbelow. The
results indicate that the highest potency was achieved by using EPO
3 and EPO 0 in both 2 and 0.5 IU/ml concentrations. TABLE-US-00004
TABLE 4 TF-1 Bioactivity IU/ml Eprex EPO 0 EPO 1 EPO 2 EPO 3 EPO 4
STD SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID EPO IU/ml NO: 16 NO: 1 NO: 2
NO: 3 NO: 4 Recormon st 2 4.93 2.32 2.13 6.91 3.55 3.44 7.40 0.5
1.60 0.76 0.53 1.34 0.84 0.87 1.53
Conclusion
[0308] As summarized in Table 4 hereinabove, different levels of
potency were exerted by EPO-CTP polypeptides, indicating
differences in receptor binding. EPO-CTP polypeptides differ by the
number of CTP cassettes and the location to which they are fused.
EPO-1 and EPO-2 contain 1 CTP sequence or 2 CTP sequences at the
C-terminal of EPO, while EPO-3 contains 1 CTP at N-terminal
terminal and 2 CTP sequences at C-terminal. EPO-4 is a dimer of two
EPO molecules linked by CTP sequence. EPO-3 demonstrated potency
level quite similar to WT-EPO, while EPO-1 and EPO-4 were about 50%
less potent than WT-EPO, and EPO-2 potency was even less than
50%.
Example 4
Evaluation of the EPO-CTP Polypeptides of the Present Invention in
a Mouse Model
[0309] The following experiment was performed in order to compare
the bio-activity of the EPO-polypeptides CTP polypeptides of the
present invention and commercial EPO
Materials and Methods
Animals:
Species/Strain: ICR or CD-1 Mice of either sex about 20-25 g
Group Size: n=7
No. Groups: 9
Total No. Animals: n=63
[0310] Experimental design of the study: The experiment was set up
as summarized in Table 5 hereinbelow. TABLE-US-00005 TABLE 5
TREATMENT No. Mice Dose Group No. per Group Compound Level Dosing
Regimen 1 n = 7 Vehicle (Control) 0 1 .times. weekly 2 MOCK 3
MOD-7010 15 .mu.g/kg 4 MOD-7011 5 MOD-7012 6 MOD-7013 7 MOD-7014 8
Commercial 15 .mu.g/kg 9 rhEPO 5 .mu.g/kg 3 .times. weekly
[0311] Animal treatment: All animals were administered with either
control or the test EPO polypeptides of the present invention by
bolus injection. The injection volume did not exceed 10 ml/kg. The
length of the experiment was 22 days. A morbidity and mortality
check was performed daily.
[0312] Reticulocyte count and hematocrit (hct) examination:
Reticulocyte count was carried out in all test animals at day 2 and
14 hrs following the 1st respective vehicle or treatment injection.
HCT was determined in all animals once prior to initial treatment
("0" Baseline control) and at 24 hrs after the 1st respective
vehicle or treatment injection, and thereafter twice weekly until
study termination (Day-22).
Results
[0313] The hematocrit results which are illustrated in FIGS. 3-5
show that EPO 3 has the highest hematocrit percentage change from
baseline compared to EPO 1, EPO 2, Recormon 1, Recormon 3, rhEPO,
and vehicle. The results demonstrating the percentage of
reticulocytes in mice treated with the EPO-CTP polypeptides are
summarized in Table 6 hereinbelow. These results show that EPO-3 is
the most potent stimulator of erythropoiesis. TABLE-US-00006 TABLE
6 % reticulocytes Days 2 14 Control 3.72 3.46 1.08 0.8 Mock 3.5
3.64 0.6 1.13 7010 SEQ ID NO: 16 3.5 3.9 0.6 1.54 7011 SEQ ID NO: 1
3.52 1.94 1.38 1.08 7012 SEQ ID NO: 2 3.82 3.0 1.02 0.88 7013 SEQ
ID NO: 3 2.66 5.20 0.97 2.96 7014 SEQ ID NO: 4 3.48 3.82 0.71 0.90
Recormon 1/W 3.23 3.27 0.73 0.59 Recormon 3/w 4.13 4.24 1.21
1.14
Conclusion
[0314] The in vivo experiment was designed to measure two
parameters; the first was to measure erythropoiesis parameters such
as percentage of reticulocytes and increase of hemoglobin, RBC and
hematocrit levels. The second was to measure the durability of the
biological activity of each variant by injecting once weekly
doses.
[0315] A superior performance of EPO-3 in its ability to stimulate
erythropoiesis was observed in normal mice.
Example 5
Comparison of the EPO-CTP Polypeptides of the Present Invention to
Aranesp
[0316] The following experiment was performed in order to compare
the biological activity of a single bolus dose of some EPO-CTP
polypeptides of the present invention, commercial EPO and Aranesp.
Aranesp is a commercial long-acting recombinant erythropoietin in
which two site mutations were introduced, resulting in two
additional N-glycosylation sites and an increase in the number of
incorporated sialic acid residues.
Materials and Methods
Animals:
Species/Strain: Female CD-1 Mice of either sex about 20-25 g
Group Size: n=3
[0317] Experimental design of the study: The experiment was set up
as summarized in Table 7 hereinbelow. TABLE-US-00007 TABLE 7 Dose
animals/ Solution Dose group/ Conc. Volume Time-Points * Group #
Test Article time-point (.mu.g/mL) (mL/kg) (hours
post-administration) 1 MOD-7010 3 1.5 10 0 (Pre-dose), 0.25, 0.5,
1, 2, 6, SEQ ID NO: 11 24, 48, 96, 168, 216, 264 and 336 hr
post-dose administration 2 MOD-7013 3 1.5 10 0.25, 0.5, 1, 2, 6,
24, 48, 96, SEQ ID NO: 3 168, 216, 264 and 336 hr post- dose
administration 3 Aranesp 3 1.5 10 0.25, 0.5, 1, 2, 6, 24, 48, 96,
168, 216, 264 and 336 hr post- dose administration
[0318] Animal treatment: All animals were administered with either
control or the test EPO polypeptides of the present invention by
bolus injection. The injection volume did not exceed 10 ml/kg. The
length of the experiment was 14 days. A morbidity and mortality
check was performed daily.
[0319] Reticulocyte count and hematocrit (hct) examination:
Reticulocyte count and hematocrit examination were performed as
described above.
Results
[0320] The results are illustrated in FIGS. 6-9. Following a single
I.V. injection of 15 .mu.g/kg of EPO 3, all three blood parameters
associated with erythropoietin i.e. number of reticulocytes,
hemoglobin level and hematocrit, were improved relative to those
obtained with similar injected dose of rhEPO or Aranesp.
Example 6
Comparison of the Pharmacokinetics of EPO-CTP Polypeptides of the
Present Invention to Aranesp
[0321] The following experiment was performed in order to compare
the pharmacokinetics of EPO-CTP polypeptide of the present
invention, commercial EPO and Aranesp.
Materials and Methods
[0322] Serum samples were analyzed in order to determine specific
concentration levels for each sample. Concentration and time-point
data were processed using WinNonLin noncompartmental analysis.
Parameters determined included: AUC, CL, Ke, t1/2, Cmax, Tmax, and
Vdz.
[0323] Serum concentrations were determined using two ELISA kits in
parallel. EPO-3 serum concentration was measured using StemCell
ELISA kit in comparison to EPO-0 and Aranesp serum concentration
which were determined using R&D system ELISA kit.
Results
[0324] The results of the pharmacokinetic analysis are summarized
in Table 8, hereinbelow. These results show that EPO 3 exhibited
favorable pharmacokinetic measures as indicated for example in AUC
measures, t1/2, and Cmax. Tmax measures were equal to EPO-0, EPO-3,
and Aranesp. TABLE-US-00008 TABLE 8 Parameters Units EPO-0 EPO-3
Aranesp AUClast hr * mIU/mL 31739 306072 178661 CL{circumflex over
( )} mL/hr/kg 1.1152 0.2188 0.1207 Ke 1/hr 0.157 0.0529 0.0639 t1/2
hr 4.4139 13.1141 10.84 Cmax mIU/mL 10766 16466 13266 Tmax Hr 0.25
0.25 0.25 Vdz mL/kg 7.1017 4.1394 1.8877
[0325] The results of the serum concentration analysis are
illustrated in FIG. 9. These results show that EPO-3 was still
detectable in the serum after about 190 hours. Both EPO-0 and
Aranesp were not detectable in the serum after about 140 hours and
50 hours, respectively.
Conclusion
[0326] Clearance of EPO-3 (MOD-7013) from the blood of CD-1 mice
was significantly slower than that for rhEPO or Aranesp. The
corresponding calculated half life times were: rhEPO-4.41 h;
Aranesp-0.84 h; and MOD-7013-13.11 h.
Example 7
Generation of hGH Constructs
Materials and Methods
[0327] Four hGH clones (variants of 20 kD protein) were
synthesized. Xba I-Not I fragments containing hGH sequences from
the four variants were ligated into the eukaryotic expression
vector pCI-dhfr previously digested with XbaI-NotI. DNA from the 4
clones (401-0, 1, 2, 3 and 4) was prepared. Another partial hGH
clone (1-242 bp) from 22 kD protein was also synthesized (0606114).
Primers were ordered from Sigma-Genosys. The primer sequences used
to generate the hGH-CTP polypeptides of the present invention are
summarized in Table 9, hereinbelow. TABLE-US-00009 TABLE 9
Restriction SEQ site Primer ID (underlined number NO sequence in
sequence) 25 27 5' CTCTAGAGGACATGGCCAC 3' Xbal 32R 28 5'
ACAGGGAGGTCTGGGGGTTCTGC A 3' 33 29 5' TGCAGAACCCCCAGACCTCCCTG TGC
3' 4R 30 5' CCAAACTCATCAATGTATCTT A 3' 25 31 5' CTCTAGAGGACATGGCCAC
3' XbaI 35R 32 5' CGAACTCCTGGTAGGTGTCAAAGG C 3' 34 33 5'
GCCTTTGACACCTACCAGGAGTTC G 3' 37R 34 5' ACGCGGCCGCATCCAGACCTTCAT
NotI CACTGAGGC 3' 39R 35 5' GCGGCCGCGGACTCATCAGAAGCC GCAGCTGCCC
3'
[0328] Construction of 402-0-p69-1 (hGH) SEQ ID NO: 36: MOD-4020 is
the wild type recombinant human growth hormone (without CTP) which
was prepared for use as control in the below described
experiments.
[0329] Three PCR reactions were performed. The first reaction was
conducted with primer 25 and primer 32.sup.R and plasmid DNA of
0606114 (partial clone of hGH 1-242 bp) as a template; as a result
of the PCR amplification, a 245 bp product was formed.
[0330] The second reaction was conducted with primer 33 and primer
4.sup.R and plasmid DNA of 401-0-p57-2 as a template; as a result
of the PCR amplification, a 542 bp product was formed.
[0331] The last reaction was conducted with primers 25 and 4.sup.R
and a mixture of the products of the previous two reactions as a
template; as a result of the PCR amplification, a 705 bp product
was formed and ligated into the TA cloning vector (Invitrogen,
catalog K2000-01). The XbaI-NotI fragment containing hGH-0 sequence
was ligated into the eukaryotic expression vector pCI-dhfr. The
vector was transfected into DG-44 CHO cells. Cells were grown in
protein-free medium.
[0332] Construction of 402-1-p83-5 (hGH-CTP)--SEQ ID NO: 37 and
402-2-p72-3 (hGH-CTPx2)--SEQ ID NO: 38: MOD-4021 is a recombinant
human growth hormone which was fused to 1 copy of the C-terminal
peptide of the beta chain of human Chorionic Gonadotropin (CTP).
The CTP cassette of MOD-4021 was attached to the C-terminus (one
cassette). MOD-4022 is a recombinant human growth hormone which was
fused to 2 copies of the C-terminal peptide of the beta chain of
human Chorionic Gonadotropin (CTP). The two CTP cassettes of
MOD-4022 were attached to the C-terminus (two cassettes).
[0333] Construction of hGH-CTP and hGH-CTP-CTP was performed in the
same way as the construction of hGH-0. pCI-dhfr-401-1-p20-1
(hGH*-ctp) and pCI-dhfr-401-2-p21-2 (hGH*-ctp x2) were used as
templates in the second PCR reaction.
[0334] MOD-4021 and MOD-4022 were expressed in DG-44 CHO cells.
Cells were grown in protein-free medium. The molecular weight of
MOD-4021 is .about.30.5 Kd since hGH has a MW of 22 Kd while each
"CTP cassette" contributes 8.5 Kd to the overall molecular weight
(see FIG. 10). The molecular weight of MOD-4022 is .about.39 Kd
(see FIG. 10).
[0335] Construction of 402-3-p81-4 (CTP-hGH-CTP-CTP)--SEQ ID NO: 39
and 402-4-p82-9 (CTP*hGH-CTP-CTP)--SEQ ID NO: 40: MOD-4023 is a
recombinant human growth hormone which was fused to 3 copies of the
C-terminal peptide of the beta chain of human Chorionic
Gonadotropin (CTP). The three CTP cassettes of MOD-4023 were
attached to both N-terminus (one cassette) and the C-terminus (two
cassettes). MOD-4024 is a recombinant human growth hormone which is
fused to 1 truncated and 2 complete copies of the C-terminal
peptide of the beta chain of human Chorionic Gonadotropin (CTP).
The truncated CTP cassette of MOD-4024 was attached to the
N-terminus and two CTP cassettes were attached to the C-terminus
(two cassettes).
[0336] Three PCR reactions were performed. The first reaction was
conducted with primer 25 and primer 35.sup.R and plasmid DNA of
p401-3-p12-5 or 401-4-p22-1 as a template; as a result of the PCR
amplification, a 265 or 220 bp product was formed. The second
reaction was conducted with primer 34 and primer 37.sup.R and
plasmid DNA of TA-hGH-2-q65-1 as a template; as a result of the PCR
amplification, a 695 bp product was formed. The last reaction was
conducted with primers 25 and 37.sup.R and a mixture of the
products of the previous two reactions as a template; as a result
of the PCR amplification, a 938 or 891 bp product was formed and
ligated into TA cloning vector (Invitrogen, catalog K2000-01). Xba
I-Not I fragment containing hGH sequence was ligated into our
eukaryotic expression vector pCI-dhfr.
[0337] MOD-4023 and MOD-4024 were expressed in DG-44 CHO cells.
Cells were grown in protein-free medium. The molecular weight of
MOD-4023 is .about.47.5 Kd (see FIG. 10) and the molecular weight
of MOD-4024 is .about.43.25 Kd (see FIG. 10).
[0338] Construction of 402-6-p95a-8 (CTP-hGH-CTP)--SEQ ID NO: 41:
Construction of hGH-6 was performed in the same way as the
construction of hGH-3. pCI-dhfr-402-1-p83-5 (hGH-ctp) was used as a
template in the second PCR reaction.
[0339] Construction of 402-5-p96-4 (CTP-hGH)--SEQ ID NO: 42: PCR
reaction was performed using primer 25 and primer 39.sup.R and
plasmid DNA of pCI-dhfr-ctp-EPO-ctp (402-6-p95a-8) as a template;
as a result of the PCR amplification, a 763 bp product was formed
and ligated into TA cloning vector (Invitrogen, catalog K2000-01).
Xba I-Not I fragment containing ctp-hGH sequence was ligated into
our eukaryotic expression vector pCI-dhfr to yield 402-5-p96-4
clone.
Example 8
In Vivo Bioactivity Tests of hGH-CTP Polypeptides of the Present
Invention
[0340] The following experiment was performed in order to test the
potential long acting biological activity of hGH-CTP polypeptides
in comparison with commercial recombinant human GH and
MOD-4020.
Materials and Methods
[0341] Female hypophysectomized rats (60-100 g) received a weekly
S.C. injection of 21.7 .mu.g hGH-CTP polypeptides or a once daily 5
.mu.g S.C. injection of control commercial rhGH.
[0342] Weight was measured in all animals before treatment, 24
hours following first injection and then every other day until the
end of the study on day 21. Each point represents the group's
average weight gain percentage ((Weight day 0-weight last
day)/Weight day 0). Average weight gain was normalized against
once-daily injection of commercial hGH. The treatment schedule is
summarized in Table 10. TABLE-US-00010 TABLE 10 Equimolar
Accumulate Treatment Dose Dosage Dose No. Drug N Route Schedule
(.mu.g/rat) (.mu.g/rat) Vol.(ml) 1 Vehicle 7 s.c. days 1, 7 NA NA
0.25 and 13; 1/W 2 Mock 7 s.c days 1, 7 NA NA 0.25 and 13; 1/W 3
MOD-4020 7 s.c days 1, 7 21.7 65 0.25 SEQ ID NO: 36 and 13; 1/W 4
MOD-4021 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 37 and 13; 1/W 5
MOD-4022 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 38 and 13; 1/W 6
MOD-4023 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 39 and 13; 1/W 7
MOD-4024 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID NO: 40 and 13; 1/W 8
Commercial 7 s.c. days 1, 7 21.7 65 0.25 hGH v.1 and 13; 1/W 9
Commercial 7 s.c. days 1-13; 5 65 0.25 hGH v.1 d/W
Results
[0343] Results are summarized in FIG. 11. These results show that
MOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced over
120% weight gain compared to commercial rhGH which induced 100%
weight gain.
Conclusion
[0344] 3 weekly doses (Days of injections; 1, 7, and 13) of 21.7
.mu.g of MOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40)
induced a 30% greater weight increase in hypophysectomised rats
compared to commercial rhGH injected at the same accumulated dose
which was administered once per day at a dose of 5 .mu.g for 13
days.
Sequence CWU 1
1
47 1 221 PRT Homo sapiens 1 Met Gly Val His Glu Cys Pro Ala Trp Leu
Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro
Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val
Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile
Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile
Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80
Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85
90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser
Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala
Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu
Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser
Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg
Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu
Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg Ser
Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 195 200 205
Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 210 215 220 2
249 PRT Homo sapiens 2 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp
Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val
Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu
Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr
Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr
Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met
Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90
95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser
100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val
Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly
Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala
Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys
Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys
Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg Ser Ser
Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 195 200 205 Arg
Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser 210 215
220 Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly
225 230 235 240 Pro Ser Asp Thr Pro Ile Leu Pro Gln 245 3 277 PRT
Homo sapiens 3 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu
Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly
Ser Ser Ser Ser Lys 20 25 30 Ala Pro Pro Pro Ser Leu Pro Ser Pro
Ser Arg Leu Pro Gly Pro Ser 35 40 45 Asp Thr Pro Ile Leu Pro Gln
Ala Pro Pro Arg Leu Ile Cys Asp Ser 50 55 60 Arg Val Leu Glu Arg
Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile 65 70 75 80 Thr Thr Gly
Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val 85 90 95 Pro
Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly 100 105
110 Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala
115 120 125 Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro
Trp Glu 130 135 140 Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly
Leu Arg Ser Leu 145 150 155 160 Thr Thr Leu Leu Arg Ala Leu Gly Ala
Gln Lys Glu Ala Ile Ser Pro 165 170 175 Pro Asp Ala Ala Ser Ala Ala
Pro Leu Arg Thr Ile Thr Ala Asp Thr 180 185 190 Phe Arg Lys Leu Phe
Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu 195 200 205 Lys Leu Tyr
Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg Ser Ser Ser 210 215 220 Ser
Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly 225 230
235 240 Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala
Pro 245 250 255 Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro
Ser Asp Thr 260 265 270 Pro Ile Leu Pro Gln 275 4 387 PRT Homo
sapiens 4 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu
Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala
Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr
Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys
Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp
Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly
Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser
Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110
Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115
120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys
Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu
Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg
Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr
Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg Ser Ser Ser Ser Lys
Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 195 200 205 Arg Leu Pro Gly
Pro Ser Asp Thr Pro Ile Leu Pro Gln Ala Pro Pro 210 215 220 Arg Leu
Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala 225 230 235
240 Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu
245 250 255 Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr
Ala Trp 260 265 270 Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val
Trp Gln Gly Leu 275 280 285 Ala Leu Leu Ser Glu Ala Val Leu Arg Gly
Gln Ala Leu Leu Val Asn 290 295 300 Ser Ser Gln Pro Trp Glu Pro Leu
Gln Leu His Val Asp Lys Ala Val 305 310 315 320 Ser Gly Leu Arg Ser
Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln 325 330 335 Lys Glu Ala
Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg 340 345 350 Thr
Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn 355 360
365 Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr
370 375 380 Gly Asp Arg 385 5 221 PRT Homo sapiens 5 Met Gly Val
His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu
Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ser Ser Ser Ser Lys 20 25
30 Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser
35 40 45 Asp Thr Pro Ile Leu Pro Gln Ala Pro Pro Arg Leu Ile Cys
Asp Ser 50 55 60 Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu
Ala Glu Asn Ile 65 70 75 80 Thr Thr Gly Cys Ala Glu His Cys Ser Leu
Asn Glu Asn Ile Thr Val 85 90 95 Pro Asp Thr Lys Val Asn Phe Tyr
Ala Trp Lys Arg Met Glu Val Gly 100 105 110 Gln Gln Ala Val Glu Val
Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala 115 120 125 Val Leu Arg Gly
Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu 130 135 140 Pro Leu
Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu 145 150 155
160 Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro
165 170 175 Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala
Asp Thr 180 185 190 Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu
Arg Gly Lys Leu 195 200 205 Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr
Gly Asp Arg 210 215 220 6 249 PRT Homo sapiens 6 Met Gly Val His
Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser
Leu Pro Leu Gly Leu Pro Val Leu Gly Ser Ser Ser Ser Lys 20 25 30
Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser 35
40 45 Asp Thr Pro Ile Leu Pro Gln Ala Pro Pro Arg Leu Ile Cys Asp
Ser 50 55 60 Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala
Glu Asn Ile 65 70 75 80 Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn
Glu Asn Ile Thr Val 85 90 95 Pro Asp Thr Lys Val Asn Phe Tyr Ala
Trp Lys Arg Met Glu Val Gly 100 105 110 Gln Gln Ala Val Glu Val Trp
Gln Gly Leu Ala Leu Leu Ser Glu Ala 115 120 125 Val Leu Arg Gly Gln
Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu 130 135 140 Pro Leu Gln
Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu 145 150 155 160
Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro 165
170 175 Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp
Thr 180 185 190 Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg
Gly Lys Leu 195 200 205 Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly
Asp Arg Ser Ser Ser 210 215 220 Ser Lys Ala Pro Pro Pro Ser Leu Pro
Ser Pro Ser Arg Leu Pro Gly 225 230 235 240 Pro Ser Asp Thr Pro Ile
Leu Pro Gln 245 7 25 DNA Artificial Artificial short sequence 7
aatctagagg tcatcatggg ggtgc 25 8 32 DNA Artificial Artificial short
sequence 8 attgcggccg cggatccaga agacctttat tg 32 9 25 DNA
Artificial Artificial short sequence 9 taaatattgg ggtgtccgag ggccc
25 10 32 DNA Artificial Artificial short sequence 10 ccaatattac
cacaagcccc accacgcctc at 32 11 35 DNA Artificial Artificial short
sequence 11 tgcggccgcg gatccttatc tgtcccctgt cctgc 35 12 17 DNA
Artificial Artificial short sequence 12 gccctgctgt cggaagc 17 13 32
DNA Artificial Artificial short sequence 13 attgcggccg cggatccaga
agacctttat tg 32 14 32 DNA Artificial Artificial short sequence 14
ctttgaggaa gaggagccca ggactgggag gc 32 15 24 DNA Artificial Was it
fun? Wierd? Tell me about it 15 cctgggctcc tcttcctcaa aggc 24 16
193 PRT Homo sapiens 16 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp
Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val
Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu
Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr
Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr
Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met
Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90
95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser
100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val
Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly
Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala
Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys
Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys
Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 17 34
PRT Artificial Artificial short sequence 17 Asp Pro Arg Phe Gln Asp
Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser 1 5 10 15 Leu Pro Ser Pro
Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu 20 25 30 Pro Gln 18
28 PRT Artificial Artificial short sequence 18 Ser Ser Ser Ser Lys
Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 1 5 10 15 Leu Pro Gly
Pro Ser Asp Thr Pro Ile Leu Pro Gln 20 25 19 27 PRT Homo sapiens 19
Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5
10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly 20 25 20 786 DNA
Homo sapiens 20 tctagaggtc atcatggggg tgcacgaatg tcctgcctgg
ctgtggcttc tcctgtccct 60 tctgtcgctc cctctgggcc tcccagtcct
gggctcctct tcctcaaagg cccctccccc 120 gagccttcca agtccatccc
gactcccggg gccctcggac accccaatat taccacaagc 180 cccaccacgc
ctcatctgtg acagccgagt cctggagagg tacctcttgg aggccaagga 240
ggccgagaat atcacgacgg gctgtgctga acactgcagc ttgaatgaga atatcactgt
300 cccagacacc aaagttaatt tctatgcctg gaagaggatg gaggtcgggc
agcaggccgt 360 agaagtctgg cagggcctgg ccctgctgtc ggaagctgtc
ctgcggggcc aggccctgtt 420 ggtcaactct tcccagccgt gggagcccct
gcagctgcat gtggataaag ccgtcagtgg 480 ccttcgcagc ctcaccactc
tgcttcgggc tctgggagcc cagaaggaag ccatctcccc 540 tccagatgcg
gcctcagctg ctccactccg aacaatcact gctgacactt tccgcaaact 600
cttccgagtc tactccaatt tcctccgggg aaagctgaag ctgtacacag gggaggcctg
660 caggacaggg gacagatcct cttcctcaaa ggcccctccc ccgagccttc
caagtccatc 720 ccgactcccg gggccctcgg acaccccgat cctcccacaa
taaaggtctt ctggatccgc 780 ggccgc 786 21 873 DNA Homo sapiens 21
tctagaggtc atcatggggg tgcacgaatg tcctgcctgg ctgtggcttc tcctgtccct
60 tctgtcgctc cctctgggcc tcccagtcct gggctcctct tcctcaaagg
cccctccccc 120 gagccttcca agtccatccc gactcccggg gccctcggac
accccaatat taccacaagc 180 cccaccacgc ctcatctgtg acagccgagt
cctggagagg tacctcttgg aggccaagga 240 ggccgagaat atcacgacgg
gctgtgctga acactgcagc ttgaatgaga atatcactgt 300 cccagacacc
aaagttaatt tctatgcctg gaagaggatg gaggtcgggc agcaggccgt 360
agaagtctgg cagggcctgg ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt
420 ggtcaactct tcccagccgt gggagcccct gcagctgcat gtggataaag
ccgtcagtgg 480 ccttcgcagc ctcaccactc tgcttcgggc tctgggagcc
cagaaggaag ccatctcccc 540 tccagatgcg gcctcagctg ctccactccg
aacaatcact gctgacactt tccgcaaact 600 cttccgagtc tactccaatt
tcctccgggg aaagctgaag ctgtacacag gggaggcctg 660 caggacaggg
gacagatcct cttcctcaaa ggcccctccc ccgagccttc caagtccatc 720
ccgactcccg gggccctccg acacaccaat cctgccacag agcagctcct ctaaggcccc
780 tcctccatcc ctgccatccc cctcccggct gcctggcccc tctgacaccc
ctatcctgcc 840 tcagtgatga aggtcttctg gatccgcggc cgc 873 22 221 PRT
Homo sapiens 22 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu
Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly
Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg
Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly
Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro
Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val
Gly Gln Gln Ala
Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val
Leu Arg Ser Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp
Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu
Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135
140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile
145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser
Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala
Cys Arg Thr Gly Asp 180 185 190 Arg Ser Ser Ser Ser Lys Ala Pro Pro
Pro Ser Leu Pro Ser Pro Ser 195 200 205 Arg Leu Pro Gly Pro Ser Asp
Thr Pro Ile Leu Pro Gln 210 215 220 23 217 PRT Homo sapiens 23 Met
Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10
15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu
20 25 30 Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu
His Gln 35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala
Tyr Ile Pro Lys 50 55 60 Val Gln Lys Tyr Ser Phe Leu Gln Asn Pro
Gln Thr Ser Leu Cys Phe 65 70 75 80 Ser Glu Ser Ile Pro Thr Pro Ser
Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95 Ser Asn Leu Glu Leu Leu
Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110 Leu Glu Pro Val
Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120 125 Tyr Gly
Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu 130 135 140
Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg 145
150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr
Asn Ser 165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu
Leu Tyr Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr Phe
Leu Arg Ile Val Gln Cys 195 200 205 Arg Ser Val Glu Gly Ser Cys Gly
Phe 210 215 24 166 PRT Homo sapiens 24 Met Ser Tyr Asn Leu Leu Gly
Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Ser Gln Lys Leu Leu
Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg
Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln
Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55
60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn
65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln
Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys
Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser Leu His Leu
Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu Lys Ala Lys
Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg Val Glu Ile
Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160 Thr Gly Tyr
Leu Arg Asn 165 25 30 PRT Homo sapiens 25 His Ala Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 26 21 PRT Homo
sapiens 26 Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys
Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser 20 27 19 DNA Artificial
Artificial short sequence 27 ctctagagga catggccac 19 28 24 DNA
Artificial Artificial short sequence 28 acagggaggt ctgggggttc tgca
24 29 26 DNA Artificial Artificial short sequence 29 tgcagaaccc
ccagacctcc ctgtgc 26 30 22 DNA Artificial Artificial short sequence
30 ccaaactcat caatgtatct ta 22 31 19 DNA Artificial Artificial
short sequence 31 ctctagagga catggccac 19 32 25 DNA Artificial
Artificial short sequence 32 cgaactcctg gtaggtgtca aaggc 25 33 25
DNA Artificial Artificial short sequence 33 gcctttgaca cctaccagga
gttcg 25 34 33 DNA Artificial Artificial short sequence 34
acgcggccgc atccagacct tcatcactga ggc 33 35 34 DNA Artificial
Artificial short sequence 35 gcggccgcgg actcatcaga agccgcagct gccc
34 36 217 PRT Homo sapiens 36 Met Ala Thr Gly Ser Arg Thr Ser Leu
Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu
Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25 30 Ser Arg Leu Phe Asp
Asn Ala Met Leu Arg Ala His Arg Leu His Gln 35 40 45 Leu Ala Phe
Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60 Glu
Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe 65 70
75 80 Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln
Lys 85 90 95 Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile
Gln Ser Trp 100 105 110 Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe
Ala Asn Ser Leu Val 115 120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr
Asp Leu Leu Lys Asp Leu Glu 130 135 140 Glu Gly Ile Gln Thr Leu Met
Gly Arg Leu Glu Asp Gly Ser Pro Arg 145 150 155 160 Thr Gly Gln Ile
Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser 165 170 175 His Asn
Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190
Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195
200 205 Arg Ser Val Glu Gly Ser Cys Gly Phe 210 215 37 245 PRT Homo
sapiens 37 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly
Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro
Thr Ile Pro Leu 20 25 30 Ser Arg Leu Phe Asp Asn Ala Met Leu Arg
Ala His Arg Leu His Gln 35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu
Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60 Glu Gln Lys Tyr Ser Phe
Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe 65 70 75 80 Ser Glu Ser Ile
Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95 Ser Asn
Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110
Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115
120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu
Glu 130 135 140 Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly
Ser Pro Arg 145 150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser
Lys Phe Asp Thr Asn Ser 165 170 175 His Asn Asp Asp Ala Leu Leu Lys
Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys
Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200 205 Arg Ser Val Glu
Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro 210 215 220 Pro Pro
Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr 225 230 235
240 Pro Ile Leu Pro Gln 245 38 273 PRT Homo sapiens 38 Met Ala Thr
Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys
Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25
30 Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln
35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile
Pro Lys 50 55 60 Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr
Ser Leu Cys Phe 65 70 75 80 Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg
Glu Glu Thr Gln Gln Lys 85 90 95 Ser Asn Leu Glu Leu Leu Arg Ile
Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110 Leu Glu Pro Val Gln Phe
Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120 125 Tyr Gly Ala Ser
Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu 130 135 140 Glu Gly
Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg 145 150 155
160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser
165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr
Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg
Ile Val Gln Cys 195 200 205 Arg Ser Val Glu Gly Ser Cys Gly Phe Ser
Ser Ser Ser Lys Ala Pro 210 215 220 Pro Pro Ser Leu Pro Ser Pro Ser
Arg Leu Pro Gly Pro Ser Asp Thr 225 230 235 240 Pro Ile Leu Pro Gln
Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 245 250 255 Pro Ser Pro
Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 260 265 270 Gln
39 301 PRT Homo sapiens 39 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu
Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly
Ser Ala Ser Ser Ser Ser Lys Ala 20 25 30 Pro Pro Pro Ser Leu Pro
Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp 35 40 45 Thr Pro Ile Leu
Pro Gln Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe 50 55 60 Asp Asn
Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp 65 70 75 80
Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr 85
90 95 Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser
Ile 100 105 110 Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser
Asn Leu Glu 115 120 125 Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser
Trp Leu Glu Pro Val 130 135 140 Gln Phe Leu Arg Ser Val Phe Ala Asn
Ser Leu Val Tyr Gly Ala Ser 145 150 155 160 Asp Ser Asn Val Tyr Asp
Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln 165 170 175 Thr Leu Met Gly
Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile 180 185 190 Phe Lys
Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp 195 200 205
Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met 210
215 220 Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val
Glu 225 230 235 240 Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys Ala Pro
Pro Pro Ser Leu 245 250 255 Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser
Asp Thr Pro Ile Leu Pro 260 265 270 Gln Ser Ser Ser Ser Lys Ala Pro
Pro Pro Ser Leu Pro Ser Pro Ser 275 280 285 Arg Leu Pro Gly Pro Ser
Asp Thr Pro Ile Leu Pro Gln 290 295 300 40 285 PRT Homo sapiens 40
Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5
10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys
Ala 20 25 30 Pro Pro Pro Ser Leu Pro Phe Pro Thr Ile Pro Leu Ser
Arg Leu Phe 35 40 45 Asp Asn Ala Met Leu Arg Ala His Arg Leu His
Gln Leu Ala Phe Asp 50 55 60 Thr Tyr Gln Glu Phe Glu Glu Ala Tyr
Ile Pro Lys Glu Gln Lys Tyr 65 70 75 80 Ser Phe Leu Gln Asn Pro Gln
Thr Ser Leu Cys Phe Ser Glu Ser Ile 85 90 95 Pro Thr Pro Ser Asn
Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu 100 105 110 Leu Leu Arg
Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val 115 120 125 Gln
Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser 130 135
140 Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln
145 150 155 160 Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr
Gly Gln Ile 165 170 175 Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn
Ser His Asn Asp Asp 180 185 190 Ala Leu Leu Lys Asn Tyr Gly Leu Leu
Tyr Cys Phe Arg Lys Asp Met 195 200 205 Asp Lys Val Glu Thr Phe Leu
Arg Ile Val Gln Cys Arg Ser Val Glu 210 215 220 Gly Ser Cys Gly Phe
Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 225 230 235 240 Pro Ser
Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 245 250 255
Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 260
265 270 Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 275 280
285 41 273 PRT Homo sapiens 41 Met Ala Thr Gly Ser Arg Thr Ser Leu
Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu
Gly Ser Ala Ser Ser Ser Ser Lys Ala 20 25 30 Pro Pro Pro Ser Leu
Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp 35 40 45 Thr Pro Ile
Leu Pro Gln Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe 50 55 60 Asp
Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp 65 70
75 80 Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys
Tyr 85 90 95 Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser
Glu Ser Ile 100 105 110 Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln
Lys Ser Asn Leu Glu 115 120 125 Leu Leu Arg Ile Ser Leu Leu Leu Ile
Gln Ser Trp Leu Glu Pro Val 130 135 140 Gln Phe Leu Arg Ser Val Phe
Ala Asn Ser Leu Val Tyr Gly Ala Ser 145 150 155 160 Asp Ser Asn Val
Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln 165 170 175 Thr Leu
Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile 180 185 190
Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp 195
200 205 Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp
Met 210 215 220 Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg
Ser Val Glu 225 230 235 240 Gly Ser Cys Gly Phe Ser Ser Ser Ser Lys
Ala Pro Pro Pro Ser Leu 245 250 255 Pro Ser Pro Ser Arg Leu Pro Gly
Pro Ser Asp Thr Pro Ile Leu Pro 260 265 270 Gln 42 245 PRT Homo
sapiens 42 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly
Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser
Ser Ser Lys Ala 20 25 30 Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg
Leu Pro Gly Pro Ser Asp 35 40 45 Thr Pro Ile Leu Pro Gln Phe Pro
Thr Ile Pro Leu Ser Arg Leu Phe 50 55 60 Asp Asn Ala Met Leu Arg
Ala His Arg Leu His Gln Leu Ala Phe Asp 65 70 75 80 Thr Tyr Gln Glu
Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr
85 90 95 Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu
Ser Ile 100 105 110 Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys
Ser Asn Leu Glu 115 120 125 Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln
Ser Trp Leu Glu Pro Val 130 135 140 Gln Phe Leu Arg Ser Val Phe Ala
Asn Ser Leu Val Tyr Gly Ala Ser 145 150 155 160 Asp Ser Asn Val Tyr
Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln 165 170 175 Thr Leu Met
Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile 180 185 190 Phe
Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp 195 200
205 Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met
210 215 220 Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser
Val Glu 225 230 235 240 Gly Ser Cys Gly Phe 245 43 12 PRT
Artificial Artificial short protein 43 Ser Ser Ser Ser Lys Ala Pro
Pro Pro Ser Leu Pro 1 5 10 44 853 DNA Homo sapiens 44 tctagaggac
atggccaccg gcagcaggac cagcctgctg ctggccttcg gcctgctgtg 60
cctgccatgg ctgcaggagg gcagcgccag ctcttcttct aaggctccac ccccatctct
120 gcccagcccc agcagactgc cgggccccag cgacacaccc attctgcccc
agttccccac 180 catccccctg agcaggctgt tcgacaacgc catgctgagg
gctcacaggc tgcaccagct 240 ggcctttgac acctaccagg agttcgagga
agcctacatc cccaaggagc agaagtacag 300 cttcctgcag aacccccaga
cctccctgtg cttcagcgag agcatcccca cccccagcaa 360 cagagaggag
acccagcaga agagcaacct ggagctgctg aggatctccc tgctgctgat 420
ccagagctgg ctggagcccg tgcagttcct gagaagcgtg ttcgccaaca gcctggtgta
480 cggcgccagc gacagcaacg tgtacgacct gctgaaggac ctggaggagg
gcatccagac 540 cctgatgggc cggctggagg acggcagccc caggaccggc
cagatcttca agcagaccta 600 cagcaagttc gacaccaaca gccacaacga
cgacgccctg ctgaagaact acgggctgct 660 gtactgcttc agaaaggaca
tggacaaggt ggagaccttc ctgaggatcg tgcagtgcag 720 aagcgtggag
ggcagctgcg gcttcagctc cagcagcaag gcccctcccc cgagcctgcc 780
ctccccaagc aggctgcctg ggccctccga cacaccaatc ctgcctcagt gatgaaggtc
840 tggatgcggc cgc 853 45 937 DNA Homo sapiens 45 tctagaggac
atggccaccg gcagcaggac cagcctgctg ctggccttcg gcctgctgtg 60
cctgccatgg ctgcaggagg gcagcgccag ctcttcttct aaggctccac ccccatctct
120 gcccagcccc agcagactgc cgggccccag cgacacaccc attctgcccc
agttccccac 180 catccccctg agcaggctgt tcgacaacgc catgctgagg
gctcacaggc tgcaccagct 240 ggcctttgac acctaccagg agttcgagga
agcctacatc cccaaggagc agaagtacag 300 cttcctgcag aacccccaga
cctccctgtg cttcagcgag agcatcccca cccccagcaa 360 cagagaggag
acccagcaga agagcaacct ggagctgctg aggatctccc tgctgctgat 420
ccagagctgg ctggagcccg tgcagttcct gagaagcgtg ttcgccaaca gcctggtgta
480 cggcgccagc gacagcaacg tgtacgacct gctgaaggac ctggaggagg
gcatccagac 540 cctgatgggc cggctggagg acggcagccc caggaccggc
cagatcttca agcagaccta 600 cagcaagttc gacaccaaca gccacaacga
cgacgccctg ctgaagaact acgggctgct 660 gtactgcttc agaaaggaca
tggacaaggt ggagaccttc ctgaggatcg tgcagtgcag 720 aagcgtggag
ggcagctgcg gcttcagctc cagcagcaag gcccctcccc cgagcctgcc 780
ctccccaagc aggctgcctg ggccctccga cacaccaatc ctgccacaga gcagctcctc
840 taaggcccct cctccatccc tgccatcccc ctcccggctg cctggcccct
ctgacacccc 900 tatcctgcct cagtgatgaa ggtctggatg cggccgc 937 46 889
DNA Homo sapiens 46 tctagaggac atggccaccg gcagcaggac cagcctgctg
ctggccttcg gcctgctgtg 60 cctgccatgg ctgcaggagg gcagcgccag
ctcttcttct aaggctccac ccccgagcct 120 gcccttcccc accatccccc
tgagcaggct gttcgacaac gccatgctga gggctcacag 180 gctgcaccag
ctggcctttg acacctacca ggagttcgag gaagcctaca tccccaagga 240
gcagaagtac agcttcctgc agaaccccca gacctccctg tgcttcagcg agagcatccc
300 cacccccagc aacagagagg agacccagca gaagagcaac ctggagctgc
tgaggatctc 360 cctgctgctg atccagagct ggctggagcc cgtgcagttc
ctgagaagcg tgttcgccaa 420 cagcctggtg tacggcgcca gcgacagcaa
cgtgtacgac ctgctgaagg acctggagga 480 gggcatccag accctgatgg
gccggctgga ggacggcagc cccaggaccg gccagatctt 540 caagcagacc
tacagcaagt tcgacaccaa cagccacaac gacgacgccc tgctgaagaa 600
ctacgggctg ctgtactgct tcagaaagga catggacaag gtggagacct tcctgaggat
660 cgtgcagtgc agaagcgtgg agggcagctg cggcttcagc tccagcagca
aggcccctcc 720 cccgagcctg ccctccccaa gcaggctgcc tgggccctcc
gacacaccaa tcctgccaca 780 gagcagctcc tctaaggccc ctcctccatc
cctgccatcc ccctcccggc tgcctggccc 840 ctctgacacc cctatcctgc
ctcagtgatg aaggtctgga tgcggccgc 889 47 217 PRT Homo sapiens 47 Met
Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10
15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu
20 25 30 Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu
His Gln 35 40 45 Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala
Tyr Ile Pro Lys 50 55 60 Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro
Gln Thr Ser Leu Cys Phe 65 70 75 80 Ser Glu Ser Ile Pro Thr Pro Ser
Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95 Ser Asn Leu Glu Leu Leu
Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110 Leu Glu Pro Val
Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120 125 Tyr Gly
Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu 130 135 140
Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg 145
150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr
Asn Ser 165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu
Leu Tyr Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr Phe
Leu Arg Ile Val Gln Cys 195 200 205 Arg Ser Val Glu Gly Ser Cys Gly
Phe 210 215
* * * * *