U.S. patent application number 17/164438 was filed with the patent office on 2021-12-02 for methods of producing long acting ctp-modified polypeptides.
This patent application is currently assigned to OPKO BIOLOGICS LTD.. The applicant listed for this patent is OPKO BIOLOGICS LTD.. Invention is credited to Oren HERSHKOVITZ, Laura MOSCHCOVICH.
Application Number | 20210371487 17/164438 |
Document ID | / |
Family ID | 1000005767924 |
Filed Date | 2021-12-02 |
United States Patent
Application |
20210371487 |
Kind Code |
A1 |
HERSHKOVITZ; Oren ; et
al. |
December 2, 2021 |
METHODS OF PRODUCING LONG ACTING CTP-MODIFIED POLYPEPTIDES
Abstract
Disclosed herein is a method for manufacturing a recombinant
polypeptide of interest modified by a CTP extension in a mammalian
cells culture system.
Inventors: |
HERSHKOVITZ; Oren; (M.P.
Shikmim, IL) ; MOSCHCOVICH; Laura; (Givat Shmuel,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OPKO BIOLOGICS LTD. |
Kiryat Gat |
|
IL |
|
|
Assignee: |
OPKO BIOLOGICS LTD.
Kiryat Gat
IL
|
Family ID: |
1000005767924 |
Appl. No.: |
17/164438 |
Filed: |
February 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14965079 |
Dec 10, 2015 |
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17164438 |
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62090124 |
Dec 10, 2014 |
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62090116 |
Dec 10, 2014 |
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62090104 |
Dec 10, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/31 20130101;
C07K 14/575 20130101; C07K 14/59 20130101; C12N 9/644 20130101;
C12Y 304/21022 20130101; C07K 14/505 20130101; C07K 14/555
20130101; C12Y 304/21021 20130101; C12N 9/6437 20130101; C07K
2319/00 20130101; C12N 15/85 20130101; C07K 14/61 20130101; A61K
38/00 20130101 |
International
Class: |
C07K 14/59 20060101
C07K014/59; C07K 14/61 20060101 C07K014/61; C07K 14/505 20060101
C07K014/505; C07K 14/555 20060101 C07K014/555; C07K 14/575 20060101
C07K014/575; C12N 9/64 20060101 C12N009/64; C12N 15/85 20060101
C12N015/85 |
Claims
1. A method of manufacturing a human CTP-modified polypeptide of
interest, the method comprising the steps of: (a) stably
transfecting a predetermined number of cells with an expression
vector comprising a coding portion encoding said CTP-modified
polypeptide of interest; (i) wherein said transfected cells express
and secrete said CTP-modified polypeptide of interest; (b)
obtaining cell clones that overexpress said CTP-modified
polypeptide of interest; (c) expanding said clones in solution to a
predetermined scale; (d) harvesting said solution containing said
clones; (e) filtering said solution containing said clones to
obtain a clarified harvest solution; and, (f) purifying said
clarified harvest solution to obtain a purified protein solution
having a desired concentration of a CTP-modified polypeptide of
interest; thereby manufacturing the CTP-modified polypeptide of
interest.
2. The method of claim 1, wherein the purity of the CTP-modified
polypeptide of interest is at least 70%.
3. The method of claim 1, wherein said CTP-modified polypeptide of
interest is highly glycosylated.
4. The method of claim 3, wherein the glycosylation pattern of each
CTP comprises at least glycosylation at 4 O-linked glycosylation
sites.
5. The method of claim 1, wherein said CTP-modified polypeptide of
interest is highly sialylated.
6. The method of claim 1, wherein when said polypeptide of interest
is an erythropoietin (EPO), an interferon (IFN), or a glucagon-like
peptide 1 (GLP-1), said CTP-modified polypeptide of interest
polypeptide consists of two CTPs attached to the carboxy terminus
of said polypeptide of interest, and one CTP attached to the amino
terminus of said polypeptide of interest.
7. The method of claim 1, wherein when said polypeptide of interest
is a coagulation factor, a coagulation Factor VII (FVII), an
activated coagulation Factor VII (FVIIa) or a coagulation Factor IX
(FIX), said CTP-modified polypeptide of interest polypeptide
consists of one to five CTPs attached to the carboxy terminus of
said polypeptide of interest.
8. The method of claim 7, wherein said CTP-modified polypeptide of
interest polypeptide consists of three CTPs attached to the carboxy
terminus of said polypeptide of interest.
9. The method of claim 1, wherein when said polypeptide of interest
is a dual GLP-1/Glucagon receptor agonist or OXM (OXM), said
CTP-modified polypeptide of interest polypeptide consists of one to
five chorionic CTPs attached to the carboxy terminus of said
polypeptide of interest and/or one to five CTPs attached to the
amino terminus of said polypeptide of interest.
10. The method of claim 1, wherein in said step (a)(i) the secreted
CTP-modified polypeptide of interest lack a signal peptide.
11. The method of claim 1, wherein said step (c) comprises
expanding clones from a working cell bank (WCB) that optimally
expresses and secretes said CTP-modified polypeptide of
interest.
12. The method of claim 1, wherein said step (c) comprises
expanding clones obtained from a master cell bank (MCB) that
optimally expresses and secretes said CTP-modified polypeptide of
interest.
13. The method of claim 1, wherein said method of manufacturing is
an animal-free process.
14. The method of claim 1, wherein at step (c) said CTP-modified
polypeptide of interest is expressed at a level of at least 30
mg/ml.
15. The method of claim 1, wherein at step (c) expanding said
clones in solution comprises expanding said clones in solution
through a series of sub-cultivating steps up to production
bioreactor level.
16. The method of claim 15, wherein said bioreactor comprises a
disposable bioreactor, a stainless steel bioreactor, a fed-batch
mode bioreactor, a batch mode bioreactor, a repeated batch mode
bioreactor, or a perfusion mode bioreactor, or any combination
thereof.
17. The method of claim 1, wherein at step (f) at least 60% of the
purified CTP-modified polypeptides of interest in said clarified
harvest comprises a high glycosylation form of said CTP-modified
polypeptide.
18. The method of claim 1, wherein said purification (step f)
comprises sequentially performing the following steps comprising:
(g) concentrating, diafiltering and purifying said clarified
harvest solution; i. wherein said concentration, diafiltration and
purifying is accomplished by sequentially passing said clarified
harvest solution through an anion exchange column and a hydrophobic
interaction column; (h) obtaining said clarified harvest obtained
following step (g) and inactivating viruses present in said
clarified harvest by incubating in a solution toxic to said
viruses; (i) obtaining said clarified harvest solution from step
(h) and concentrating, diafiltering, and purifying said clarified
harvest solution, i. wherein said concentration, diafiltration and
purifying is followed by sequentially passing said clarified
harvest solution through a multimodal or mixed-mode protein
chromatography column and a cation exchange column; (j) obtaining
said clarified harvest solution following step (i) and physically
removing said clarified harvest solution from viruses by
nanofiltration; and (k) obtaining said clarified harvest solution
following step (j) and concentrating and diafiltering said
clarified harvest solution to arrive at a maximally purified
clarified harvest containing said highly glycosylated form of
CTP-modified polypeptide of interest.
19. The method of claim 1, wherein said method achieves at least a
20% recovery rate of highly glycosylated CTP-modified polypeptide
of interest.
20. The method of claim 18, wherein said viral clearance shows a
viral log reduction factor (LRF) of more than 12.
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/965,079, filed Dec. 10, 2015, which claims the benefit
of U.S. Provisional Application Ser. Nos. 62/090,104, 62/090,116,
and 62/090,124, all filed Dec. 10, 2014 and which are incorporated
by reference herein in their entirety.
FIELD OF INVENTION
[0002] The application describes a method for manufacturing a
recombinant protein or peptide of interest modified by CTP
extensions in a mammalian cell culture system.
BACKGROUND
[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.
[0005] Addressed herein is the problem of providing long-lasting
therapeutic polypeptides, by providing methods of production of
long-acting CTP-modified polypeptides of interest. Use of these
long-lasting therapeutic polypeptides of interest may obviate the
need for the numerous injections that may be required for a
therapeutic, prophylactic or chronic treatment of a subject in
need. This technology is based on the use of a natural peptide, the
C-terminal peptide (CTP) of the beta chain of human chorionic
gonadotropin (hCG), which provides hCG with the required longevity
to maintain pregnancy (initial T.sub.1/2.about.10 h, terminal
T.sub.1/2.about.37 h). CTP has 28 amino acids and the capacity for
glycosylation at four to six O-linked sugar chains, which are all
at serine residue. The beta chain of luteinizing hormone (LH), a
fertility hormone that triggers ovulation, is almost identical to
hCG but does not include the CTP. As a result, LH has a
significantly shorter half-life in blood (initial T.sub.1/2.about.1
h, terminal T.sub.1/2.about.10 h).
SUMMARY OF THE INVENTION
[0006] In one aspect, disclosed herein is a method of manufacturing
a human chorionic gonadotropin carboxy terminal peptide
(CTP)-modified polypeptide of interest, the method comprising the
steps of: (a) stably transfecting a predetermined number of cells
with an expression vector comprising a coding portion encoding said
CTP-modified polypeptide of interest, wherein said transfected cell
expresses and secretes said CTP-modified polypeptide of interest;
(b) obtaining cell clones that overexpress said CTP-modified
polypeptide of interest; (c) expanding said clones in solution to a
predetermined scale; (d) harvesting said solution containing said
clones; (e) filtering said solution containing said clones to
obtain a clarified harvest solution; and, (f) purifying said
clarified harvest solution to obtain a purified protein solution
having a desired concentration of a CTP-modified polypeptide of
interest, thereby manufacturing a human chorionic gonadotropin
peptide (CTP)-modified polypeptide of interest.
[0007] In a related aspect, said CTP-modified polypeptide of
interest is highly glycosylated. In another related aspect, each
CTP comprised in a CTP-modified polypeptide comprises glycosylation
at more than 4 O-linked glycosylation sites. In another related
aspect, the CTP-modified polypeptide of interest is highly
sialylated.
[0008] In a related aspect, the CTP-modified polypeptide of
interest comprises a CTP-polypeptide of interest-CTP-CTP
polypeptide. In another related aspect, the CTP-modified
polypeptide of interest comprises a polypeptide of
interest-CTP.sub.(3) polypeptide. In another related aspect, the
CTP-modified polypeptide of interest comprises a
CTP.sub.(1-5)-polypeptide of interest-CTP.sub.(1-5)
polypeptide.
[0009] Other features and advantages of the polypeptides and
methods of manufacturing the same, disclosed herein, will become
apparent from the following detailed description examples and
figures. It should be understood, however, that the detailed
description and the specific examples while indicating embodiments
of polypeptide, compositions comprising the same and methods of
making the same, are given by way of illustration only, since
various changes and modifications within the spirit and scope of
the disclosure herein, will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The subject matter disclosed herein for the polypeptides,
methods of making the same and method of using the same, is
particularly pointed out and distinctly claimed in the concluding
portion of the specification. However, both as to the organization
and the method of operation, together with objects, features, and
advantages thereof, these may best be understood by reference to
the following detailed description when read with the accompanying
drawings in which:
[0011] FIGS. 1A-1F are diagrams illustrating six EPO-CTP
constructs. FIG. 1A is a diagram of the polypeptide of SEQ ID NO:
1. FIG. 1B is a diagram of the polypeptide of SEQ ID NO: 2. FIG. 1C
is a diagram of the polypeptide of SEQ ID NO: 3. FIG. 1D is a
diagram of the polypeptide of SEQ ID NO: 4. FIG. 1E is a diagram of
the polypeptide of SEQ ID NO: 5. FIG. 1F is a diagram of the
polypeptide of SEQ ID NO: 6.
[0012] 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.
[0013] FIG. 3 is a Western blot illustrating the molecular weight
& identity of commercial interferon-.beta.1a (AVONEX), MOD-9013
(SEQ ID NO: 9), MOD-9016 (SEQ ID NO: 10), MOD-9015 (SEQ ID NO: 11),
MOD-9012 (SEQ ID NO: 12), MOD-9011 (SEQ ID NO: 13) and Mock. PAGE
SDS gel was blotted and stained using monoclonal anti-IFN-.beta.1A
antibodies (B). The photograph indicates that like commercial
interferon .beta.1a (AVONEX), MOD-901X variants are recognized by
anti IFN-.beta.1A antibodies.
[0014] FIG. 4. Shows a graph showing the PK profile of purified
FIX-CTP-CTP, rhFIX, harvest of FIX-CTP-CTP, and harvest of
FIX-CTP.
[0015] FIG. 5. Shows anti-CTP and anti-gamma carboxylation
antibodies Western blots of FIX fused to three, four or five CTPs.
FIX-CTP.sub.3, FIX-CTP.sub.4, and FIX-CTP.sub.5 harvests were
loaded on 12% Tris-Glycine gel using Precision plus dual color
protein marker (Bio-Rad). The SDS-PAGE analysis was performed by
Western immuno-blot using anti-CTP polyclonal Ab (Adar Biotech
Production) or anti-Gla Ab (American Diagnostica).
[0016] FIG. 6. Shows a Coomassie blue detection of FIX-CTP.sub.3,
FIX-CTP.sub.4, and FIX-CTP.sub.5. After a purification process
utilizing a Jacalin column (immunoaffinity purification of
glycosylated proteins), FIX-CTP.sub.3, FIX-CTP.sub.4, and
FIX-CTP.sub.5 were loaded on 12% Tris-Glycine gel using Precision
Plus Dual Color Protein Marker (Bio-Rad). The SDS-PAGE was stained
by Coomassie blue dye for sample detection.
[0017] FIG. 7. Shows FIX Chromogenic activity. A comparative
assessment of the in vitro potency of fully purified (HA column)
FIX-CTP.sub.3 FIX-CTP.sub.4 and FIX-CTP.sub.5 versus human pool
normal plasma was performed using a commercially available
chromogenic activity test kit, BIOPHEN (Hyphen BioMed 221802). All
samples were serially diluted and the potency was assessed by
comparing a dose response curve to a reference preparation
consisting of normal human plasma.
[0018] FIG. 8. Shows the comparative pharmacokinetic (PK) profile
of FIX-CTP.sub.3 FIX-CTP.sub.4 and FIX-CTP.sub.5. FIX concentration
in plasma samples were quantified using human FIX Elisa kits
(Affinity Biologicals). Pharmacokinetic profile was calculated and
is the mean of 3 animals at each time point. Terminal half-lives
were calculated using PK Solutions 2.0 software.
[0019] FIGS. 9A-9D show diagrams of rFVII and rFIX-CTP constructs:
a diagram illustrating the rFVII-CTP construct (FIG. 9A),
rFVII-CTP-CTP construct (FIG. 9B), rFIX-CTP construct (FIG. 9C),
and rFIX-CTP-CTP construct (FIG. 9D).
[0020] FIGS. 10A-10D. FIG. 10A shows a bar graph showing harvests
limited diluted clone transfected and selected cells with FVII-CTP
variants in the presence of 5 .mu.g/ml of Vitamin K3. The level of
FVII was quantified using FVII ELISA (AssayPro). FIG. 10B shows a
bar graph showing harvests of limited diluted transfected and
selected cells with FVII-CTP variants in the presence of 5 .mu.g of
Vitamin K3.activity. FVII activity was quantified using FVII
chromogenic activity assay (AssayPro). FIG. 10C shows a bar graph
showing harvests of limited diluted transfected and selected cells
with FVII-CTP variants in the presence of 5 .mu.g of Vitamin K3.
The specific activity of FVII was calculated for each version by
dividing the activity value by the harvest FVII concentration. FIG.
10D shows a graph showing PK profile of FVII, FVII-CTP-CTP, and
FVII-CTP harvests.
[0021] FIGS. 11A-11B show PAGE and Western Blot (WB) analysis of
purified OXM-CTP variants: OXM-CTP-CTP-CTP, OXM-CTP 4X, OXM-CTP 5X.
FIG. 11A: Coomassie staining of OXM samples and OXM-CTP variants
(10 and 2 .mu.g protein/lane). FIG. 11B: WB analysis of OXM-CTP
variants using anti OXM.
[0022] FIG. 12 shows PAGE analysis of samples from purification
process of OXM-CTP variants: CTP-OXM-CTP, CTP-OXM-CTP-CTP,
OXM-CTP-CTP and CTP-CTP-OXM.
[0023] FIG. 13 shows PK profiles of three sequential experiments of
OXM peptide and OXM-CTP variants in SD-1 rats.
[0024] FIG. 14 shows the results of glucose tolerance test of OXM
peptide and OXM-CTP variants as measured in C57BL/6 mice.
[0025] FIG. 15 Shows upstream process flow production chart of
CTP-modified polypeptides.
[0026] FIG. 16. Presents a flow chart of the purification process
of CTP-modified polypeptides.
[0027] It will be appreciated that for simplicity and clarity of
illustration, elements shown in the figures have not necessarily
been drawn to scale. For example, the dimensions of some of the
elements may be exaggerated relative to other elements for clarity.
Further, where considered appropriate, reference numerals may be
repeated among the figures to indicate corresponding or analogous
elements.
DETAILED DESCRIPTION
[0028] In the following detailed description, numerous specific
details are set forth in order to provide a thorough understanding
of the long-acting CTP-modified polypeptides disclosed herein and
methods of manufacturing the same. However, it will be understood
by those skilled in the art that the long-acting CTP-modified
polypeptides and methods of manufacturing disclosed herein may be
practiced without these specific details. In other instances,
well-known methods, procedures, and components have not been
described in detail so as not to obscure the long-acting
polypeptides and methods of manufacturing disclosed herein.
[0029] In one embodiment, disclosed herein are long-acting
polypeptides and methods of manufacturing the same. In another
embodiment, long-acting polypeptides comprise at least one carboxy
terminal peptide (CTP) of human Chorionic Gonadotropin (hCG). In
another embodiment, CTP acts as a protectant against degradation of
proteins or peptides of interest. In another embodiment, CTP
extends circulatory half-lives of proteins or peptides of interest.
In some embodiments, CTP enhances the potency of proteins or
peptides of interest.
[0030] A skilled artisan would recognize that the terms "CTP
peptide", "carboxy terminal peptide" and "CTP sequence" may be used
interchangeably herein having the same qualities and meanings. In
another embodiment, the carboxy terminal peptide is a full-length
CTP. In another embodiment, the carboxy terminal peptide is a
truncated CTP.
[0031] A skilled artisan would recognize that the terms "signal
sequence" and "signal peptide" may be used interchangeably herein.
A skilled artisan would recognize that the term "sequence" when in
reference to a polynucleotide may encompass a coding portion of
that sequence.
[0032] A skilled artisan would recognize that the terms
"polypeptide of interest", "peptide of interest", "peptide", and
"polypeptide sequence of interest" may be used interchangeably
herein. In another embodiment, the polypeptide of interest is a
full-length protein. In another embodiment, the polypeptide of
interest is a protein fragment. In one embodiment, a polypeptide of
interest is a peptide. In one embodiment, a polypeptide of interest
is an amino acid (AA) sequence. In some embodiments, a "polypeptide
of interest" is a recombinant polypeptide.
[0033] In one embodiment, a polypeptide of interest comprises an
erythropoietin (EPO). In another embodiment, a polypeptide of
interest comprises an interferon (IFN). In another embodiment, a
polypeptide of interest comprises a cytokine. In another
embodiment, a polypeptide of interest comprises a coagulation
factor. In another embodiment, a coagulation factor comprises
Factor VII (FVII), activated Factor VIIa (FVIIa), or Factor IX
(FIX). In another embodiment, a polypeptide of interest comprises
an oxyntomodulin (OXM) peptide or portion thereof. In another
embodiment, a polypeptide of interest comprises a glucagon-like
peptide 1 (GLP-1) peptide. In another embodiment, a polypeptide of
interest comprises a dual GLP-1/Glucagon receptor agonist. In
another embodiment, a polypeptide of interest is not a growth
hormone. In another embodiment, a polypeptide of interest is not a
human growth hormone. CTP-modified polypeptides of interest are
described, for example, in U.S. Pat. Nos. 8,110,376; 8,323,636;
8,426,166; International Application Publication No. WO
2013/121416; International Application Publication No. WO
2013/157002; and International Application Publication WO
2007/094985, each of which are incorporated herein in full.
[0034] In one embodiment, disclosed herein is a method of
manufacturing a human chorionic gonadotropin carboxy terminal
peptide (CTP)-modified polypeptide of interest, the method
comprising the steps of: (a) stably transfecting a predetermined
number of cells with an expression vector comprising a coding
portion encoding said CTP-modified polypeptide of interest, wherein
said transfected cell expresses and secretes said CTP-modified
polypeptide of interest; (b) obtaining cell clones that overexpress
said CTP-modified polypeptide of interest; (c) expanding said
clones in solution to a predetermined scale; (d) harvesting said
solution containing said clones; (e) filtering said solution
containing said clones to obtain a clarified harvest solution; and,
(f) purifying said clarified harvest solution to obtain a purified
protein solution having a desired concentration of a CTP-modified
polypeptide of interest, thereby manufacturing a CTP-modified
polypeptide of interest polypeptide. In another embodiment, a
method of manufacturing a human chorionic gonadotropin carboxy
terminal peptide (CTP)-modified polypeptide of interest comprises
the steps of: (a) stably transfecting a predetermined number of
cells with an expression vector comprising a coding portion
encoding said CTP-modified polypeptide of interest, wherein said
transfected cell expresses said CTP-modified polypeptide of
interest; (b) obtaining cell clones that overexpress said
CTP-modified polypeptide of interest; (c) expanding said clones in
solution to a predetermined scale; (d) harvesting said solution
containing said clones; (e) filtering said solution containing said
clones to obtain a clarified harvest solution; and, (f) purifying
said clarified harvest solution to obtain a purified protein
solution having a desired concentration of a CTP-modified
polypeptide of interest, thereby manufacturing a CTP-modified
polypeptide of interest polypeptide. In another embodiment, said
expressed CTP-modified polypeptide of interest is isolated from the
cell clones directly.
[0035] In another embodiment, disclosed herein is a method of
manufacturing a CTP-modified polypeptide of interest with increased
glycosylation content. In another embodiment, a CTP-modified
polypeptide of interest manufactured by the methods disclosed
herein, has increased occupancy of O-linked glycosylation
sites.
Human Chorionic Gonadotropin Carboxy Terminal Peptide
(CTP)-Modified Polypeptides
[0036] A skilled artisan would recognize that the phrase
"polypeptide" or "polypeptide sequence of interest" may encompass
any polypeptide or peptide sequence, such as one comprising a
biological activity. In another embodiment, the polypeptide of
interest is glycosylated. In another embodiment, the polypeptide of
interest is not glycosylated. Examples of polypeptides and peptides
of interest, which benefit from an extension in their circulatory
half-life include, but are not limited to erythropoietin (EPO),
interferon (IFN), glucagon-like peptide-1 (GLP-1), cytokine,
coagulation factor, FVII, FVIIa, FIX, OXM, and GLP-1/Glucagon
receptor agonist. In another embodiment, a polypeptide of interest
comprises a cytokine polypeptide, an erythropoietin polypeptide, an
interferon polypeptide, a coagulation factor polypeptide, a Factor
VII polypeptide, an activated Factor VII polypeptide, a Factor IX
polypeptide, a glucagon-like peptide 1 polypeptide, a dual
GLP-1/Glucagon receptor agonist polypeptide, or an oxyntomodulin
polypeptide. Each possibility represents an embodiment disclosed
herein.
[0037] In one embodiment, long-acting polypeptides disclosed herein
comprise at least one carboxy terminal peptide (CTP) of human
Chorionic Gonadotropin (hCG). In another embodiment, an at least
one CTP acts as a protectant against degradation of proteins or
peptides derived therefrom. In another embodiment, an at least one
CTP extends circulatory half-lives of proteins or peptides derived
therefrom. In some embodiments, an at least one CTP enhances the
potency of proteins or peptides derived therefrom.
[0038] In one embodiment, disclosed herein are long-acting
erythropoietin (EPO) polypeptides and methods of producing or
manufacturing and using same. In another embodiment, disclosed
herein are long-acting interferon (IFN) polypeptides and methods of
producing or manufacturing and using same. In another embodiment,
disclosed herein are long-acting cytokine polypeptides and methods
of producing or manufacturing and using same. In another
embodiment, disclosed herein are long-acting coagulation factor
polypeptides and methods of producing or manufacturing and using
same. In another embodiment, disclosed herein are long-acting
coagulation Factor VII (FVII) polypeptides and methods of producing
or manufacturing and using same. In another embodiment, disclosed
herein are long-acting activated coagulation Factor VIIa (FVIIa)
polypeptides and methods of producing or manufacturing and using
same. In another embodiment, disclosed herein are long-acting
oxyntomodulin (OXM) polypeptides and methods of producing or
manufacturing and using same. In another embodiment, disclosed
herein is a long-acting glucagon-like peptide 1 (GLP-1)
polypeptides and methods of producing or manufacturing and using
same. In another embodiment, disclosed herein is a long-acting dual
GLP-1/Glucagon receptor agonist polypeptides and methods of
producing or manufacturing and using same.
[0039] In another embodiment, a polypeptide comprising at least two
carboxy-terminal peptide (CTP) sequences of chorionic gonadotropin
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 gonadotropin.
[0040] In another embodiment, the 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] In another embodiment, the CTP is fused to a protein. In
another embodiment, the CTP is fused to a glycoprotein. In another
embodiment, the CTP is fused to a glycoprotein hormone. In another
embodiment, the CTP is fused to a peptide derived from a
glycoprotein hormone. In some embodiments, glycoprotein hormones
comprise EPO, FSH, or TSH and peptides derived therefrom.
[0042] 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.
[0043] 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.
[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 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.
[0045] In one embodiment, a CTP sequence comprises an amino acid
sequence selected from the sequences set forth in SEQ ID NO: 14 and
SEQ ID NO: 15. In another embodiment, SEQ ID NO: 14 comprise the
following amino acid (AA) sequence:
TABLE-US-00001 (SEQ ID NO: 14)
DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL.
In another embodiment, SEQ ID NO: 15 comprise the following amino
acid (AA) sequence:
TABLE-US-00002 (SEQ ID NO: 15) SSSSKAPPPSLPSPSRLPGPSDTPILPQ.
[0046] In another embodiment, the CTP peptide comprises the amino
acid sequence from amino acid 112 to position 145 of human
chorionic gonadotropin, as set forth in SEQ ID NO: 14. In another
embodiment, the CTP sequence comprises the amino acid sequence from
amino acid 118 to position 145 of human chorionic gonadotropin, as
set forth in SEQ ID NO: 15. In another embodiment, the CTP sequence
also commences from any position between positions 112-118 and
terminates at position 145 of human chorionic gonadotropin. 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 truncated CTP comprises the first
10 amino acids of SEQ ID NO: 15. In another embodiment, the
truncated CTP comprises the first 5 amino acids of SEQ ID NO: 15,
the first 6 amino acids, the first 7 amino acids, the first 8 amino
acids, the first 9 amino acids, the first 11 amino acids, the first
12 amino acids, the first 13 amino acids, or the first 14 amino
acids of SEQ ID NO: 15. Each possibility represents an embodiment
disclosed herein.
[0048] In another embodiment, the CTP peptide is a variant of
chorionic gonadotropin 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 gonadotropin CTP which differs from the native CTP by 1
conservative amino acid substitution, by 2 conservative amino acid
substitutions, by 3 conservative amino acid substitutions, by 4
conservative amino acid substitutions, or by 5 conservative amino
acid substitutions. Each possibility represents an embodiment
disclosed herein.
[0049] In another embodiment, the CTP peptide amino acid sequence
is at least 70% homologous to the native CTP amino acid sequence or
a peptide thereof, is at least 80%, is at least 90%, is at least
95%, or is at least 98% homologous to the native CTP amino acid
sequence or a peptide thereof. Each possibility represents an
embodiment disclosed herein.
[0050] In one embodiment, the CTP peptide DNA sequence encodes a
peptide that is at least 70% homologous to the native CTP DNA
sequence or a peptide thereof. In another embodiment, the CTP
peptide DNA sequence encodes a peptide that is at least 80%
homologous to the native CTP DNA sequence or a peptide thereof, is
at least 90%, is at least 95% homologous, is at least 98%
homologous to the native CTP DNA sequence or peptide thereof. Each
possibility represents an embodiment disclosed herein.
[0051] In one embodiment, at least one of the chorionic
gonadotropin CTP amino acid sequences is truncated. In another
embodiment, both of the chorionic gonadotropin CTP amino acid
sequences are truncated. In another embodiment, 2 of the chorionic
gonadotropin CTP amino acid sequences are truncated. In another
embodiment, 2 or more of the chorionic gonadotropin CTP amino acid
sequences are truncated. In another embodiment, all of the
chorionic gonadotropin CTP amino acid sequences are truncated. In
one embodiment, the truncated CTP comprises the first 10 amino
acids of SEQ ID NO: 16. In one embodiment, the truncated CTP
comprises the first 11 amino acids of SEQ ID NO: 16. In one
embodiment, the truncated CTP comprises the first 12 amino acids of
SEQ ID NO: 16. In another embodiment, SEQ ID NO: 16 comprises the
amino acid (AA) sequence: SSSSKAPPPSLP.
[0052] In one embodiment, at least one of the CTP amino acid
sequences is glycosylated. In another embodiment, both of the CTP
amino acid sequences are glycosylated. In another embodiment, 2 of
the CTP amino acid sequences are glycosylated. In another
embodiment, 3 of the CTP amino acid sequences are glycosylated. In
another embodiment, 4 of the CTP amino acid sequences are
glycosylated. In another embodiment, 5 of the CTP amino acid
sequences are glycosylated. In another embodiment, 2 or more of the
CTP amino acid sequences are glycosylated. In another embodiment,
all of the CTP amino acid sequences are glycosylated.
[0053] In one embodiment, the CTP sequence disclosed herein
comprises at least one glycosylation site. In another embodiment,
the CTP sequence disclosed herein comprises 2 glycosylation sites,
3 glycosylation sites, 4 glycosylation sites, 5 glycosylation
sites, or 6 glycosylation sites. In one embodiment, one or more of
the CTP amino acid sequences is fully glycosylated. In another
embodiment, one or more of the CTP amino acid sequences is
partially glycosylated. In one embodiment, partially glycosylated
indicates that one of the CTP glycosylation sites is glycosylated.
In another embodiment, two of the CTP glycosylation sites are
glycosylated. In another embodiment, three of the CTP glycosylation
sites are glycosylated.
[0054] In some embodiments, the CTP sequence modification is
advantageous in permitting the usage of lower dosages. In some
embodiments, the CTP sequences modification is advantageous in
permitting fewer dosages. In some embodiments, the CTP sequences
modification is advantageous in permitting a safe, long-acting
effect.
[0055] In another embodiment, disclosed herein is a polypeptide
consisting of a cytokine, a single CTP attached to the amino
terminus of the cytokine, and two CTPs attached to the carboxy
terminus of the cytokine. In another embodiment, disclosed herein
is a polypeptide consisting of a cytokine, a single CTP attached to
the amino terminus of the cytokine, two CTPs attached to the
carboxy terminus of the cytokine, and a signal peptide attached to
the amino terminus of one chorionic gonadotropin carboxy terminal
peptide.
[0056] In another embodiment, a cytokine is a low molecular weight
protein. In another embodiment, a cytokine is a protein secreted by
a cell. In another embodiment, a cytokine induces and/or regulates
an immune response. In another embodiment, a cytokine has a high
affinity binding to a specific receptor or receptors. In another
embodiment, cytokines as described herein include mimetics of
cytokines that can be used to inhibit or potentiate their effects
in vivo. In another embodiment, a cytokine comprises an autocrine
activity. In another embodiment, a cytokine comprises a paracrine
activity. In another embodiment, a cytokine comprises an endocrine
activity.
[0057] In another embodiment, the cytokine is a Hematopoietin
cytokine. In another embodiment, the cytokine is an Interferon
cytokine. In another embodiment, the cytokine is a chemokine. In
another embodiment, the cytokine is a Tumor Necrosis Factor
cytokine. In another embodiment, a cytokine as used herein
comprises biological activity and clinical efficacy. In another
embodiment, a cytokine as used herein is a therapeutic protein.
[0058] In another embodiment, a CTP-modified cytokine as described
herein has enhanced biological activity in vivo compared the same
cytokine without CTPs. In another embodiment, a cytokine comprising
at least one CTP attached to its amino terminus and at least two
CTPs attached to its carboxy terminus has enhanced biological
activity in vivo compared the same cytokine without CTPs. In
another embodiment, a cytokine comprising one CTP attached to its
amino terminus and two CTPs attached to its carboxy terminus has
enhanced biological in vivo activity compared the same cytokine in
without CTPs.
[0059] In another embodiment, a CTP-modified cytokine is used to
facilitate organ transplantation. In another embodiment, a
CTP-modified cytokine is used to reduce inflammation. In another
embodiment, a CTP-modified cytokine is used to induce
erythropoiesis. In another embodiment, a CTP-modified cytokine is
used to induce growth. In another embodiment, a CTP-modified
cytokine is used to induce weight gain. In another embodiment, a
CTP-modified cytokine is used in cancer therapy as will be readily
understood by one of average skill in the art. In another
embodiment, a CTP-modified cytokine is used to induce an immune
response. In another embodiment, a CTP-modified cytokine is used in
infectious disease therapy as will be readily understood by one of
average skill in the art. In another embodiment, a CTP-modified
cytokine is used in treating allergy as will be readily understood
by one of average skill in the art.
[0060] In another embodiment, a CTP-cytokine-CTP-CTP as described
herein comprises a cytokine or an active fragment thereof connected
via a peptide bond to at least one CTP unit. In another embodiment,
a CTP-cytokine-CTP-CTP as described herein comprises a cytokine or
an active fragment thereof connected via a peptide bond to at least
one CTP unit which is connected to an additional CTP unit via a
peptide bond. In another embodiment, a polypeptide as described
herein comprising a cytokine fragments thereof and CTP units and/or
fragments thereof are interconnected via a peptide bond. In another
embodiment, one nucleic acid molecule encodes a polypeptide as
described herein comprising a cytokine and/or fragments thereof and
CTP units and/or fragments thereof.
[0061] A skilled artisan would appreciate that a polypeptide of
interest disclosed herein may encompass a homologue. In one
embodiment, a homologue comprises a deletion, insertion, or
substitution variant, including an amino acid substitution, thereof
and biologically active polypeptide fragments thereof. In another
embodiment, homologues comprise polypeptides which are at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 87%, at least 89%,
at least 91%, at least 93%, at least 95%, at least 96%, at least
97%, at least 98%, or at least 99% homologous to a polypeptide of
interest as determined using BlastP software of the National Center
of Biotechnology Information (NCBI) using default parameters. Each
possibility represents an embodiment disclosed herein.
[0062] In one embodiment, the cytokine is a homologue.
[0063] In another embodiment, disclosed herein is a polypeptide
consisting of a cytokine antagonist, a single chorionic
gonadotropin carboxy terminal peptide attached to the amino
terminus of the cytokine antagonist, and two chorionic gonadotropin
carboxy terminal peptides attached to the carboxy terminus of the
cytokine antagonist. In another embodiment, disclosed herein is a
polypeptide consisting of a cytokine antagonist, a single chorionic
gonadotropin carboxy terminal peptide attached to the amino
terminus of the cytokine antagonist, two chorionic gonadotropin
carboxy terminal peptides attached to the carboxy terminus of the
cytokine antagonist, and a signal peptide attached to the amino
terminus of one chorionic gonadotropin carboxy terminal
peptide.
[0064] In another embodiment, a cytokine antagonist modified with
CTPs is applied as an anti-cytokine strategy. In another
embodiment, a cytokine antagonist modified with CTPs is effective
in decrease an inflammatory response. In another embodiment, a
cytokine antagonist modified with CTPs is more effective in
decreasing an inflammatory response compared to an unmodified
cytokine antagonist. In another embodiment, a cytokine antagonist
modified with CTPs is more stable than an unmodified cytokine
antagonist. In another embodiment, a cytokine antagonist modified
with CTPs is more stable in-vivo than an unmodified cytokine
antagonist. In another embodiment, a cytokine antagonist modified
with CTPs is more bioactive than an unmodified cytokine
antagonist.
[0065] In another embodiment, a cytokine antagonist is a cytokine
homologue. In another embodiment, a cytokine antagonist is a
soluble fragment of a cytokine receptor. In another embodiment, a
cytokine antagonist is a chemokine receptor homologue.
[0066] In another embodiment, a cytokine as described herein is
involved in cytokine signaling cascade comprising Ras-MAP kinase
pathway. In another embodiment, a cytokine as described herein is
involved in induction of JNK. In another embodiment, a cytokine as
described herein is involved in induction of p38MAP. In another
embodiment, a cytokine as described herein induces cell
proliferation. In another embodiment, a cytokine as described
herein is involved in cytokine signaling cascade comprising the
JAK/STAT pathway. In another embodiment, a cytokine as described
herein induces cell growth inhibition. In another embodiment, a
cytokine as described herein induces differentiation.
[0067] In another embodiment, a cytokine as described herein is a
four .alpha.-helix bundle cytokine. In another embodiment, a
cytokine as described herein is a long-chain 4-helix bundle
cytokine. In another embodiment, a cytokine as described herein is
a short-chain 4-helix bundle cytokine.
[0068] In another embodiment, a cytokine as described herein is a
beta-trefoil cytokine. In another embodiment, a cytokine as
described herein is a beta-sandwich cytokine. In another
embodiment, a cytokine as described herein is an EGF-like cytokine.
In another embodiment, a cytokine as described herein comprises a
cystine knot dimerization domain. In another embodiment, a cytokine
as described herein comprises both alpha and beta chains. In
another embodiment, a cytokine as described herein is an alpha
superfamily cytokine such as IL-2, IL-4, IL-5, GM-CSF, IL-3,
IFN-alpha, or IL-13. In another embodiment, a cytokine as described
herein is a dimeric 4-helix bundles cytokine. In another
embodiment, a cytokine as described herein is a member of the IL
family of cytokines.
[0069] In another embodiment, a cytokine as described herein is a
long-chain 4-helix bundle superfamily cytokine such as G-CSF,
Myelomonocytic growth factor, IL-6, IL-3, IL-7, LIF, Oncostatin M,
Ciliary neurotrophic factor (CNTF), or cholinergic differentiation
factor (CDF). In another embodiment, a cytokine as described herein
is a short-chain 4-helix bundle superfamily cytokine such as IL-2,
IL-4, IL-13, IFN-alpha, IL-5, GM-CSF, IL-3, Macrophage
colony-stimulating factor (M-CSF). In another embodiment, a
cytokine as described herein is a dimeric 4-helix bundles such as
IFN-Gamma, IL-10, or IFN-Beta.
[0070] In another embodiment, a cytokine as described herein is a
Beta-Trefoil cytokine such as IL1-A, IL1-B, or FGF. In another
embodiment, a cytokine as described herein is a Beta-sandwich
cytokine such as TNF-alpha, or TNF-Beta. In another embodiment, a
cytokine as described herein is an EGF-like cytokine such as
TGF-Alpha. In another embodiment, a cytokine as described herein
comprises cystine knot dimerization domains. In another embodiment,
Gonadotropin, Nerve Growth Factor (NGF), Platelet-derived growth
factor (PDGF), and TGF-Beta2 comprise cystine knot dimerization
domains. In another embodiment, a cytokine as described herein
comprises both alpha and beta chains. In another embodiment IL-8,
IP10, platelet factor 4 (PF-4), bTG, GRO, 9E3, HLA-A2, macrophage
inflammatory protein 1 alpha (MIP-1 alpha), macrophage inflammatory
protein 1 beta (MIP-1 beta), and Melanoma growth stimulating
activity (MGSA) comprise both alpha and beta chains. Each
possibility represents an embodiment disclosed herein.
[0071] In another embodiment, a cytokine as described herein binds
a hematopoietin-receptor family member (also called the class I
cytokine receptor family. In another embodiment, a cytokine as
described herein binds a class II cytokine receptor (interferons or
interferon-like cytokines). In another embodiment, a cytokine as
described herein binds a tumor necrosis factor-receptor (TNFR). In
another embodiment, a cytokine as described herein binds a,
chemokine-receptor. In another embodiment, a cytokine as described
herein binds a G protein-coupled receptor.
[0072] In another embodiment, a cytokine as described herein is an
IL-2 cytokine, is an interferon, is an IL-10 cytokine, is
erythropoietin (EPO), thrombopoietin (THPO), is IL-1, IL-18, or
IL-17. In another embodiment, a cytokine as described herein
promotes proliferation of T-cells. Each possibility represents an
embodiment disclosed herein.
[0073] In another embodiment, a cytokine as described herein is
close to the cluster formed by ciliary neurotrophic factor and
granulocyte colony-stimulating factor (CSF)
[0074] In another embodiment, a cytokine as described herein
enhances cytokine responses, type 1 (IFN-.gamma., TGF-.beta. etc.).
In another embodiment, a cytokine as described herein enhances
antibody responses, type 2 (IL-4, IL-10, IL-13, etc).
[0075] In another embodiment, a cytokine is a peptide. In another
embodiment, the cytokine is glycosylated. In another embodiment, a
cytokine is a polypeptide. In another embodiment, a cytokine as
described herein is a modified cytokine comprising at least one CTP
peptide attached to an amino terminus of said cytokine and at least
two chorionic gonadotropin carboxy terminal peptides attached to a
carboxy terminus of said cytokine. In another embodiment, a
cytokine as described herein is a modified cytokine consisting of a
cytokine, one CTP peptide attached to an amino terminus of the
cytokine, and at least two chorionic gonadotropin carboxy terminal
peptides attached to a carboxy terminus of the cytokine. In another
embodiment, a cytokine as described herein is a modified cytokine
consisting a cytokine, at least one CTP peptide attached to an
amino terminus of the cytokine, and two chorionic gonadotropin
carboxy terminal peptides attached to a carboxy terminus of the
cytokine. In another embodiment, a cytokine as described herein is
a modified cytokine consisting a cytokine, one CTP peptide attached
to an amino terminus of the cytokine, and two chorionic
gonadotropin carboxy terminal peptides attached to a carboxy
terminus of the cytokine.
[0076] In another embodiment, the carboxy-terminal peptide (CTP) is
attached to the cytokine via a linker.
[0077] In one embodiment, the polynucleotides disclosed herein
further comprise a signal sequence encoding a signal peptide for
the secretion of the CTP-modified polypeptide of interest disclosed
herein. 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. In another
embodiment, the signal sequence is N-terminal to the polypeptide of
interest sequence that is in turn N-terminal to at least one CTP
sequence. In another embodiment, following expression and secretion
of a CTP-modified polypeptide of interest, the signal peptide is
cleaved from the precursor proteins resulting in the mature
proteins. In another embodiment, a CTP-modified polypeptide of
interest is expressed as a precursor protein including a signal
peptide, wherein upon secretion from the cell the signal peptide is
cleaved from the precursor protein resulting in secretion of a
mature form of the CTP-modified polypeptide of interest.
[0078] 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: 108. 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.
[0079] In some embodiments, a CTP sequences at both the amino
terminal end of a cytokine and at the carboxy terminal end of the
cytokine provide enhanced protection against degradation of a
cytokine. In another embodiment, at least one CTP sequence at the
amino terminal end of a cytokine and two CTP units in the carboxy
terminal end of a cytokine provide enhanced protection against
clearance. In another embodiment, at least one CTP sequence at the
amino terminal end of a cytokine and two CTP units in the carboxy
terminal end of a cytokine provide prolonged clearance time. In
another embodiment, at least one CTP sequence at the amino terminal
end of a cytokine and two CTP units in the carboxy terminal end of
a cytokine enhance Cmax of a cytokine. In another embodiment, at
least one CTP sequence at the amino terminal end of a cytokine and
two CTP units in the carboxy terminal end of a cytokine enhance
Tmax of a cytokine. In another embodiment, at least one CTP
sequence at the amino terminal end of a cytokine and two CTP units
in the carboxy terminal end of a cytokine enhanced T1/2.
[0080] In some embodiments, CTP sequences at both the amino
terminal end of a cytokine and at the carboxy terminal end of the
cytokine extend the half-life of the attached cytokine. In another
embodiment, at least a single CTP sequence at the amino terminal
end of a cytokine and at least two CTP sequences at the carboxy
terminal end of the cytokine provide extended half-life to the
modified cytokine. In another embodiment, a single CTP sequence at
the amino terminal end of a cytokine and two CTP sequences at the
carboxy terminal end of the cytokine provide extended half-life to
the attached cytokine. In another embodiment, a single CTP sequence
at the amino terminal end of a cytokine and two CTP sequences in
tandem at the carboxy terminal end of the cytokine provide extended
half-life to the cytokine.
[0081] In some embodiments, a CTP sequence at the amino terminal
end of the cytokine a CTP sequence at the carboxy terminal end of
the cytokine, and at least one additional CTP sequence attached in
tandem to the CTP sequence at the carboxy terminus provide enhanced
protection against degradation to a cytokine. In some embodiments,
a CTP sequence at the amino terminal end of a cytokine, a CTP
sequence at the carboxy terminal end of the cytokine, and at least
one additional CTP sequence attached in tandem to the CTP sequence
at the carboxy terminus extend the half-life of the cytokine. In
some embodiments, a CTP sequence at the amino terminal end of a
cytokine, a CTP sequence at the carboxy terminal end of the
cytokine, and at least one additional CTP sequence attached in
tandem to the CTP sequence at the carboxy terminus enhance the
biological activity of the cytokine.
[0082] In another embodiment, CTP conjugated cytokines disclosed
herein are used in the same manner as unmodified cytokines. In
another embodiment, conjugated cytokines disclosed herein have an
increased circulating half-life and plasma residence time,
decreased clearance, and increased clinical activity in vivo. In
another embodiment, due to the improved properties of the
conjugated cytokines as described herein, these conjugates are
administered less frequently than unmodified cytokines. In another
embodiment, conjugated cytokines as described herein is
administered once a week instead of the three times a week for
unmodified cytokines. In another embodiment, decreased frequency of
administration will result in improved patient compliance leading
to improved treatment outcomes, as well as improved patient quality
of life. In another embodiment, compared to conventional conjugates
of cytokines linked to poly(ethylene glycol) it has been found that
conjugates having the molecular weight and linker structure of the
conjugates disclosed herein have an improved potency, improved
stability, elevated AUC levels, enhanced circulating half-life. In
another embodiment, compared to conventional conjugates of
cytokines linked to poly(ethylene glycol) it has been found that
EPO having the molecular weight and linker structure of the
conjugates disclosed herein have an improved potency, improved
stability, elevated AUC levels, enhanced circulating half-life. In
another embodiment, a therapeutically effective amount of a
conjugated cytokine is the amount of conjugate necessary for the in
vivo measurable expected biological activity.
[0083] In another embodiment, a therapeutically effective amount of
a conjugated EPO is the amount of EPO conjugate necessary for the
in biological activity of inducing bone marrow cells to increase
production of reticulocytes and red blood cells. In another
embodiment, a therapeutically effective amount of a conjugated
cytokine is determined according to factors as the exact type of
condition being treated, the condition of the patient being
treated, as well as the other ingredients in the composition. In
another embodiment, a therapeutically effective amount of a
conjugated cytokine is 0.01 to 10 .mu.g per kg body weight
administered once a week. In another embodiment, a therapeutically
effective amount of a conjugated cytokine is 0.1 to 1 .mu.g per kg
body weight, administered once a week. In another embodiment, a
pharmaceutical composition comprising a conjugated cytokine is
formulated at a strength effective for administration by various
means to a human patient.
[0084] In another embodiment, a polypeptide comprising a cytokine
and CTP units as described herein is used as an anti-tumor agent.
In another embodiment, a polypeptide comprising an IFN .alpha. and
CTP units as described herein is used as an anti-tumor agent. In
another embodiment, a polypeptide comprising an IFN .alpha. and CTP
units is formulated in a pharmaceutical composition that is
administered to a patient afflicted with cancer.
[0085] In another embodiment, a polypeptide comprising an IFN
protein and CTP units as described herein is used equivalently to a
regular or a recombinant interferon as known to one of average
skill in the art. In another embodiment, a polypeptide comprising
an IFN protein and CTP units is formulated equivalently to a
regular or a recombinant interferon as known to one of average
skill in the art.
[0086] In another embodiment, a polypeptide comprising a cytokine
and CTP units as described herein modulates an immune response. In
another embodiment, a polypeptide comprising a cytokine and CTP
units as described herein modulates a cellular immune response. In
another embodiment, a polypeptide comprising a cytokine and CTP
units as described herein modulates an antibody immune response. In
another embodiment, a polypeptide comprising a cytokine and CTP
units as described herein inhibits an immune response as described
herein. In another embodiment, a polypeptide comprising a cytokine
and CTP units as described herein trigger an immune response as
described herein.
[0087] In another embodiment, a polypeptide comprising an IFN
inhibits the activity of T-cells, while simultaneously reducing the
production cytokines that operate in the inflammatory response to
infection and injury. In another embodiment, a polypeptide
comprising an IFN protein and CTP units as described herein
enhances the activity of T-cells, while simultaneously reducing the
production cytokines that operate in the inflammatory response to
infection and injury. In another embodiment, a polypeptide
comprising an IFN protein and CTP units is formulated in a
pharmaceutical composition that is administered to a patient in
need of T-cells activity enhancement. In another embodiment, a
polypeptide comprising an IFN protein and CTP units is formulated
in a pharmaceutical composition that is administered to a patient
afflicted with multiple sclerosis. In another embodiment, a
polypeptide comprising an IFN protein and CTP units is formulated
in a pharmaceutical composition that is administered to a patient
afflicted with hepatitis C infection.
[0088] In another embodiment, a cytokine is an interferon (IFN). In
another embodiment, a cytokine is a type I interferon. In another
embodiment, the interferon (IFN) is IFN-.alpha.2a. In another
embodiment, the interferon (IFN) is IFN-.alpha.2b In another
embodiment, the interferon (IFN) is IFN-.beta.1a. In another
embodiment, the interferon (IFN) is IFN-.alpha.2b In another
embodiment, the interferon (IFN) is IFN-.beta.1b. In another
embodiment, a signal peptide of IFN-.beta.1 is set forth in SEQ ID
NO: 108: MTNKCLLQIALLLCFSTTALS (SEQ ID NO: 108).
[0089] A skilled artisan would appreciate that the term
erythropoietin (EPO) may encompass the terms "EPO peptide" and "EPO
sequence" and may be used interchangeably herein. In another
embodiment, the EPO peptide is an EPO protein.
[0090] In some embodiments, erythropoietin (EPO) is utilized
according to the teachings disclosed herein. In some embodiments,
any EPO encoding amino acid sequence is an EPO sequence. In some
embodiments, any EPO encoding nucleic acid sequence is an EPO
sequence In some embodiments, the attachment of CTP sequence to
both the amino and carboxy termini of the EPO protein results in
increased potency at stimulating erythropoiesis, 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 U.S. Pat. No. 8,110,376
incorporated herein in full, which demonstrates that EPO attached
to three CTP sequences is equally effective at stimulating
proliferation of TF-1 cells as wild-type EPO. In some embodiments
CTP-modified EPO polypeptides disclosed herein are set forth in SEQ
ID NO: 3 and SEQ ID NO: 6.
[0091] A skilled artisan would appreciate that the term
"erythropoietin" may encompass mammalian erythropoietin.
[0092] A skilled artisan would appreciate that the term "EPO
sequence" disclosed herein may also encompass homologues. In one
embodiment, the erythropoietin amino acid sequence disclosed herein
is at least 50% homologous to an EPO sequence disclosed herein, as
determined using BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters). In
another embodiment, the EPO amino acid sequence disclosed herein is
at least 60%, is at least 70% homologous, is at least 80%, is at
least 90%, or is at least 95% homologous to an EPO sequence
disclosed herein, as determined using BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters). In one embodiment, an EPO substitution variant
comprises a glycine in position 104 of EPO amino acid sequence is
substituted by a serine (SEQ ID NO: 7).
[0093] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein manufacture 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
disclosed herein manufacture 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, SEQ
ID NO: 1 comprises the following amino acid (AA) sequence:
TABLE-US-00003 MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEA
ENITTGCAEHCSLNENITVPDTVNFYAWKRMEVGQQAVEVWQGLALLSE
AVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISP
PDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRSSSS
KAPPPSLPSPSRLPGPSDTPILPQ.
In another embodiment, the methods disclosed herein manufacture 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.
[0094] In another embodiment, the methods disclosed herein
manufacture 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, SEQ ID NO: 2 comprises
the following amino acid (AA) sequence:
TABLE-US-00004 MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEA
ENITTGCAEHCSLNENITVPDTVNFYAWKRMEVGQQAVEVWQGLALLSE
AVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISP
PDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRSSSS
KAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPI LPQ.
[0095] In another embodiment, the methods disclosed herein
manufacture 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 one embodiment, SEQ ID NO: 3 comprises the
following amino acid (AA) sequence:
TABLE-US-00005 MGVHECPAWLWLLLSLLSLPLGLPVLGSSSSKAPPPSLPSPSRLPGPSD
TPILPQAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPD
TKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQ
LHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKL
IRVYSNFLRGKLKLYTGEACRTGDRSSSSKAPPPSLPSPSRLPGPSDTP
ILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ.
[0096] In one embodiment, following expression a mature
CTP-modified EPO polypeptide is secreted, the signal peptide having
been cleaved from the precursor protein resulting in a mature
protein. For example, in the precursor CTP-modified EPO polypeptide
set forth in SEQ ID NO: 3, amino acids 1-27:
MGVHECPAWLWLLLSLLSLPLGLPVLG (SEQ ID NO: 19) represent the signal
peptide of the precursor CTP-modified EPO polypeptide, and amino
acids
TABLE-US-00006 (SEQ ID NO: 109)
SSSSKAPPPSLPSPSRLPGPSDTPILPQAPPRLICDSRVLERYLLEAKE
AENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALL
SEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAI
SPPDAASAAPLRTITADTPRKLFRVYSNFLRGKLKLYTGEACRTGDRSS
SSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDT FILPQ
represent the mature engineered CTP-modified EPO polypeptide
lacking the signal peptide. In one embodiment, the amino acid
sequence of CTP-modified EPO without the signal peptide is set
forth in SEQ ID NO: 109. In another embodiment, the signal peptide
of CTP-modified EPO is set forth in SEQ ID NO: 19.
[0097] In another embodiment, the methods disclosed herein
manufacture 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 one embodiment, SEQ ID NO: 4 comprises the
following amino acid (AA) sequence
TABLE-US-00007 MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEA
ENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLS
EAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAIS
PPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRSSS
SKAPPPSLPSPSRLPGPSDTPILPQAPPRLICDSRVLERYLLEAKEAEN
ITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEA
VLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPP
DAASAAPLRTITADTIRKLIRVYSNFLRGKLKLYTGEACRTGDR.
[0098] In another embodiment, the methods disclosed herein
manufacture 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, SEQ ID NO: 5 comprises
the following amino acid (AA) sequence:
TABLE-US-00008 MGVHECPAWLWLLLSLLSLPLGLPVLGSSSSKAPPPSLPSPSRLPGPSD
TPILPQAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPD
TKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQ
LHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKL
IRVYSNFLRGKLKLYTGEACRTGDR.
[0099] In another embodiment, the methods disclosed herein
manufacture 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 one embodiment, SEQ ID NO: 6 comprises the
following amino acid (AA) sequence
TABLE-US-00009 MGVHECPAWLWLLLSLLSLPLGLPVLGSSSSKAPPPSLPSPSRLPGPSD
TPILPQAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPD
TKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQ
LHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKL
FRVYSNFLRGKLKLYTGEACRTGDRSSSSKAPPPSLPSPSRLPGPSDTP ILPQ.
[0100] In another embodiment, the methods disclosed herein
manufacture an EPO peptide set forth in SEQ ID NO: 7 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 one embodiment, SEQ ID NO: 7 comprises the
following amino acid (AA) sequence
TABLE-US-00010 MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEA
ENITTGCAEHCSLNENITVPDTVNFYAWKRMEVGQQAVEVWQGLALLSE
AVLRSQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISP
PDAASAAPLRTITADTPRKLFRVYSNFLRGKLKLYTGEACRTGDRSSSS
KAPPPSLPSPSRLPGPSDTPILPQ.
[0101] In another embodiment, the methods disclosed herein
manufacture an EPO peptide set forth in SEQ ID NO: 8 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 one embodiment, SEQ ID NO: 8 comprises the
following amino acid (AA) sequence
TABLE-US-00011 MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEA
ENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLS
EAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAIS
PPDAASAAPLRTITADTFRKLPRVYSNFLRGKLKLYTGEACRTGDR.
[0102] In another embodiment, the methods disclosed herein utilize
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 disclosed herein utilize
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 disclosed herein utilize a nucleic acid set
forth in SEQ ID NO: 17 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, SEQ ID NO: 17 comprises the nucleotide (nt) sequence:
tctagaggtc atcatggggg tgcacgaatg tcctgcctgg ctgtggcttc tcctgtccct
tctgtcgctc cctctgggcc tcccagtcct gggctcctct tcctcaaagg cccctccccc
gagccttcca agtccatccc gactcccggg gccctcggac accccaatat taccacaagc
cccaccacgc ctcatctgtg acagccgagt cctggagagg tacctcttgg aggccaagga
ggccgagaat atcacgacgg gctgtgctga acactgcagc ttgaatgaga atatcactgt
cccagacacc aaagttaatt tctatgcctg gaagaggatg gaggtcgggc agcaggccgt
agaagtctgg cagggcctgg ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt
ggtcaactct tcccagccgt gggagcccct gcagctgcat gtggataaag ccgtcagtgg
ccttcgcagc ctcaccactc tgcttcgggc tctgggagcc cagaaggaag ccatctcccc
tccagatgcg gcctcagctg ctccactccg aacaatcact gctgacactt tccgcaaact
cttccgagtc tactccaatt tcctccgggg aaagctgaag ctgtacacag gggaggcctg
caggacaggg gacagatcct cttcctcaaa ggcccctccc ccgagccttc caagtccatc
ccgactcccg gggccctcgg acaccccgat cctcccacaa taaaggtctt ctggatccgc
ggccgc.
[0103] In another embodiment, the methods disclosed herein utilize
a nucleic acid set forth in SEQ ID NO: 18 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. In
another embodiment, SEQ ID NO: 18 comprises a nt sequences:
tctagaggtc atcatggggg tgcacgaatg tcctgcctgg ctgtggcttc tcctgtccct
tctgtcgctc cctctgggcc tcccagtcct gggctcctct tcctcaaagg cccctccccc
gagccttcca agtccatccc gactcccggg gccctcggac accccaatat taccacaagc
cccaccacgc ctcatctgtg acagccgagt cctggagagg tacctcttgg aggccaagga
ggccgagaat atcacgacgg gctgtgctga acactgcagc ttgaatgaga atatcactgt
cccagacacc aaagttaatt tctatgcctg gaagaggatg gaggtcgggc agcaggccgt
agaagtctgg cagggcctgg ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt
ggtcaactct tcccagccgt gggagcccct gcagctgcat gtggataaag ccgtcagtgg
ccttcgcagc ctcaccactc tgcttcgggc tctgggagcc cagaaggaag ccatctcccc
tccagatgcg gcctcagctg ctccactccg aacaatcact gctgacactt tccgcaaact
cttccgagtc tactccaatt tcctccgggg aaagctgaag ctgtacacag gggaggcctg
caggacaggg gacagatcct cttcctcaaa ggcccctccc ccgagccttc caagtccatc
ccgactcccg gggccctccg acacaccaat cctgccacag agcagctcct ctaaggcccc
tcctccatcc ctgccatccc cctcccggct gcctggcccc tctgacaccc ctatcctgcc
tcagtgatga aggtcttctg gatccgcggc cgc.
[0104] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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 disclosed
herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture an EPO peptide set forth in
SEQ ID NO: 8 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 disclosed herein manufacture an EPO peptide set forth in
SEQ ID NO: 7 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 disclosed herein manufacture a CTP-modified EPO having the
amino acid sequence set forth in SEQ ID NO: 109. In another
embodiment, a CTP-modified EPO having the amino acid sequence set
forth in SEQ ID NO: 109 is for inhibiting anemia. In another
embodiment, a CTP-modified EPO having the amino acid sequence set
forth in SEQ ID NO: 109 is for treating anemia.
[0105] In another embodiment, the methods disclosed herein utilize
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 disclosed herein utilize 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 disclosed herein utilize a nucleic acid set forth in SEQ ID
NO: 17 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 disclosed herein utilize a nucleic acid set forth in SEQ ID
NO: 18 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.
[0106] In another embodiment, the methods disclosed herein
manufacture 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
disclosed herein manufacture 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 disclosed herein
manufacture 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 disclosed herein manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture an EPO peptide
set forth in SEQ ID NO: 8 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
disclosed herein manufacture an EPO peptide set forth in SEQ ID NO:
7 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, a CTP-modified EPO having the amino acid sequence set
forth in SEQ ID NO: 109 is for treating tumor associated
anemia.
[0107] In another embodiment, the methods disclosed herein utilize
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
disclosed herein utilize 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 disclosed herein utilize a nucleic acid set forth in
SEQ ID NO: 17 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
disclosed herein utilize a nucleic acid set forth in SEQ ID NO: 18
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.
[0108] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein
manufacture 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 disclosed herein manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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 disclosed herein manufacture an EPO peptide set forth in
SEQ ID NO: 8 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 disclosed herein manufacture an EPO
peptide set forth in SEQ ID NO: 7 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, a CTP-modified EPO having the amino
acid sequence set forth in SEQ ID NO: 109 is for inhibiting tumor
associated anemia.
[0109] In another embodiment, the methods disclosed herein utilize
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
disclosed herein utilize 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 disclosed herein utilize a nucleic acid set forth in SEQ ID
NO: 17 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 disclosed herein utilize a nucleic acid set
forth in SEQ ID NO: 18 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.
[0110] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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
disclosed herein manufacture an EPO peptide set forth in SEQ ID NO:
8 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 disclosed herein manufacture an EPO peptide
set forth in SEQ ID NO: 7 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, a CTP-modified EPO having the amino acid
sequence set forth in SEQ ID NO: 109 is for treating tumor
hypoxia.
[0111] In another embodiment, the methods disclosed herein utilize
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 disclosed herein
utilize 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 disclosed herein
utilize a nucleic acid set forth in SEQ ID NO: 17 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
disclosed herein utilize a nucleic acid set forth in SEQ ID NO: 18
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.
[0112] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein
manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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
disclosed herein manufacture an EPO peptide set forth in SEQ ID NO:
8 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 disclosed herein manufacture an EPO peptide
set forth in SEQ ID NO: 7 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, a CTP-modified EPO having the
amino acid sequence set forth in SEQ ID NO: 109 is for the
treatment of chronic infections such as HIV, inflammatory bowel
disease or septic episodes.
[0113] In another embodiment, the methods disclosed herein utilize
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 disclosed
herein utilize 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 disclosed
herein utilize a nucleic acid set forth in SEQ ID NO: 17 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
disclosed herein utilize a nucleic acid set forth in SEQ ID NO: 18
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.
[0114] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture an EPO
peptide set forth in SEQ ID NO: 8 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 disclosed herein manufacture an EPO
peptide set forth in SEQ ID NO: 7 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, a CTP-modified EPO having the amino acid
sequence set forth in SEQ ID NO: 109 is for the inhibition of
chronic infections such as HIV, inflammatory bowel disease or
septic episodes.
[0115] In another embodiment, the methods disclosed herein utilize
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 disclosed herein
utilize 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 disclosed herein utilize a nucleic acid set
forth in SEQ ID NO: 17 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 disclosed herein utilize a nucleic acid set
forth in SEQ ID NO: 18 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.
[0116] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed
herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed
herein manufacture 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 disclosed herein manufacture an EPO
peptide set forth in SEQ ID NO: 8 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 disclosed herein manufacture an EPO peptide set forth in
SEQ ID NO: 7 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, a CTP-modified EPO
having the amino acid sequence set forth in SEQ ID NO: 109 is for
the treatment of fatigue syndrome following cancer
chemotherapy.
[0117] In another embodiment, the methods disclosed herein utilize
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 disclosed herein utilize 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 disclosed
herein utilize a nucleic acid set forth in SEQ ID NO: 17 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 disclosed herein utilize a nucleic
acid set forth in SEQ ID NO: 18 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.
[0118] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein manufacture 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 engraftment. In another embodiment, the methods
disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein
manufacture 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
disclosed herein manufacture an EPO peptide set forth in SEQ ID NO:
8 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 disclosed herein manufacture an EPO peptide
set forth in SEQ ID NO: 7 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, a CTP-modified EPO having the amino acid
sequence set forth in SEQ ID NO: 109 is for improving stem cell
engraftment.
[0119] In another embodiment, the methods disclosed herein utilize
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 disclosed
herein utilize 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 engraftment. In another embodiment, the methods disclosed
herein utilize a nucleic acid set forth in SEQ ID NO: 17 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 disclosed
herein utilize a nucleic acid set forth in SEQ ID NO: 18 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.
[0120] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein manufacture 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
disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture 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 disclosed herein manufacture an EPO
peptide set forth in SEQ ID NO: 8 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 disclosed herein manufacture an EPO
peptide set forth in SEQ ID NO: 7 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, a CTP-modified EPO having the amino acid
sequence set forth in SEQ ID NO: 109 is for increasing th survival
rate of a patient with aplastic anemia or myelodysplastic
syndrome.
[0121] In another embodiment, the methods disclosed herein utilize
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 disclosed herein
utilize 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 disclosed herein
utilize a nucleic acid set forth in SEQ ID NO: 17 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
disclosed herein utilize a nucleic acid set forth in SEQ ID NO: 18
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.
[0122] In another embodiment, the cytokine further comprises a
signal peptide for its secretion. In some embodiments, signal
sequences include, but are not limited to the endogenous signal
sequence for IFN. In some embodiments, signal sequences include,
but are not limited to the endogenous signal sequence of any known
cytokine. In another embodiment, the polypeptides and methods
disclosed herein manufacture a cytokine having additionally a
signal peptide of SEQ ID NO: 19 and at least one CTP peptide on the
N-terminus and at least one CTP peptide on the C-terminus. In
another embodiment, the polypeptides and methods disclosed herein
manufacture a cytokine having additionally on the N-terminus the
signal peptide of SEQ ID NO: 19 and at least one CTP peptide on the
N-terminus and at least two CTP peptides on the C-terminus. In
another embodiment, the polypeptides and methods disclosed herein
manufacture a cytokine having additionally on the N-terminus the
signal peptide of SEQ ID NO: 19 and a single CTP peptide on the
N-terminus and two CTP peptides on the C-terminus. In another
embodiment, SEQ ID NO: 19 comprise the following amino acid (AA)
sequence: MTNKCLLQIALLLCFSTTALS (SEQ ID NO: 19).
[0123] In another embodiment, the cytokine is an interferon. In
some embodiments, interferon is utilized according to the teachings
disclosed herein. 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.
[0124] A skilled artisan would appreciate that the term
"interferon" may encompass a mammalian interferon polypeptide Type
I. In another embodiment, "interferon" comprises 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.2a. In one embodiment, the
interferon (IFN) subspecies is IFN-.beta.2b.
[0125] In one embodiment, interferon disclosed herein exhibits
interferon activity, such as antiviral or antiproliferative
activity. In some embodiments, non-limiting examples of interferons
are listed in Table 1 below.
[0126] A skilled artisan would appreciate that the term
"interferon" may also encompass homologues. In one embodiment,
interferon amino acid sequence disclosed herein is at least 50%
homologous to interferon sequences listed in Table 1 as determined
using BlastP software of the National Center of Biotechnology
Information (NCBI) using default parameters). In another
embodiment, interferon amino acid sequence disclosed herein is at
least 60%, is at least 70%, is at least 80%, is at least 90%, or is
at least 95% homologous interferon sequences listed in Table 1 as
determined using BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters). A
skilled artisan would appreciate that homology may 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: 20). In
one embodiment, SEQ ID NO: 20 comprises the amino acid (AA)
sequence:
TABLE-US-00012 MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNIDIPEEIKQLQQ
FQKEDAALTIYEMLQNIFAIPRQDSSSTGWNETIVENLLANVYHQINHL
KTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVR
VEILRNFYFINRLTGYLRN.
[0127] Table 1 below lists examples of interferons with their
respective NCBI sequence numbers.
TABLE-US-00013 TABLE 1 Interferon name interferon, .alpha. 1
interferon, .alpha. 10 interferon, .alpha. 13 interferon, .alpha.
14 interferon, .alpha. 16 interferon, .alpha. 17 interferon,
.alpha. 2 interferon, .alpha. 21 interferon, .alpha. 4 interferon,
.alpha. 5 interferon, .alpha. 6 interferon, .alpha. 7 interferon,
.alpha. 8 interferon, .beta. 1 interferon, .epsilon.1 interferon,
.gamma. interferon, .epsilon. interferon, .OMEGA.1
[0128] In another embodiment, a method of treating or reducing a
disease treatable or reducible by a cytokine or a pharmaceutical
formulation comprising the same, in a subject, comprises the step
of administering to a subject a therapeutically effective amount of
the polypeptide comprising a cytokine and CTP units as described
herein, thereby treating or reducing a disease treatable or
reducible by a cytokine in a subject.
[0129] In another embodiment, the methods disclosed herein
manufacture 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 disclosed
herein manufacture 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 disclosed herein
manufacture an interferon beta 1 peptide set forth in SEQ ID NO: 20
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 disclosed herein manufacture an interferon beta 1
peptide set forth in SEQ ID NO: 20 having additionally on the
N-terminus the signal peptide of SEQ ID NO: 19 and at least one CTP
amino acid peptide on the N-terminus of SEQ ID NO: 19 and at least
one CTP amino acid peptide on the C-terminus of SEQ ID NO: 20 for
treating or inhibiting multiple sclerosis.
[0130] In another embodiment, the cytokine as described herein
comprises a cytokine and at least three CTP units. In another
embodiment, the polypeptide as described herein comprises an
interferon (IFN) peptide and three CTP units. In another
embodiment, the cytokine as described herein comprises an
interferon (IFN) peptide--CTP polypeptide encoded by an amino acid
sequence comprising the amino acid sequence set forth in SEQ ID NO:
13. In another embodiment, SEQ ID NO: 13 comprises the following
amino acid (AA) sequence:
TABLE-US-00014 (SEQ ID NO: 13, MOD-9011)
MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRL
EYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSST
GWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYY
GRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNSSSSKAPPP
SLPSPSRLPGPSDTPILPQ.
[0131] In another embodiment, the cytokine as described herein
comprising an interferon (IFN) peptide--and CTP is encoded by a
nucleic acid molecule set forth in SEQ ID NO: 22. In another
embodiment, SEQ ID NO: 22 comprises the following nucleotide acid
(NA) sequence:
tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcncagcaccaccgccctgagca-
tgagctacaacctgctgg
gcttcctgcagaggtccagcaacttccagtgccagaagctgctgtggcagctgaacggcaggctggaatactg-
cctgaaggacaggatgaa
cttcgacatcccagaggaaatcaagcagctgcagcagttccagaaggaggacgccgccctgaccatctacgag-
atgctgcagaacatatc
gccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctgctggccaacgtgt-
accaccagatcaaccac
ctgaaaaccgtgctggaagagaagctggaaaaggaggacttcaccaggggcaagctgatgagcagcctgcacc-
tgaagaggtactacgg
cagaatcctgcactacctgaaggccaaggagtacagccactgcgcctggaccatcgtgagggtggagatcctg-
aggaacttctacttcatca
acaggctgaccggctacctgaggaacagctccagcagcaaggcccctccaccttccctgcccagtccaagccg-
actccctgggccctccg atacaccaattctgccacagtgatga (SEQ ID NO: 22,
MOD-9011).
[0132] In another embodiment, the cytokine as described herein
comprises an interferon (IFN) peptide and two CTP units attached to
its carboxy terminus. In another embodiment, the polypeptide as
described herein comprises an interferon (IFN) peptide--CTP (x2)
encoded by an amino acid sequence comprising the amino acid
sequence set forth in SEQ ID NO: 12. In another embodiment, SEQ ID
NO: 12 comprises the following amino acid (AA) sequence:
TABLE-US-00015 (SEQ ID NO: 12, MOD-9012)
MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRL
EYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSST
GWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYY
GRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNSSSSKAPPP
SLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ.
[0133] In another embodiment, the cytokine as described herein
comprising an interferon (IFN) peptide--and two CTP units attached
to its carboxy terminus is encoded by a nucleic acid molecule set
forth in SEQ ID NO: 23. In another embodiment, SEQ ID NO: 23
comprises the following nucleotide acid (NA) sequence:
TABLE-US-00016 (SEQ ID NO: 23, MOD-9012)
tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctg
tgcttcagcaccaccgccctgagcatgagctacaacctgctgggcttcc
tgcagaggtccagcaacttccagtgccagaagctgctgtggcagctgaa
cggcaggctggaatactgcctgaaggacaggatgaacttcgacatccca
gaggaaatcaagcagctgcagcagttccagaaggaggacgccgccctga
ccatctacgagatgctgcagaacatcttcgccatcttcaggcaggacag
cagcagcaccggctggaacgagaccatcgtggagaacctgctggccaac
gtgtaccaccagatcaaccacctgaaaaccgtgctggaagagaagctgg
aaaaggaggacttcaccaggggcaagctgatgagcagcctgcacctgaa
gaggtactacggcagaatcctgcactacctgaaggccaaggagtacagc
cactgcgcctggaccatcgtgagggtggagatcctgaggaacttctact
tcatcaacaggctgaccggctacctgaggaacagctccagcagcaaggc
ccctccaccttccctgcccagtccaagccgactccctgggccctccgac
acaccaatcctgccacagagcagctcctctaaggcccctcctccatccc
tgccatccccctcccggctgcctggcccctctgacacccctatcctgcc
tcagtgatgaaggtctggatccgcggccgc.
[0134] In another embodiment, the cytokine as described herein
comprises an interferon (IFN) peptide, a single CTP unit attached
to the IFN's amino terminus, and two CTP units attached to the
IFN's carboxy terminus. In another embodiment, the polypeptide as
described herein comprises an interferon (IFN) peptide, a single
CTP unit attached to the IFN's amino terminus and two CTP units
attached in tandem to the IFN's carboxy terminus. In another
embodiment, the polypeptide as described herein comprises (from
amino to carboxy termini): CTP (x1)-interferon (IFN) peptide--CTP
(x2) comprising an amino acid sequence set forth in SEQ ID NO: 9.
In another embodiment, SEQ ID NO: 9 comprises the following amino
acid (AA) sequence:
TABLE-US-00017 (SEQ ID NO: 9, MOD-9013)
MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQ
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQ
FQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHL
KTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVR
VEILRNFYFINRLTGYLRNSSSSKAPPPSLPSPSRLPGPSDTPILPQSS
SSKAPPPSLPSPSRLPGPSDTPILPQ.
[0135] In another embodiment, the cytokine as described herein
comprising an interferon (IFN) peptide, a single CTP unit attached
to the IFN's amino terminus and two CTP units attached to the IFN's
carboxy terminus is encoded by a nucleic acid molecule set forth in
SEQ ID NO: 24. In another embodiment, SEQ ID NO: 24 comprises the
following nucleotide acid (NA) sequence:
TABLE-US-00018 (SEQ ID NO: 24, MOD-9013)
tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctg
tgcttcagcaccaccgccctgagcagcagcagctccaaggccccacccc
ccagcctgcccagccccagcagactgccaggccccagcgacacccccat
cctgccccagatgagctacaacctgctgggcttcctgcagaggtccagc
aacttccagtgccagaagctgctgtggcagctgaacggcaggctggaat
actgcctgaaggacaggatgaacttcgacatcccagaggaaatcaagca
gctgcagcagttccagaaggaggacgccgccctgaccatctacgagatg
ctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggct
ggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagat
caaccacctgaaaaccgtgctggaagagaagctggaaaaggaggacttc
accaggggcaagctgatgagcagcctgcacctgaagaggtactacggca
gaatcctgcactacctgaaggccaaggagtacagccactgcgcctggac
catcgtgagggtggagatcctgaggaacttctacttcatcaacaggctg
accggctacctgaggaacagctccagcagcaaggcccctccaccttccc
tgcccagtccaagccgactccctgggccctccgacacaccaatcctgcc
acagagcagctcctctaaggcccctcctccatccctgccatccccctcc
cggctgcctggcccctctgacacccctatcctgcctcagtgatgaaggt
ctggatccgcggccgc.
[0136] In another embodiment, the cytokine as described herein
comprises an interferon (IFN) peptide, a single CTP attached to the
IFN's amino terminus, and a single CTP located within an IFN coding
sequence. In another embodiment, the polypeptide as described
herein comprises (from amino to carboxy termini): CTP
(x1)-interferon (IFN) peptide (fragment 1)--CTP-interferon (IFN)
peptide (fragment 2) comprising an amino acid sequence set forth in
SEQ ID NO: 25. In another embodiment, SEQ ID NO: 25 comprises the
following amino acid (AA) sequence:
TABLE-US-00019 (SEQ ID NO: 25, MOD-9014)
MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQ
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQ
FQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHL
KTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVR
VEILRNFYFINRLTGYLRNSSSSKAPPPSLPSPSRLPGPSDTPILPQMS
YNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQ
KEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKT
VLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVE
ILRNFYFINRLTGYLRN.
[0137] In another embodiment, the cytokine as described herein
comprising an interferon (IFN) peptide, a single CTP unit attached
to the IFN's amino terminus, and a single CTP unit located within
the IFN coding sequence is encoded by a nucleic acid molecule set
forth in SEQ ID NO: 26. In another embodiment, SEQ ID NO: 26
comprises the following nucleotide acid (NA) sequence:
TABLE-US-00020 (SEQ ID NO: 26, MOD-9014)
tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctg
tgcttcagcaccaccgccctgagcagcagcagctccaaggccccacccc
ccagcctgcccagccccagcaggctgccaggccccagcgacacccccat
cctgccccagatgagctacaacctgctgggcttcctgcagaggtccagc
aacttccagtgccagaaactgctgtggcagctgaacggcaggctggaat
actgcctgaaggaccggatgaacttcgacatccccgaagagatcaagca
gctgcagcagttccagaaagaggacgccgccctgaccatctacgagatg
ctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggct
ggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagat
caaccacctgaaaaccgtgctggaagagaagctggaaaaagaggacttc
accaggggcaagctgatgagcagcctgcacctgaagaggtactacggca
gaatcctgcactacctgaaggccaaagagtacagccactgcgcctggac
catcgtgagggtggagatcctgcggaacttctacttcatcaacaggctg
accggctacctgaggaacagctccagcagcaaggcccctccaccctccc
tgccctccccaagcagactgcccggaccctccgacacaccaattctgcc
acagatgtcctacaatctgctcggatttctgcagcgctcctccaacttt
cagtgtcagaagctcctctggcagctcaatggccgcctggaatattgtc
tgaaagacagaatgaattttgacatcccagaggaaattaaacagctcca
gcagtttcagaaagaagatgctgctctcacaatctatgaaatgctccag
aatatctttgcaatctttcgccaggacagctcctccaccgggtggaatg
agacaattgtcgagaatctgctcgccaatgtctatcatcagatcaatca
cctcaagacagtcctcgaagaaaaactcgaaaaagaagatttcacacgc
ggcaaactgatgtcctccctgcatctgaagcgctactatgggcgcatcc
tgcattatctgaaagctaaagaatactcccactgtgcttggacaattgt
gcgcgtcgagatcctgagaaacttttatttcattaaccgcctgacagga
tacctgcgcaactgatgaaggtctggatgcggccgc.
[0138] In another embodiment, the cytokine as described herein
comprises an interferon (IFN) peptide and a single CTP unit
attached to its amino terminus. In another embodiment, the
polypeptide as described herein comprises an interferon (IFN)
peptide--CTP comprising an amino acid sequence set forth in SEQ ID
NO: 11. In another embodiment, SEQ ID NO: 11 comprises the
following amino acid (AA) sequence:
TABLE-US-00021 (SEQ ID NO: 11, MOD-9015)
MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQ
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQ
FQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHL
KTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVR
VEILRNFYFINRLTGYLRN*.
[0139] In another embodiment, the polypeptide as described herein
comprising an interferon (IFN) peptide--and a single CTP attached
to its amino terminus is encoded by a nucleic acid molecule set
forth in SEQ ID NO: 27. In another embodiment, SEQ ID NO: 27
comprises the following nucleotide acid (NA) sequence:
TABLE-US-00022 (SEQ ID NO: 27, MOD-9015)
tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctg
tgcttcagcaccaccgccctgagcagcagcagctccaaggccccacccc
ccagcctgcccagccccagcaggctgccaggccccagcgacacccccat
cctgccccagatgagctacaacctgctgggcttcctgcagaggtccagc
aacttccagtgccagaaactgctgtggcagctgaacggcaggctggaat
actgcctgaaggaccggatgaacttcgacatccccgaagagatcaagca
gctgcagcagttccagaaagaggacgccgccctgaccatctacgagatg
ctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggct
ggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagat
caaccacctgaaaaccgtgctggaagagaagctggaaaaagaggacttc
accaggggcaagctgatgagcagcctgcacctgaagaggtactacggca
gaatcctgcactacctgaaggccaaagagtacagccactgcgcctggac
catcgtgagggtggagatcctgcggaacttctacttcatcaacaggctg
accggctacctgaggaactgatgagtccgcggccgc.
[0140] In another embodiment, the polypeptide as described herein
comprises an interferon (IFN) peptide, a single CTP unit attached
to its amino terminus, and a single CTP unit attached to its
carboxy terminus. In another embodiment, the polypeptide as
described herein comprises an interferon (IFN) peptide--CTP
comprising an amino acid sequence set forth in SEQ ID NO: 10. In
another embodiment, SEQ ID NO: 10 comprises the following amino
acid (AA) sequence:
TABLE-US-00023 (SEQ ID NO: 10, MOD-9016)
MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQ
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNHAPEEIKQLQQF
QKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLK
TVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRV
EILRNFYFINRLTGYLRNSSSSKAPPPSLPSPSRLPGPSDTPILPQ.
[0141] In another embodiment, the cytokine as described herein
comprising an interferon (IFN) peptide, a single CTP unit attached
to its amino terminus, and a single CTP unit attached to its
carboxy terminus is encoded by a nucleic acid molecule set forth in
SEQ ID NO: 28. In another embodiment, SEQ ID NO: 28 comprises the
following nucleotide acid (NA) sequence:
TABLE-US-00024 (SEQ ID NO: 28, MOD-9016)
tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctg
tgcttcagcaccaccgccctgagcagcagcagctccaaggccccacccc
ccagcctgcccagccccagcagactgccaggccccagcgacacccccat
cctgccccagatgagctacaacctgctgggcttcctgcagaggtccagc
aacttccagtgccagaagctgctgtggcagctgaacggcaggctggaat
actgcctgaaggacaggatgaacttcgacatcccagaggaaatcaagca
gctgcagcagttccagaaggaggacgccgccctgaccatctacgagatg
ctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggct
ggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagat
caaccacctgaaaaccgtgctggaagagaagctggaaaaggaggacttc
accaggggcaagctgatgagcagcctgcacctgaagaggtactacggca
gaatcctgcactacctgaaggccaaggagtacagccactgcgcctggac
catcgtgagggtggagatcctgaggaacttctacttcatcaacaggctg
accggctacctgaggaacagctccagcagcaaggcccctccaccttccc
tgcccagtccaagccgactccctgggccctccgatacaccaattctgcc
acagtgatgaaggtctggatgcggccgc.
[0142] In another embodiment, an interferon .beta. peptide
comprises SEQ ID NO: 29 comprising the following amino acid (AA)
sequence:
TABLE-US-00025 (SEQ ID NO: 29)
MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQ
FQKEDAALTIYEMLQNWAIFRQDSSSTGWNETIVENLLANVYHQINHLK
TVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRV
EILRNFYFINRLTGYLRN.
[0143] In another embodiment, the polypeptide of interest comprises
a cytokine 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 cytokine having additionally at least
one CTP amino acid peptide on the N-terminus and two CTP amino acid
peptides on the C-terminus is selected from lymphokines, monokines,
chemokine, and interleukin. In another embodiment, the cytokine
comprises an autocrine action activity. In another embodiment, the
cytokine comprises a paracrine action activity. In another
embodiment, the cytokine comprises an endocrine action
activity.
[0144] In some embodiments, glucagon-like peptide-1 is utilized
according to the teachings disclosed herein. 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.
[0145] A skilled artisan would appreciate that the
term"glucagon-like peptide-1" (GLP-1) may encompass a mammalian
polypeptide. Further, the skilled artisan would appreciate that the
term "glucagon-like peptide-1" (GLP-1) may encompass a human
polypeptide. In some embodiments, GLP-1 is cleaved from a glucagon
preproprotein that has the ability to bind to the GLP-1 receptor
and initiate a signal transduction pathway resulting in
insulinotropic activity. A skilled artisan would appreciate that
the term "insulinotropic activity" may encompass 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.
[0146] In one embodiment, the amino acid sequence of GLP-1
comprises the sequence set forth in SEQ ID NO: 21:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR. A skilled artisan would recognize
that a GLP-1 disclosed herein may also encompass a GLP-1 homologue.
In one embodiment, GLP-1 amino acid sequence disclosed herein is at
least 50% homologous to GLP-1 sequences set forth in SEQ ID NO: 21
as determined using BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters). In
another embodiment, GLP-1 amino acid sequence disclosed herein is
at least 60% homologous to GLP-1 sequences set forth in SEQ ID NO:
21 as determined using BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters), is at
least 70% homologous to GLP-1 sequences set forth in SEQ ID NO: 21,
is at least 80% homologous to GLP-1 sequences set forth in SEQ ID
NO: 21, is at least 90% homologous to GLP-1 sequences set forth in
SEQ ID NO: 21, is at least 95% homologous to GLP-1 sequences set
forth in SEQ ID NO: 21 as determined using BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters).
[0147] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein
manufacture 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 disclosed herein manufacture a GLP-1
peptide set forth in SEQ ID NO: 21 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.
[0148] In one embodiment, disclosed herein are long-acting
coagulation factors and methods of producing and using same. In
another embodiment, long-acting coagulation factors comprise a
carboxy terminal peptide (CTP, also referred to as CTP unit). In
another embodiment, long-acting polypeptides which comprise a
coagulation factor further comprise a carboxy terminal peptide
(CTP) of human Chorionic Gonadotropin (hCG). In another embodiment,
CTP acts as a protectant against the degradation of a coagulation
factor. In another embodiment, CTP extends the C.sub.max of a
coagulation factor. In another embodiment, CTP extends the
T.sub.max of a coagulation factor. In another embodiment, CTP
extends the circulatory half-life of a coagulation factor. In some
embodiments, CTP enhances the potency of a coagulation factor.
[0149] In another embodiment, disclosed herein is a method of
extending the biological half-life of a coagulation factor,
comprising the step of attaching one to ten CTPs to the carboxy
terminus of the coagulation factor, thereby extending the
biological half-life of the coagulation factor. In another
embodiment, disclosed herein is a method of extending the
biological half-life of a coagulation factor, comprising the step
of attaching one to five CTPs to the carboxy terminus of the
coagulation factor, thereby extending the biological half-life of
the coagulation factor. In another embodiment, disclosed herein is
a method for extending the circulatory half-life of a coagulation
factor. In another embodiment, disclosed herein is a method for
increasing the half-life of a coagulation factor. In another
embodiment, disclosed herein is a method for extending the
half-life of a coagulation factor.
[0150] Coagulation Factor VII (FVII) is a 444 amino acid
glycoprotein (50 KDa) secreted by hepatocytes into the bloodstream
as an inactive pro-enzyme. Upon tissue injury and exposure to
circulating blood, FVII forms a complex with Tissue Factor (TF)
which is a true receptor protein to FVII and is expressed by
various cells localized in the deeper layers of the vessel wall.
The formation of this FVII-TF complex leads to activation of FVII.
Activated FVII (FVIIa) initiates the extrinsic coagulation pathway
by activating Factor IX and Factor X.
[0151] FVII belong to a group of Vitamin K-dependent glycoproteins
associated with the coagulation system. Besides FVII, this group
consists of Factor IX, Factor X, Protein C and prothrombin. These
proteins have similar domain organizations and are synthesized as
precursors with an N-terminal propeptide followed by a mature amino
acid sequence. The propeptide contains a docking site for gamma
carboxylase which converts glutamic acids (Glu) into gamma carboxy
glutamic acids (Gla). This domain is followed by two epidermal
growth factor-like (EGF) domains, a connecting region (CR) and a
C-terminal serine protease domain Prior to secretion, FVII
propeptide is cleaved forming a 406 amino acid single chain zymogen
FVII glycoprotein. After secretion, the protein can be activated
into a disulfide-linked two chain heterodimer, FVIIa, by cleavage
in the CR. The plasma concentration of FVII is 10 nM and
approximately 1% circulates in the active form in healthy
individuals.
[0152] Factor IX (FIX) is a 415 Amino acid (55 KDa) glycoprotein;
it belongs to a group of vitamin K dependent glycoproteins
associated with the coagulation system. FIX has a similar domain
organization as factor FVII, Factor X, Protein C and prothrombin
that are synthesized as precursors with an N-terminal propeptide
followed by a mature amino acid sequence.
[0153] FIX is secreted as a single chain molecule that undergoes
complex post-transcriptional modifications, many of which are
critical to its biochemical and pharmacokinetic properties. Among
all the post-transcriptional modifications, 12 glutamic acid
residues near the amino terminus of FIX that are gamma carboxylated
by the vitamin K-dependent gamma carboxylase are the most crucial
ones. Carboxylation is required for the interaction of FIX with the
phospholipid surfaces and for optimal FIX activity. The amino
terminus propeptide serves as a recognition site for the gamma
carboxylase and thus, following gamma carboxylation, it is cleaved
off by the Golgi apparatus serine protease known as Paired basic
Amino acid Cleave Enzyme (PACE/Furin). Four additional
post-transcriptional modifications might occur at the Golgi
apparatus: sulfation of tyrosine 155, phosphorylation of serine
158, O-glycosylation on Ser 63 and on 61 and finally,
N-glycosylation on Asn 157 and 16, but were shown not to be
necessary for proper activity of FIX.
[0154] FIX circulates in the plasma (average concentration of 5
.mu.g/ml) as a single chain inactive zymogen. Upon proteolytic
cleavage at two peptide bonds: Arg 145 and Arg 180 by either one or
two physiological activators, FVIIa-TF complex or FIXa, the
activation peptide is removed, converting FIX to a fully active
enzyme consisting of a light and heavy chain held together by a
single disulfide bond. The N-terminal light chain contains the
non-catalytic gamma carboxyglutamic acid (Gla) and two epidermal
growth factor-like domains, while the C-terminal heavy chain
contains the trypsin-like catalytic domain of the molecule. FIXa
alone is characterized by poor catalytic activity. However when
complexed with FVIII, its proteolytic activity increase by 4-5
orders of magnitude towards its natural substrate FX.
[0155] In another embodiment, disclosed herein is a method of
extending the biological half-life or a method of improving the
area under the curve (AUC) of a coagulation factor, comprising the
step of attaching one to ten CTPs to the carboxy terminus of the
coagulation factor, thereby extending the biological half-life or
improving the AUC of the coagulation factor. In another embodiment,
disclosed herein is a method of extending the biological half-life
or a method of improving the area under the curve (AUC) of a
coagulation factor, comprising the step of attaching one to five
CTPs to the carboxy terminus of the coagulation factor, thereby
extending the biological half-life or improving the AUC of the
coagulation factor. In another embodiment, disclosed herein is a
method of extending the biological half-life or a method of
improving the area under the curve (AUC) of FIX, comprising the
step of attaching one to five CTPs to the carboxy terminus of the
FIX, thereby extending the biological half-life or improving the
AUC of the FIX. In another embodiment, disclosed herein is a method
of extending the biological half-life or a method of improving the
area under the curve (AUC) of FVII or FVIIa, comprising the step of
attaching one to five CTPs to the carboxy terminus of FVII or
FVIIa, thereby extending the biological half-life or improving the
AUC of FVII or FVIIa.
[0156] In another embodiment, disclosed herein is a method of
extending the biological half-life of a Factor IX (FIX)
polypeptide, comprising the step of attaching three chorionic
gonadotropin carboxy terminal peptides (CTPs) to the carboxy
terminus of said FIX polypeptide, thereby extending the biological
half-life of said FIX polypeptide. In another embodiment a method
of extending the biological half-life of a Factor Vita (FVIIa)
polypeptide, comprises the step of attaching up to five chorionic
gonadotropin carboxy terminal peptides (CTPs) to the carboxy
terminus of said FVIIa polypeptide, thereby extending the
biological half-life of said FVIIa polypeptide. In one embodiment,
three chorionic gonadotropin carboxy terminal peptides (CTPs) are
attached to the carboxy terminus of said FVIIa polypeptide. In
another embodiment, four chorionic gonadotropin carboxy terminal
peptides (CTPs) are attached to the carboxy terminus of said FVIIa
polypeptide. In another embodiment, five chorionic gonadotropin
carboxy terminal peptides (CTPs) are attached to the carboxy
terminus of said FVIIa polypeptide.
[0157] In another embodiment, disclosed herein is a method of
improving the area under the curve (AUC) of a Factor IX (FIX)
polypeptide, comprising the step of attaching three chorionic
gonadotropin carboxy terminal peptides (CTPs) to the carboxy
terminus of said FIX polypeptide, thereby improving the AUC of said
FIX polypeptide. In another embodiment, disclosed herein is a
method of improving the area under the curve (AUC) of a Factor Vita
(FVIIa) polypeptide, comprising the step of attaching up to five
chorionic gonadotropin carboxy terminal peptides (CTPs) to the
carboxy terminus of said FVIIa polypeptide, thereby improving the
AUC of said FVIIa polypeptide. In one embodiment, three chorionic
gonadotropin carboxy terminal peptides (CTPs) are attached to the
carboxy terminus of said FVIIa polypeptide. In another embodiment,
four chorionic gonadotropin carboxy terminal peptides (CTPs) are
attached to the carboxy terminus of said FVIIa polypeptide. In
another embodiment, five chorionic gonadotropin carboxy terminal
peptides (CTPs) are attached to the carboxy terminus of said FVIIa
polypeptide.
[0158] In another embodiment, a coagulation factor disclosed herein
is a protein. In another embodiment, a coagulation factor disclosed
herein is a peptide. In another embodiment, a coagulation factor
disclosed herein is a polypeptide. In another embodiment, the
coagulation factor is an enzyme. In another embodiment, the
coagulation factor is a serine protease. In another embodiment, the
coagulation factor is a glycoprotein. In another embodiment, the
coagulation factor is a transglutaminase. In another embodiment,
the coagulation factor is an inactive zymogen. In another
embodiment, the coagulation factor is any coagulation factor known
to one of skill in the art.
[0159] In another embodiment, the coagulation factor is Factor VIII
(FVIII). In another embodiment, the coagulation factor is Factor V
(FV). In another embodiment, the coagulation factor is Factor XIII
(FXIII). In another embodiment, the coagulation factor is Factor X
(FX). In another embodiment, the coagulation factor is fibrin.
[0160] In another embodiment, the coagulation factor is Factor VIIa
(FVIIa). In another embodiment, the coagulation factor is Factor
VII (FVII). In another embodiment, the coagulation factor is Factor
IX (FIX). In another embodiment, the coagulation factor is Factor X
(FX). In another embodiment, the coagulation factor is Factor XIa
(FXIa). In another embodiment, the coagulation factor is Factor XII
(FXII). In another embodiment, the coagulation factor is Factor Xa
(FXa). In another embodiment, the coagulation factor is Factor Va
(FVa). In another embodiment, the coagulation factor is
prothrombin. In another embodiment, the coagulation factor is
thrombin. In another embodiment, the coagulation factor is Factor
XI (FXI). In another embodiment, the coagulation factor is Von
Willebrand factor (vWF). In another embodiment, the coagulation
factor is activated Factor VIIIa (FVIIIa). In another embodiment,
the coagulation factor is B-deleted Domain FVIII (FVIIIBDD). In
another embodiment, the coagulation factor is B domain-deleted
FVIII (FVIIIBDD). In another embodiment, the coagulation factor is
Beta domain-deleted FVIII (FVIIIBDD). In another embodiment, the
coagulation factor is Factor IXa (FIXa). In another embodiment, the
coagulation factor is prekallikrein. In another embodiment, the
coagulation factor is kallikrein. In another embodiment, the
coagulation factor is Factor XIIa (FXIIa). In another embodiment,
the coagulation factor is Fibrinogen. In another embodiment, the
coagulation factor is thrombomodulin. In another embodiment, the
coagulation factor is Factor II (FII).
[0161] In another embodiment, the coagulation factor is a
glycoprotein. In another embodiment, the coagulation factor is a
vitamin K-dependent glycoprotein. In another embodiment, the
coagulation factor is a vitamin K-independent glycoprotein.
[0162] In another embodiment, the coagulation factor is a
recombinant protein. In another embodiment, the coagulation factor
is a recombinant glycoprotein. In another embodiment, the
coagulation factor is a recombinant glycoprotein FV. In another
embodiment, the coagulation factor is a recombinant FVI. In another
embodiment, the coagulation factor is a recombinant FVII. In
another embodiment, the coagulation factor is a recombinant FVIII.
In another embodiment, the coagulation factor is a recombinant FIX.
In another embodiment, the coagulation factor is a recombinant FX.
In another embodiment, the coagulation factor is a recombinant FXI.
In another embodiment, the coagulation factor is a recombinant
FXII. In another embodiment, the coagulation factor is a
recombinant FvW. In another embodiment, the coagulation factor is a
recombinant HI. In another embodiment, the coagulation factor is a
recombinant FIXa. In another embodiment, the coagulation factor is
a recombinant FXIa. In another embodiment, the coagulation factor
is a recombinant fibrin. In another embodiment, the coagulation
factor is a recombinant FVIIa. In another embodiment, the
coagulation factor is a recombinant FXa. In another embodiment, the
coagulation factor is a recombinant FVa. In another embodiment, the
coagulation factor is a recombinant prothrombin. In another
embodiment, the coagulation factor is a recombinant thrombin. In
another embodiment, the coagulation factor is a recombinant FVIIIa.
In another embodiment, the coagulation factor is a recombinant
prekallikrein. In another embodiment, the coagulation factor is a
recombinant kallikrein. In another embodiment, the coagulation
factor is a recombinant FXIIa. In another embodiment, the
coagulation factor is any known recombinant coagulation factor. In
another embodiment, the coagulation factor comprising a signal
peptide is any known recombinant coagulation factor.
[0163] In another embodiment, a coagulation factor comprises 1-10
CTP repeats attached to the C-terminus and no CTPs attached to the
N-terminus. In another embodiment, a coagulation factor comprises
at least one CTP attached to the C-terminus and no CTPs attached to
the N-terminus. In another embodiment, a coagulation factor
comprising 1-10 CTP repeats attached to the C-terminus and no CTPs
attached to the N-terminus is an engineered coagulation factor. In
another embodiment, a coagulation factor comprising at least one
CTP attached to the C-terminus and no CTPs attached to the
N-terminus is an engineered coagulation factor. In another
embodiment, a coagulation factor comprising 1-10 CTP repeats
attached to the C-terminus and no CTPs attached to the N-terminus
is a conjugated coagulation factor. In another embodiment, a
coagulation factor comprising at least one CTP attached to the
C-terminus and no CTPs attached to the N-terminus is a conjugated
coagulation factor.
[0164] In one embodiment, disclosed herein is a CTP-modified Factor
IX (FIX) polypeptide consisting of a FIX polypeptide and three
gonadotropin carboxy terminal peptides (CTPs) attached to the
carboxy terminus of said CTP-modified FIX polypeptide.
[0165] In another embodiment, a CTP-modified Factor VIIa (FVIIa)
polypeptide disclosed herein consists of a FVIIa polypeptide and
five gonadotropin carboxy terminal peptides (CTPs) attached to the
carboxy terminus of said FVIIa.
[0166] In another embodiment, the coagulation factor is a
coagulation factor comprising a domain organization similar or
identical to the domain organization of FIX, FVII, Factor X,
Protein C, or prothrombin. In another embodiment, the coagulation
factor is synthesized as a precursor with an N-terminal propeptide.
In another embodiment, the coagulation factor as used herein is in
an inactive pro-enzyme form. In another embodiment, the coagulation
factor is produced in hepatocytes. In another embodiment, the
coagulation factor comprises a docking site for gammacarboxylase
which converts glutamic acids (Glu) into gamma carboxy glutamic
acids (Gla). In another embodiment, the coagulation factor as used
herein is a commercially available coagulation factor.
[0167] In one embodiment, the nucleic acid sequence encoding Factor
VII comprises the following nucleic acid sequence:
TABLE-US-00026 (SEQ ID NO: 30)
ctcgaggacatggtctcccaggccctcaggctcctctgccuctgcttgg
gcttcagggctgcctggctgcagtcttcgtaacccaggaggaagcccac
ggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgc
ggccgggctccctggagagggagtgcaaggaggagcagtgctccttcga
ggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctgg
atttcttacagtgatggggaccagtgtgcctcaagtccatgccagaatg
ggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccc
tgccttcgagggccggaactgtgagacgcacaaggatgaccagctgatc
tgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgg
gcaccaagcgctcctgtcggtgccacgaggggtactctctgctggcaga
cggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacct
attctagaaaaaagaaatgccagcaaaccccaaggccgaattgtggggg
gcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttggt
gaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtg
gtctccgcggcccactgtttcgacaaaatcaagaactggaggaacctga
tcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagca
gagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccgggc
accaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcc
tcactgaccatgtggtgcccctctgcctgcccgaacggacgttctctga
gaggacgctggccttcgtgcgcttctcattggtcagcggctggggccag
ctgctggaccgtggcgccacggccctggagctcatggtcctcaacgtgc
cccggctgatgacccaggactgcctgcagcagtcacggaaggtgggaga
ctccccaaatatcacggagtacatgttctgtgccggctactcggatggc
agcaaggactcctgcaagggggacagtggaggcccacatgccacccact
accggggcacgtggtacctgacgggcatcgtcagctggggccagggctg
cgcaaccgtgggccactttggggtgtacaccagggtctcccagtacatc
gagtggctgcaaaagctcatgcgctcagagccacgcccaggagtcctcc
tgcgagccccatttccctgaggatgcggccgc.
[0168] In another embodiment, the amino acid sequence of Factor VII
comprises the following amino acid sequence:
TABLE-US-00027 (SEQ ID NO: 31)
MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPG
SLERECKEEQCSFEEAREWKDAERTKLFWISYSDGDQCASSPCQNGGSC
KDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKR
SCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVC
PKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVL
GEHDLSEHDGDEQSRRVAQVRPSTYVPGTTNHDIALLRLHQPVVLTDHV
VPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMT
QDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTW
YLTGIVSWGQGCATVGHEGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPF P.
[0169] In another embodiment, the amino acid sequence of Factor VII
comprises the following amino acid sequence:
TABLE-US-00028 (SEQ ID NO: 32)
MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPG
SLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGS
CKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTK
RSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKV
CPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAV
LGEHDLSEHDGDEQSRRVAQVRPSTYVPGTTNHDIALLRLHQPVVLTDH
VVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLM
TQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGT
WYLTGIVSWGQGCATVGHEGVYTRVSQYIEWLQKLMRSEPRPGVLLRAP FP*GCGR.
[0170] In another embodiment, the nucleic acid sequence encoding
Factor VII-CTP (attached to the carboxy terminus) comprises the
following nucleic acid sequence:
TABLE-US-00029 (SEQ ID NO: 33)
ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttg
ggcttcagggctgcctggctgcagtcttcgtaacccaggaggaagccca
cggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctg
cggccgggctccctggagagggagtgcaaggaggagcagtgctccttcg
aggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctg
gatttcttacagtgatggggaccagtgtgcctcaagtccatgccagaat
gggggctcctgcaaggaccagctccagtcctatatctgcttctgcctcc
ctgccttcgagggccggaactgtgagacgcacaaggatgaccagctgat
ctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacg
ggcaccaagcgctcctgtcggtgccacgaggggtactctctgctggcag
acggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacc
tattctagaaaaaagaaatgccagcaaaccccaaggccgaattgtgggg
ggcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttgg
tgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggt
ggtctccgcggcccactgtttcgacaaaatcaagaactggaggaacctg
atcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagc
agagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccggg
caccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtc
ctcactgaccatgtggtgcccctctgcctgcccgaacggacgttctctg
agaggacgctggccttcgtgcgcttctcattggtcagcggctggggcca
gctgctggaccgtggcgccacggccctggagctcatggtcctcaacgtg
ccccggctgatgacccaggactgcctgcagcagtcacggaaggtgggag
actccccaaatatcacggagtacatgttctgtgccggctactcggatgg
cagcaaggactcctgcaagggggacagtggaggcccacatgccacccac
taccggggcacgtggtacctgaccggcatcgtgagctggggccagggct
gcgccaccgtgggccacttcggcgtgtacaccagggtgtcccagtacat
cgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctg
ctgagagcccccttccccagcagcagctccaaggcccctccccctagcc
tgcccagccctagcagactgcctgggcccagcgacacccccatcctgcc
ccagtgaggatccgcggccgc.
[0171] In another embodiment, the amino acid sequence of Factor
VII-CTP (attached to the carboxy terminus) comprises the following
amino acid sequence:
TABLE-US-00030 (SEQ ID NO: 34)
MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPG
SLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGS
CKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTK
RSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKV
CPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAV
LGEHDLSEHDGDEQSRRVAQVIEPSTYVPGTTNHDIALLRLHQPVVLTD
HVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRL
MTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRG
TWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRA
PFPSSSSKAPPPSLPSPSRLPGPSDTPILPQ*.
[0172] In another embodiment, the nucleic acid sequence encoding
Factor VII-CTP-CTP (attached to the carboxy terminus) comprises the
following nucleic acid sequence:
TABLE-US-00031 (SEQ ID NO: 35)
ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttg
ggcttcagggctgcctggctgcagtcttcgtaacccaggaggaagccca
cggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctg
cggccgggctccctggagagggagtgcaaggaggagcagtgctccttcg
aggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctg
gatttcttacagtgatggggaccagtgtgcctcaagtccatgccagaat
gggggctcctgcaaggaccagctccagtcctatatctgcttctgcctcc
ctgccttcgagggccggaactgtgagacgcacaaggatgaccagctgat
ctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacg
ggcaccaagcgctcctgtcggtgccacgaggggtactctctgctggcag
acggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacc
tattctagaaaaaagaaatgccagcaaaccccaaggccgaattgtgggg
ggcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttgg
tgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggt
ggtctccgcggcccactgtttcgacaaaatcaagaactggaggaacctg
atcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagc
agagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccggg
caccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtc
ctcactgaccatgtggtgcccctctgcctgcccgaacggacgttctctg
agaggacgctggccttcgtgcgcttctcattggtcagcggctggggcca
gctgctggaccgtggcgccacggccctggagctcatggtcctcaacgtg
ccccggctgatgacccaggactgcctgcagcagtcacggaaggtgggag
actccccaaatatcacggagtacatgttctgtgccggctactcggatgg
cagcaaggactcctgcaagggggacagtggaggcccacatgccacccac
taccggggcacgtggtacctgaccggcatcgtgagctggggccagggct
gcgccaccgtgggccacttcggcgtgtacaccagggtgtcccagtacat
cgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctg
ctgagagcccccttccccagcagcagctccaaggcccctccccctagcc
tgcccagccctagcagactgcctgggccctccgacacaccaatcctgcc
acagagcagctcctctaaggcccctcctccatccctgccatccccctcc
cggctgccaggcccctctgacacccctatcctgcctcagtgatgaaggt
ctggatccgcggccgc.
[0173] In another embodiment, the amino acid sequence of Factor
VII-CTP-CTP (attached to the carboxy terminus) comprises the
following amino acid sequence:
TABLE-US-00032 (SEQ ID NO: 36)
MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRAN
AFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWI
SYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCE
THKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLA
DGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKG
ECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWR
NLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHD
IALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVS
GWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPN
ITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIV
SWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLR
APFPSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPP PSLPSPSRLPGPSDTPILPQ.
[0174] In one embodiment, a Factor VII-CTP-CTP-CTP is an activated
Factor VII-CTP-CTP-CTP (FVIIa-CTP-CTP-CTP).
[0175] In another embodiment, the nucleic acid sequence encoding
Factor VII-CTP-CTP-CTP (attached to the carboxy terminus) comprises
the following nucleic acid sequence:
TABLE-US-00033 (SEQ ID NO: 37)
ctcgaggacatggtctcccaggccctcaggctcctctgcct
tctgcttgggcttcagggctgcctggctgcagtcttcgta
acccaggaggaagcccacggcgtcctgcaccggcgccggc
gcgccaacgcgacctggaggagctgcggccgggctccctg
gagagggagtgcaaggaggagcagtgctccacgaggaggc
ccgggagatcacaaggacgcggagaggacgaagctgactg
gatttcttacagtgatggggaccagtgtgcctcaagtcca
tgccagaatgggggctcctgcaaggaccagctccagtcct
atatctgcactgcctccctgccacgagggccggaactgtg
agacgcacaaggatgaccagctgatctgtgtgaacgagaa
cggcggctgtgagcagtactgcagtgaccacacgggcacc
aagcgctcctgtcggtgccacgaggggtactctctgctgg
cagacggggtgtcctgcacacccacagttgaatatccatg
tggaaaaatacctattctagaaaaaagaaatgccagcaaa
ccccaaggccgaattgtggggggcaaggtgtgccccaaag
gggagtgtccatggcaggtcctgagaggtgaatggagctc
agagtgtggggggaccctgatcaacaccatctgggtggtc
tccgcggcccactgatcgacaaaatcaagaactggaggaa
cctgatcgcggtgctgggcgagcacgacctcagcgagcac
gacggggatgagcagagccggcgggtggcgcaggtcatca
tccccagcacgtacgtcccgggcaccaccaaccacgacat
cgcgctgctccgcctgcaccagcccgtggtcctcactgac
catgtggtgcccctctgcctgcccgaacggacgactctga
gaggacgctggccacgtgcgcactcattggtcagcggctg
gggccagctgctggaccgtggcgccacggccctggagctc
atggtcctcaacgtgccccggctgatgacccaggactgcc
tgcagcagtcacggaaggtgggagactccccaaatatcac
ggagtacatgactgtgccggctactcggatggcagcaagg
actcctgcaagggggacagtggaggcccacatgccaccca
ctaccggggcacgtggtacctgaccggcatcgtgagctgg
ggccagggctgcgccaccgtgggccacttcggcgtgtaca
ccagggtgtcccagtacatcgagtggctgcagaaactgat
gagaagcgagcccagacccggcgtgctgctgagagccccc
accccagcagcagctccaaggcccctccccctagcctgcc
cagccctagcagactgcctgggcccagtgacacccctatc
ctgcctcagtccagctccagcaaggccccaccccctagcc
tgccactccactcggctgcctggccccagcgatactccaa
ttctgccccagtcctccagcagtaaggctccccctccatc
tctgccatcccccagcagactgccaggcccactgatacac
ccatcctcccacagtgatgaggatccgcggccgcttaatt aa.
[0176] In another embodiment, the amino acid sequence of Factor
VII-CTP-CTP-CTP (attached to the carboxy terminus) comprises the
following amino acid sequence:
TABLE-US-00034 (SEQ ID NO: 38)
MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRAN
AFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWI
SYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCE
THKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLA
DGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKG
ECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWR
NLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHD
IALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVS
GWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPN
ITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIV
SWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLR
APFPSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPP
PSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGP SDTPILPQ.
[0177] In one embodiment, following expression a mature
CTP-modified FVII polypeptide is secreted, the signal peptide
having been cleaved from the precursor protein resulting in a
mature protein. For example, in the precursor CTP-modified FVII
polypeptide set forth in SEQ ID NO: 38, amino acids 1-38:
MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRR (SEQ ID NO: 110) represent
the signal peptide of the precursor CTP-modified FVII polypeptide,
and amino acids:
TABLE-US-00035 (SEQ ID NO: 111)
ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLF
WISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRN
CETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSL
LADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCP
KGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKN
WRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTN
HDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSL
VSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDS
PNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTG
IVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVL
LRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKA
PPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLP GPSDTPILPQ
represent the mature engineered CTP-modified FVII polypeptide
lacking the signal peptide. In one embodiment, the amino acid
sequence of CTP-modified FVII without the signal peptide is set
forth in SEQ ID NO: 111. In another embodiment, the amino acid
sequence of activated CTP-modified FVII without the signal peptide
is set forth in SEQ ID NO: 111. In another embodiment, the signal
peptide of CTP-modified FVII is set forth in SEQ ID NO: 110.
[0178] In another embodiment, the amino acid sequence of activated
Factor VII-CTP-CTP-CTP (attached to the carboxy terminus)
(FVIIa-CTP3) lacks a signal peptide and comprises the amino acid
sequence as put forth in SEQ ID NO: 111. Similarly, while the term
MOD-5014 is interchangeable with the term FVIIa-CTP3, i.e.,
represents the active form of the CTP-modified coagulation factor,
in certain instances the term MOD-5014 may be used to denote an
active form of FVII or a nucleotide sequence encoding a FVII-CTP3,
which will then be expressed and secreted from a cell, and purified
and activated in vitro, resulting in the active form of FVIIa being
present in a MOD-5014 molecule.
[0179] In another embodiment, the nucleic acid sequence encoding
Factor VII-(CTP).sub.4 (attached to the carboxy terminus) comprises
the following nucleic acid sequence:
TABLE-US-00036 (SEQ ID NO: 39)
ctcgaggacatggtctcccaggccctcaggctcctctgcc
ttctgcttgggcttcagggctgcctggctgcagtcacgta
acccaggaggaagcccacggcgtcctgcaccggcgccggc
gcgccaacgcgacctggaggagctgcggccgggctccctg
gagagggagtgcaaggaggagcagtgctccacgaggaggc
ccgggagatcacaaggacgcggagaggacgaagctgactg
gatttcttacagtgatggggaccagtgtgcctcaagtcca
tgccagaatgggggctcctgcaaggaccagctccagtcct
atatctgcactgcctccctgccacgagggccggaactgtg
agacgcacaaggatgaccagctgatctgtgtgaacgagaa
cggcggctgtgagcagtactgcagtgaccacacgggcacc
aagcgctcctgtcggtgccacgaggggtactctctgctgg
cagacggggtgtcctgcacacccacagttgaatatccatg
tggaaaaatacctattctagaaaaaagaaatgccagcaaa
ccccaaggccgaattgtggggggcaaggtgtgccccaaag
gggagtgtccatggcaggtcctgagaggtgaatggagctc
agagtgtggggggaccctgatcaacaccatctgggtggtc
tccgcggcccactgatcgacaaaatcaagaactggaggaa
cctgatcgcggtgctgggcgagcacgacctcagcgagcac
gacggggatgagcagagccggcgggtggcgcaggtcatca
tccccagcacgtacgtcccgggcaccaccaaccacgacat
cgcgctgctccgcctgcaccagcccgtggtcctcactgac
catgtggtgcccctctgcctgcccgaacggacgactctga
gaggacgctggccacgtgcgcactcattggtcagcggctg
gggccagctgctggaccgtggcgccacggccctggagctc
atggtcctcaacgtgccccggctgatgacccaggactgcc
tgcagcagtcacggaaggtgggagactccccaaatatcac
ggagtacatgactgtgccggctactcggatggcagcaagg
actcctgcaagggggacagtggaggcccacatgccaccca
ctaccggggcacgtggtacctgaccggcatcgtgagctgg
ggccagggctgcgccaccgtgggccacttcggcgtgtaca
ccagggtgtcccagtacatcgagtggctgcagaaactgat
gagaagcgagcccagacccggcgtgctgctgagagccccc
accccagcagcagctccaaggcccctccccctagcctgcc
cagccctagcagactgcctgggcccagtgacacccctatc
ctgcctcagtccagctccagcaaggccccaccccctagcc
tgccactccactcggctgcctggccccagcgatactccaa
ttctgccccagtcctccagcagtaaggctccccctccatc
tctgccatcccccagcagactgccaggcccactgatacac
ccatcctcccacagtgatgaggatccgc.
[0180] In another embodiment, the amino acid sequence of Factor
VII-(CTP).sub.4 (attached to the carboxy terminus) comprises the
following amino acid sequence:
TABLE-US-00037 (SEQ ID NO: 40)
LEDMVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRR
RANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKL
FWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGR
NCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYS
LLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVC
PKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIK
NWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTT
NHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSL
VSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDS
PNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTG
IVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVL
LRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKA
PPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLP GPSDTPILPQG
[0181] In another embodiment, the nucleic acid sequence encoding
Factor VII-(CTP).sub.5 (attached to the carboxy terminus) comprises
the following nucleic acid sequence:
TABLE-US-00038 (SEQ ID NO: 41)
ctcgaggacatggtctcccaggccctcaggctcctctgcc
ttctgcttgggcttcagggctgcctggctgcagtcacgta
acccaggaggaagcccacggcgtcctgcaccggcgccggc
gcgccaacgcgacctggaggagctgcggccgggctccctg
gagagggagtgcaaggaggagcagtgctccacgaggaggc
ccgggagatcacaaggacgcggagaggacgaagctgactg
gatacttacagtgatggggaccagtgtgcctcaagtccat
gccagaatgggggctcctgcaaggaccagctccagtccta
tatctgcttctgcctccctgccacgagggccggaactgtg
agacgcacaaggatgaccagctgatctgtgtgaacgagaa
cggcggctgtgagcagtactgcagtgaccacacgggcacc
aagcgctcctgtcggtgccacgaggggtactctctgctgg
cagacggggtgtcctgcacacccacagttgaatatccatg
tggaaaaatacctattctagaaaaaagaaatgccagcaaa
ccccaaggccgaattgtggggggcaaggtgtgccccaaag
gggagtgtccatggcaggtcctgagaggtgaatggagctc
agagtgtggggggaccctgatcaacaccatctgggtggtc
tccgcggcccactgatcgacaaaatcaagaactggaggaa
cctgatcgcggtgctgggcgagcacgacctcagcgagcac
gacggggatgagcagagccggcgggtggcgcaggtcatca
tccccagcacgtacgtcccgggcaccaccaaccacgacat
cgcgctgctccgcctgcaccagcccgtggtcctcactgac
catgtggtgcccctctgcctgcccgaacggacgactctga
gaggacgctggccacgtgcgcactcattggtcagcggctg
gggccagctgctggaccgtggcgccacggccctggagctc
atggtcctcaacgtgccccggctgatgacccaggactgcc
tgcagcagtcacggaaggtgggagactccccaaatatcac
ggagtacatgactgtgccggctactcggatggcagcaagg
actcctgcaagggggacagtggaggcccacatgccaccca
ctaccggggcacgtggtacctgaccggcatcgtgagctgg
ggccagggctgcgccaccgtgggccacttcggcgtgtaca
ccagggtgtcccagtacatcgagtggctgcagaaactgat
gagaagcgagcccagacccggcgtgctgctgagagccccc
accccagcagcagctccaaggcccctccccctagcctgcc
cagccctagcagactgcctgggccctctgacacccctatc
ctgcctcagtccagctcctctaaggctccaccaccaccct
gcctagcccacaagactgccaggccctagcgatacaccaa
ttctgccccagtcctccagcagcaaggctcccccacctag
cctgccactccatcaaggctgcctggcccatccgataccc
caattagcctcagagcagctctagcaaggcacctcccccc
agtctgccctctccaagcagactccctggcccacagacac
tccaatcctcccacagtcctctagctctaaagctccacct
cccagcctgcccagccctagtagactccccggaccttctg
atacccccatcttgccccagtgatgaggatccgc.
[0182] In another embodiment, the amino acid sequence of Factor
VII-(CTP).sub.5 (attached to the carboxy terminus) comprises the
following amino acid sequence:
TABLE-US-00039 (SEQ ID NO: 42)
LEDMVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRR
RANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKL
FWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGR
NCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYS
LLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVC
PKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIK
NWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTT
NHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFS
LVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGD
SPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLT
GIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGV
LLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSK
APPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRL
PGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQS
SSSKAPPPSLPSPSRLPGPSDTPILPQGS.
[0183] In another embodiment, the nucleic acid sequence encoding
Factor IX comprises the following nucleic acid sequence:
TABLE-US-00040 (SEQ ID NO: 43)
gcgatcgccatgcagcgcgtgaacatgatcatggcagaa
tcaccaggcctcatcaccattgccattaggatatctact
cagtgctgaatgtacagatacttgatcatgaaaacgcca
acaaaattctgaatcggccaaagaggtataattcaggta
aattggaagagatgacaagggaaccagagagagaatgta
tggaagaaaagtgtagtatgaagaagcacgagaagtatt
gaaaacactgaaagaacaactgaattaggaagcagtatg
agatggagatcagtgtgagtccaatccatgataaatggc
ggcagagcaaggatgacattaattcctatgaatgaggtg
tccctaggatttgaaggaaagaactgtgaattagatgta
acatgtaacattaagaatggcagatgcgagcagattgta
aaaatagtgctgataacaaggtggatgctcctgtactga
gggatatcgacttgcagaaaaccagaagtcctgtgaacc
agcagtgccataccatgtggaagagtactgatcacaaac
actaagctcacccgtgctgagactgatacctgatgtgga
ctatgtaaattctactgaagctgaaaccattaggataac
atcactcaaagcacccaatcatttaatgacttcactcga
gttgaggtggagaagatgccaaaccaggtcaattcccag
gcaggagattgaatggtaaagttgatgcattctgtggag
gctctatcgttaatgaaaaatggattgtaactgctgccc
actgtgagaaactggtgttaaaattacagagtcgcaggt
gaacataatattgaggagacagaacatacagagcaaaag
cgaaatgtgattcgaattattcctcaccacaactacaat
gcagctattaataagtacaaccatgacattgcccactgg
aactggacgaacccttagtgctaaacagctacgttacac
ctatttgcattgctgacaaggaatacacgaacatcacct
caaataggatctggctatgtaagtggctggggaagagtc
accacaaagggagatcagcatagactccagtaccttaga
gaccacttgagaccgagccacatgtcacgatctacaaag
ttcaccatctataacaacatgactgtgctggcttccatg
aaggaggtagagattcatgtcaaggagatagtgggggac
cccatgttactgaagtggaagggaccagtacttaactgg
aattattagctggggtgaagagtgtgcaatgaaaggcaa
atatggaatatataccaaggtatcccggtatgtcaactg
gattaaggaaaaaacaaagctcacttgaacgcggccgc.
[0184] In another embodiment, the amino acid sequence of Factor IX
comprises the following amino acid sequence:
TABLE-US-00041 (SEQ ID NO: 44)
MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKI
LNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFEN
TERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWC
PFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCT
EGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFP
DVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPG
QFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKI
TVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNH
DLALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYV
SGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYN
NMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISW
GEECAMKGKYGIYTKVSRYVNWIKEKTKLT.
[0185] In another embodiment, the nucleic acid sequence encoding
Factor IX-CTP (attached to the carboxy terminus) comprises the
following nucleic acid sequence:
TABLE-US-00042 (SEQ ID NO: 45)
gcgatcgccatgcagcgcgtgaacatgatcatggcagaa
tcaccaggcctcatcaccatctgccattaggatatctac
tcagtgctgaatgtacagatacttgatcatgaaaacgcc
aacaaaattctgaatcggccaaagaggtataattcaggt
aaattggaagagatgacaagggaaccagagagagaatgt
atggaagaaaagtgtagattgaagaagcacgagaagtat
tgaaaacactgaaagaacaactgaattaggaagcagtat
gagatggagatcagtgtgagtccaatccatgataaatgg
cggcagagcaaggatgacattaattcctatgaatgaggt
gtccctaggatttgaaggaaagaactgtgaattagatgt
aacatgtaacattaagaatggcagatgcgagcagattgt
aaaaatagtgctgataacaaggtggatgctcctgtactg
agggatatcgacttgcagaaaaccagaagtcctgtgaac
cagcagtgccataccatgtggaagagtactgatcacaaa
cactaagctcacccgtgctgagactgatacctgatgtgg
actatgtaaattctactgaagctgaaaccattaggataa
catcactcaaagcacccaatcatttaatgacttcactcg
agagaggtggagaagatgccaaaccaggtcaattcccag
gcaggagattgaatggtaaagagatgcattctgtggagg
ctctatcgttaatgaaaaatggattgtaactgctgccca
ctgtgagaaactggtgttaaaattacagagtcgcaggtg
aacataatattgaggagacagaacatacagagcaaaagc
gaaatgtgattcgaattattcctcaccacaactacaatg
cagctattaataagtacaaccatgacattgcccactgga
actggacgaacccttagtgctaaacagctacgttacacc
tatttgcattgctgacaaggaatacacgaacatcacctc
aaataggatctggctatgtaagtggctggggaagagtca
ccacaaagggagatcagcatagacttcagtaccttagag
accacttgagaccgagccacatgtcacgatctacaaagt
tcaccatctataacaacatgactgtgctggcttccatga
aggaggtagagattcatgtcaaggagatagtgggggacc
ccatgttactgaagtggaagggaccagtacttaactgga
attattagctggggtgaagagtgtgcaatgaaaggcaaa
tatggaatatataccaaggtatcccggtatgtcaactgg
attaaggaaaaaacaaagctcactagctccagcagcaag
gcccctcccccgagcctgccctccccaagcaggctgcct
gggccctccgacacaccaatcctgccacagtgatgaagg tctggatccgcggccgc.
[0186] In another embodiment, the amino acid sequence of Factor
IX-CTP (attached to the carboxy terminus) comprises the following
amino acid sequence:
TABLE-US-00043 (SEQ ID NO: 46)
MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANK
ILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVF
ENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC
WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCS
CTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETV
FPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAK
PGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGV
KITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKY
NHDLALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSG
YVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTI
YNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGII
SWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAP PPSLPSPSRLPGPSDTPILPQ.
[0187] In another embodiment, the nucleic acid sequence encoding
Factor IX-CTP-CTP (attached to the carboxy terminus) comprises the
following nucleic acid sequence:
TABLE-US-00044 (SEQ ID NO: 47)
gcgatcgccatgcagcgcgtgaacatgatcatggcagaa
tcaccaggcctcatcaccatctgccattaggatatctac
tcagtgctgaatgtacagatacttgatcatgaaaacgcc
aacaaaattctgaatcggccaaagaggtataattcaggt
aaattggaagagatgacaagggaaccttgagagagaatg
tatggaagaaaagtgtagattgaagaagcacgagaagta
ttgaaaacactgaaagaacaactgaattaggaagcagta
tgagatggagatcagtgtgagtccaatccatgataaatg
gcggcagagcaaggatgacattaattcctatgaatgagg
tgtccctaggatttgaaggaaagaactgtgaattagatg
taacatgtaacattaagaatggcagatgcgagcagattg
taaaaatagtgctgataacaaggtggatgctcctgtact
gagggatatcgacttgcagaaaaccagaagtcctgtgaa
ccagcagtgccataccatgtggaagagtactgatcacaa
acactaagctcacccgtgctgagactgatacctgatgtg
gactatgtaaattctactgaagctgaaaccattaggata
acatcactcaaagcacccaatcatttaatgacttcactc
gagttgaggtggagaagatgccaaaccaggtcaattccc
aggcaggagattgaatggtaaagttgatgcattctgtgg
aggctctatcgttaatgaaaaatggattgtaactgctgc
ccactgtgagaaactggtgttaaaattacagagtcgcag
gtgaacataatattgaggagacagaacatacagagcaaa
agcgaaatgtgattcgaattattcctcaccacaactaca
atgcagctattaataagtacaaccatgacattgcccact
ggaactggacgaacccttagtgctaaacagctacgttac
acctatttgcattgctacaaggaatacacgaacatcacc
tcaaataggatctggctatgtaagtggctggggaagagt
caccacaaagggagatcagcatagacttcagtaccttag
agaccacttgagaccgagccacatgtcttcgatctacaa
agttcaccatctataacaacatgactgtgctggcaccat
gaaggaggtagagattcatgtcaaggagatagtggggga
ccccatgttactgaagtggaagggaccagtacttaactg
gaattattagctggggtgaagagtgtgcaatgaaaggca
aatatggaatatataccaaggtatcccggtatgtcaact
ggattaaggaaaaaacaaagctcactagctccagcagca
aggcccctcccccgagcctgccctccccaagcaggctgc
ctgggccctccgacacaccaatcctgccacagagcagct
cctctaaggcccctcctccatccctgccatccccctccc
ggctgcctggcccctctgacacccctatcctgcctcagt
gatgaaggtctggatccgcggccgc.
[0188] In another embodiment, the amino acid sequence of Factor
IX-CTP-CTP (attached to the carboxy terminus) comprises the
following amino acid sequence:
TABLE-US-00045 (SEQ ID NO: 48)
MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANK
ILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVF
ENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYEC
WCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCS
CTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETV
FPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAK
PGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGV
KITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKY
NHDLALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSG
YVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTI
YNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGII
SWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAP
PPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLP GPSDTPILPQ.
[0189] In another embodiment, the nucleic acid sequence encoding
Factor IX-(CTP).sub.3 (attached to the carboxy terminus) comprises
the following nucleic acid sequence:
TABLE-US-00046 (SEQ ID NO: 49)
tctagagtcgaccccgccatgcagcgcgtgaacatgatc
atggcagaatcaccaggcctcatcaccatctgcctatag
gatatctactcagtgctgaatgtacagatacttgatcat
gaaaacgccaacaaaattctgaatcggccaaagaggtat
aattcaggtaaattggaagagatgacaagggaaccttga
gagagaatgtatggaagaaaagtgtagattgaagaagca
cgagaagtattgaaaacactgaaagaacaactgaattag
gaagcagtatgagatggagatcagtgtgagtccaatcca
tgataaatggcggcagagcaaggatgacattaattccta
tgaatgaggtgtcccatggatttgaaggaaagaactgtg
aattagatgtaacatgtaacattaagaatggcagatgcg
agcagattgtaaaaatagtgctgataacaaggtggatgc
tcctgtactgagggatatcgacttgcagaaaaccagaag
tcctgtgaaccagcagtgccataccatgtggaagagtac
tgatcacaaacactaagctcacccgtgctgaggcagtat
tcctgatgtggactatgtaaattctactgaagctgaaac
cattaggataacatcactcaaagcacccaatcatttaat
gacttcactcgagttgaggtggagaagatgccaaaccag
gtcaattcccaggcaggagattgaatggtaaagttgatg
cattctgtggaggctctatcgttaatgaaaaatggattg
taactgctgcccactgtgagaaactggtgttaaaattac
agagtcgcaggtgaacataatattgaggagacagaacat
acagagcaaaagcgaaatgtgattcgaattattcctcac
cacaactacaatgcagctattaataagtacaaccatgac
attgcccactggaactggacgaacccttagtgctaaaca
gctacgttacacctatttgcattgctgacaaggaataca
cgaacatcacctcaaataggatctggctatgtaagtggc
tggggaagagtcaccacaaagggagatcagcatagactt
cagtaccttagagaccacttgagaccgagccacatgtca
cgatctacaaagttcaccatctataacaacatgactgtg
ctggcttccatgaaggaggtagagattcatgtcaaggag
atagtgggggaccccatgttactgaagtggaagggacca
gtacttaactggaattattagctggggtgaagagtgtgc
aatgaaaggcaaatatggaatatataccaaggtatcccg
gtatgtcaactggattaaggaaaaaacaaagctcactag
ctccagcagcaaggcccctcccccgagcctgccctcccc
aagcaggctgcctgggcccagtgacacccctatcctgcc
tcagtccagctccagcaaggccccaccccctagcctgcc
ttctccactcggctgcctggccccagcgatactccaatt
ctgccccagtcctccagcagtaaggctccccctccatct
ctgccatcccccagcagactgccaggcccactgatacac
ccatcctcccacagtgatgaggatccgcggccgc.
[0190] In another embodiment, the amino acid sequence of Factor
IX-(CTP).sub.3 (attached to the carboxy terminus) comprises the
following amino acid sequence:
TABLE-US-00047 (SEQ ID NO: 50)
MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENA
NKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEA
REVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDD
INSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNS
ADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQ
TSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFN
DFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNE
KWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNV
IRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTP
ICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQ
YLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSC
QGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIY
TKVSRYVNWIKEKTKLTSSSSKAPPPSLPSPSRLPGP
SDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQS
SSSKAPPPSLPSPSRLPGPSDTPILPQ.
[0191] In one embodiment, following expression a mature
CTP-modified FIX polypeptide is secreted, the signal peptide having
been cleaved from the precursor protein resulting in a mature
protein. For example, in the precursor CTP-modified FIX polypeptide
set forth in SEQ ID NO: 50 amino acids: 1-46:
MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKR (SEQ ID NO: 112)
represent the signal peptide of the precursor CTP-modified FIX
polypeptide, and amino acids:
TABLE-US-00048 (SEQ ID NO: 113)
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTER
TTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCP
FGFEGKNCELDVTCNIKNGRCEQFCKNSADNKWCSCT
EGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAV
FPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGED
AKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCV
ETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYN
AAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTN
IFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRA
TCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVT
EVEGTSFLTGnSWGEECAMKGKYGIYTKVSRYVNWIK
EKTKLTSSSSKAPPPSLPSPSRLPGPSDTPILPQSSS
SKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSRL PSPSRLPGPSDTPILPQ,
represent the mature engineered CTP-modified FIX polypeptide
lacking the signal peptide. In one embodiment, the amino acid
sequence of CTP-modified FIX without the signal peptide is set
forth in SEQ ID NO: 113. In another embodiment, the signal peptide
of CTP-modified FIX is set forth in SEQ ID NO: 112.
[0192] In another embodiment, the nucleic acid sequence encoding
Factor IX-(CTP).sub.4 (attached to the carboxy terminus) comprises
the following nucleic acid sequence:
TABLE-US-00049 (SEQ ID NO: 51) tctagagtcgaccccgccatgcagcgcgtgaacatg
atcatggcagaatcaccaggcctcatcaccatctgc
ctataggatatctactcagtgctgaatgtacagata
cttgatcatgaaaacgccaacaaaattctgaatcgg
ccaaagaggtataattcaggtaaattggaagagatg
acaagggaaccttgagagagaatgtatggaagaaaa
gtgtagattgaagaagcacgagaagtattgaaaaca
ctgaaagaacaactgaattaggaagcagtatgagat
ggagatcagtgtgagtccaatccatgataaatggcg
gcagagcaaggatgacattaattcctatgaatgagg
tgtcccatggatttgaaggaaagaactgtgaattag
atgtaacatgtaacattaagaatggcagatgcgagc
agattgtaaaaatagtgctgataacaaggtggatgc
tcctgtactgagggatatcgacttgcagaaaaccag
aagtcctgtgaaccagcagtgccataccatgtggaa
gagtactgatcacaaacactaagctcacccgtgctg
aggcagtattcctgatgtggactatgtaaattctac
tgaagctgaaaccattaggataacatcactcaaagc
acccaatcatttaatgacttcactcgagttgaggtg
gagaagatgccaaaccaggtcaattcccaggcagga
gattgaatggtaaagttgatgcattctgtggaggct
ctatcgttaatgaaaaatggattgtaactgctgccc
actgtgagaaactggtgttaaaattacagagtcgca
ggtgaacataatattgaggagacagaacatacagag
caaaagcgaaatgtgattcgaattattcctcaccac
aactacaatgcagctattaataagtacaaccatgac
attgcccactggaactggacgaacccttagtgctaa
acagctacgttacacctatttgcattgctgacaagg
aatacacgaacatcacctcaaataggatctggctat
gtaagtggctggggaagagtcaccacaaagggagat
cagcatagacttcagtaccttagagaccacttgaga
ccgagccacatgtcacgatctacaaagttcaccatc
tataacaacatgactgtgctggcttccatgaaggag
gtagagattcatgtcaaggagatagtgggggacccc
atgttactgaagtggaagggaccagtacttaactgg
aattattagctggggtgaagagtgtgcaatgaaagg
caaatatggaatatataccaaggtatcccggtatgt
caactggattaaggaaaaaacaaagctcactagctc
cagcagcaaggcccctcccccgagcctgccctcccc
aagcaggctgcctgggccctctgacacccctatcct
gcctcagtccagctcctctaaggccccaccaccacc
ctgcctagcccacaagactgccaggccctagcgata
caccaattctgccccagtcctccagcagcaaggctc
ccccacctagcctgccactccatcaaggctgcctgg
cccatccgataccccaattagcctcagagcagctct
agcaaggcacctccccccagtctgccctctccaagc
agactccctggcccttcagacactcccattctgcca cagtgatgaggatccgcggccgc.
[0193] In another embodiment, the amino acid sequence of Factor
IX-(CTP).sub.4 (attached to the carboxy terminus) comprises the
following amino acid sequence:
TABLE-US-00050 (SEQ ID NO: 52) SRVDPAMQRVNMIMAESPGLITICLLGYLLSAECTV
FLDHENANKILNRPKRYNSGKLEEFVQGNLERECME
EKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPC
LNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKN
GRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAV
PFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETI
LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNG
KVDAFCGGSrVNEKWIVTAAHCVETGVKITWAGEHN
IEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALL
ELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSG
WGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIY
NNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTG
IISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSS
SSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPS
LPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLP
GPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPIL PQGSAA.
[0194] In another embodiment, the nucleic acid sequence encoding
Factor IX-(CTP).sub.5 (attached to the carboxy terminus) comprises
the following nucleic acid sequence:
TABLE-US-00051 (SEQ ID NO: 53)
ctagagtcgaccccgccatgcagcgcgtgaacatgat
catggcagaatcaccaggcctcatcaccatctgccat
taggatatctactcagtgctgaatgtacagatacttg
atcatgaaaacgccaacaaaattctgaatcggccaaa
gaggtataattcaggtaaattggaagagatgacaagg
gaaccttgagagagaatgtatggaagaaaagtgtaga
ttgaagaagcacgagaagtattgaaaacactgaaaga
acaactgaattaggaagcagtatgagatggagatcag
tgtgagtccaatccatgataaatggcggcagagcaag
gatgacattaattcctatgaatgaggtgtcccatgga
tttgaaggaaagaactgtgaattagatgtaacatgta
acattaagaatggcagatgcgagcagattgtaaaaat
agtgctgataacaaggtggatgctcctgtactgaggg
atatcgacttgcagaaaaccagaagtcctgtgaacca
gcagtgccataccatgtggaagagtactgatcacaaa
cactaagctcacccgtgctgaggcagtattcctgatg
tggactatgtaaattctactgaagctgaaaccattag
gataacatcactcaaagcacccaatcatttaatgact
tcactcgagttgaggtggagaagatgccaaaccaggt
caattcccaggcaggagattgaatggtaaagttgatg
cattctgtggaggctctatcgttaatgaaaaatggat
tgtaactgctgcccactgtgagaaactggtgttaaaa
ttacagagtcgcaggtgaacataatattgaggagaca
gaacatacagagcaaaagcgaaatgtgattcgaatta
ttcctcaccacaactacaatgcagctattaataagta
caaccatgacattgcccactggaactggacgaaccct
tagtgctaaacagctacgttacacctatttgcattgc
tgacaaggaatacacgaacatcacctcaaataggatc
tggctatgtaagtggctggggaagagtcaccacaaag
ggagatcagcatagacttcagtaccttagagaccact
tgagaccgagccacatgtcacgatctacaaagttcac
catctataacaacatgactgtgctggcttccatgaag
gaggtagagattcatgtcaaggagatagtgggggacc
ccatgttactgaagtggaagggaccagtacttaactg
gaattattagctggggtgaagagtgtgcaatgaaagg
caaatatggaatatataccaaggtatcccggtatgtc
aactggattaaggaaaaaacaaagctcactagctcca
gcagcaaggcccctcccccgagcctgccctccccaag
caggctgcctgggccctctgacacccctatcctgcct
cagtccagctcctctaaggctccaccaccaccctgcc
tagcccacaagactgccaggccctagcgatacaccaa
ttctgccccagtcctccagcagcaaggctcccccacc
tagcctgccactccatcaaggctgcctggcccatccg
ataccccaattagcctcagagcagctctagcaaggca
cctccccccagtctgccctctccaagcagactccctg
gcccacagacactccaatcctcccacagtcctctagc
tctaaagctccacctcccagcctgcccagccctagta
gactccccggaccactgatacccccatcttgccccag tgatgaggatccgcggccgc.
[0195] In another embodiment, the amino acid sequence of Factor
IX-(CTP).sub.5 (attached to the carboxy terminus) comprises the
following amino acid sequence:
TABLE-US-00052 (SEQ ID NO: 54)
RVDPAMQRVNMIMAESPGLITICLLGYLLSAECTVFL
DHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKC
SFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGG
SCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQ
FCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGR
VSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQS
TQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGG
SIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTE
QKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLN
SYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRS
ALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEG
GRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKG
KYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPSPS
RLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTP
ILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSK
APPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSP SRLPGPSDTPILPQGSAA.
[0196] A skilled artisan would appreciate that the term coagulation
factor may encompass a homologue of a known coagulation factor. In
one embodiment, the homologue has a coagulating activity. In
another embodiment, a homologue of a coagulation factor is a
variant comprising conservative substitutions, or deletions,
insertions, or substitutions that do not significantly alter the
three dimensional structure of the coagulation factor. In another
embodiment, the deletion, insertion, or substitution does not alter
the function of interest of the coagulation factor, which in one
embodiment, is binding to a particular binding partner. In another
embodiment, disclosed herein is a homologue of a coagulation factor
having functional binding.
[0197] In another embodiment, furin is added to a cell expressing
the coagulation factor-CTP disclosed herein. In another embodiment,
furin increases the production efficiency of a coagulation
factor-CTP disclosed herein in a cell. In another embodiment, furin
is co-transfected with the vector comprising the coding sequence of
the coagulation factor-CTP disclosed herein. In another embodiment,
furin is encoded by a separate vector. In another embodiment, furin
and a coagulation factor-CTP are encoded by one vector. In another
embodiment, the coding sequence of furin is inserted into pCI-DHFR.
In another embodiment, the coding sequence of furin is engineered
in pCI-dhfr/smaI+NotI, Furin/AsisI F.I.+NotI.
In another embodiment, the nucleic acid sequence encoding furin
comprises the following nucleic acid sequence:
TABLE-US-00053 (SEQ ID NO: 55)
tctagagtcgaccccgccatggagctgaggccctggt
tgctatgggtggtagcagcaacaggaaccttggtcct
gctagcagctgatgctcagggccagaaggtcttcacc
aacacgtgggctgtgcgcatccctggaggcccagcgg
tggccaacagtgtggcacggaagcatgggttcctcaa
cctgggccagatcttcggggactattaccacttctgg
catcgaggagtgacgaagcggtccctgtcgcctcacc
gcccgcggcacagccggctgcagagggagcctcaagt
acagtggctggaacagcaggtggcaaagcgacggact
aaacgggacgtgtaccaggagcccacagaccccaagt
ttcctcagcagtggtacctgtctggtgtcactcagcg
ggacctgaatgtgaaggcggcctgggcgcagggctac
acagggcacggcattgtggtctccattctggacgatg
gcatcgagaagaaccacccggacttggcaggcaatta
tgatcciggggccagttttgatgtcaatgaccaggac
cctgacccccagcctcggtacacacagatgaatgaca
acaggcacggcacacggtgtgcgggggaagtggctgc
ggtggccaacaacggtgtctgtggtgtaggtgtggcc
tacaacgcccgcattggaggggtgcgcatgctggatg
gcgaggtgacagatgcagtggaggcacgctcgctggg
cctgaaccccaaccacatccacatctacagtgccagc
tggggccccgaggatgacggcaagacagtggatgggc
cagcccgcctcgccgaggaggccttcttccgtggggt
tagccagggccgaggggggctgggctccatctttgtc
tgggcctcggggaacgggggccgggaacatgacagct
gcaactgcgacggctacaccaacagtatctacacgct
gtccatcagcagcgccacgcagtttggcaacgtgccg
tggtacagcgaggcctgctcgtccacactggccacga
cctacagcagtggcaaccagaatgagaagcagatcgt
gacgactgacttgcggcagaagtgcacggagtctcac
acgggcacctcagcctctgcccccttagcagccggca
tcattgctctcaccctggaggccaataagaacctcac
atggcgggacatgcaacacctggtggtacagacctcg
aagccagcccacctcaatgccaacgactgggccacca
atggtgtgggccggaaagtgagccactcatatggcta
cgggcttttggacgcaggcgccatggtggccctggcc
cagaattggaccacagtggccccccagcggaagtgca
tcatcgacatcctcaccgagcccaaagacatcgggaa
acggctcgaggtgcggaagaccgtgaccgcgtgcctg
ggcgagcccaaccacatcactcggctggagcacgctc
aggcgcggctcaccctgtcctataatcgccgtggcga
cctggccatccacctggtcagccccatgggcacccgc
tccaccctgctggcagccaggccacatgactactccg
cagatgggtttaatgactgggccttcatgacaactca
ttcctgggatgaggatccctctggcgagtgggtccta
gagattgaaaacaccagcgaagccaacaactatggga
cgctgaccaagttcaccctcgtactctatggcaccgc
ccctgaggggctgcccgtacctccagaaagcagtggc
tgcaagaccctcacgtccagtcaggcctgtgtggtgt
gcgaggaaggcttctccctgcaccagaagagctgtgt
ccagcactgccctccaggcttcgccccccaagtcctc
gatacgcactatagcaccgagaatgacgtggagacca
tccgggccagcgtctgcgccccctgccacgcctcatg
tgccacatgccaggggccggccctgacagactgcctc
agctgccccagccacgcctccttggaccctgtggagc
agacttgctcccggcaaagccagagcagccgagagtc
cccgccacagcagcagccacctcggctgcccccggag
gtggaggcggggcaacggctgcgggcagggctgctgc
cctcacacctgcctgaggtggtggccggcctcagctg
cgccttcatcgtgctggtcttcgtcactgtcttcctg
gtcctgcagctgcgctctggctttagttttcgggggg
tgaaggtgtacaccatggaccgtggcctcatctccta
caaggggctgccccctgaagcctggcaggaggagtgc
ccgtctgactcagaagaggacgagggccggggcgaga
ggaccgcctttatcaaagaccagagcgccctctgaac gcggccgc.
In another embodiment, the amino acid sequence of furin comprises
the following amino acid sequence:
TABLE-US-00054 (SEQ ID NO: 56)
MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVR
IPGGPAVANSVARKHGFLNLGQEGDYYHFWHRGVTKR
SLSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDVYQE
PTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVV
SEDDGEKNHPDLAGNYDPGASFDVNDQDPDPQPRYTQ
MNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGVR
MLDGEVTDAVEARSLGLNPNHIHIYSASWGPEDDGKT
VDGPARLAEEAFFRGVSQGRGGLGSEVWASGNGGREH
DSCNCDGYTNSIYTLSISSATQFGNVPWYSEACSSTL
ATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLA
AGIIALTLEANKNLTWRDMQHLVVQTSKPAHLNANDW
ATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTVAPQR
KCIIDILTEPKDIGKRLEVRKTVTACLGEPNHITRLE
HAQARLTLSYNRRGDLAIHLVSPMGTRSTLLAARPHD
YSADGFNDWAFMTTHSWDEDPSGEWVLEENTSEANNY
GTLTKFTLVLYGTAPEGLPVPPESSGCKTLTSSQACW
CEEGFSLHQKSCVQHCPPGFAPQVLDTHYSTENDVET
EASVCAPCHASCATCQGPALTDCLSCPSHASLDPVEQ
TCSRQSQSSRESPPQQQPPRLPPEVEAGQRLRAGLLP
SHLPEVVAGLSCAFIVLVFVTVFLVLQLRSGFSFRGV
KVYTMDRGLISYKGLPPEAWQEECPSDSEEDEGRGER TAFIKDQSAL.
[0198] In another embodiment, disclosed herein is homologues of
furin. In another embodiment, disclosed herein is homologues of
furin maintaining a function of interest, which in one embodiment
is cleaving of a precursor protein. In another embodiment,
homologues e.g., polypeptides which are at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 87%, at least 89%, at least 91%, at
least 93%, at least 95%, at least 96%, at least 97%, at least 98%,
or at least 99% homologous to a furin as determined using BlastP
software of the National Center of Biotechnology Information (NCBI)
using default parameters.
[0199] In another embodiment, disclosed herein is a polypeptide
comprising a coagulation factor and one to ten CTPs attached to the
carboxy terminus of the coagulation factor. In another embodiment,
disclosed herein is a polypeptide comprising a coagulation factor
and one to three gonadotropin carboxy terminal peptides (CTPs)
attached to the carboxy terminus of the coagulation factor. In
another embodiment, disclosed herein is a polypeptide comprising a
coagulation factor and one to five gonadotropin carboxy terminal
peptides (CTPs) attached to the carboxy terminus of the coagulation
factor. In another embodiment, disclosed herein is a polypeptide
comprising a coagulation factor having at least one CTP on its
carboxy terminus.
[0200] In another embodiment, disclosed herein is a polypeptide
consisting of a coagulation factor and one to five CTPs attached to
the carboxy terminus of the coagulation factor. In another
embodiment, disclosed herein is a polypeptide consisting
essentially of a coagulation factor and one to five CTPs attached
to the carboxy terminus of the coagulation factor.
[0201] In one embodiment, disclosed herein is a polypeptide
comprising a coagulation factor and two CTPs attached to the
carboxy terminus of the coagulation factor, two to three CTPs
attached to the carboxy terminus of the coagulation factor, two to
four CTPs attached to the carboxy terminus of the coagulation, two
to five CTPs attached to the carboxy terminus of the coagulation
factor, two to six CTPs attached to the carboxy terminus of the
coagulation factor, two to seven CTPs attached to the carboxy
terminus of the coagulation factor, two to eight CTPs attached to
the carboxy terminus of the coagulation factor, two to nine CTPs
attached to the carboxy terminus of the coagulation factor, or two
to ten CTPs attached to the carboxy terminus of the coagulation
factor.
[0202] In one embodiment, disclosed herein is a polypeptide
comprising a coagulation factor and three CTPs attached to the
carboxy terminus of the coagulation factor, three to four CTPs
attached to the carboxy terminus of the coagulation factor, three
to five CTPs attached to the carboxy terminus of the coagulation
factor, three to six CTPs attached to the carboxy terminus of the
coagulation factor, three to seven CTPs attached to the carboxy
terminus of the coagulation factor, three to eight CTPs attached to
the carboxy terminus of the coagulation factor, three to nine CTPs
attached to the carboxy terminus of the coagulation factor, or
three to ten CTPs attached to the carboxy terminus of the
coagulation factor.
[0203] In one embodiment, disclosed herein is a polypeptide
comprising a coagulation factor and four CTPs attached to the
carboxy terminus of the coagulation factor, four to five CTPs
attached to the carboxy terminus of the coagulation factor, four to
six CTPs attached to the carboxy terminus of the coagulation
factor, four to seven CTPs attached to the carboxy terminus of the
coagulation factor, four to eight CTPs attached to the carboxy
terminus of the coagulation factor, four to nine CTPs attached to
the carboxy terminus of the coagulation factor, four to ten CTPs
attached to the carboxy terminus of the coagulation factor.
[0204] In one embodiment, disclosed herein is a polypeptide
comprising a coagulation factor and five CTPs attached to the
carboxy terminus of the coagulation factor, five to six CTPs
attached to the carboxy terminus of the coagulation factor, five to
seven CTPs attached to the carboxy terminus of the coagulation
factor, five to eight CTPs attached to the carboxy terminus of the
coagulation factor, five to nine CTPs attached to the carboxy
terminus of the coagulation factor, five to ten CTPs attached to
the carboxy terminus of the coagulation factor.
[0205] In one embodiment, disclosed herein is a polypeptide
consisting of a coagulation factor and two CTPs attached to the
carboxy terminus of the coagulation factor, two to three CTPs
attached to the carboxy terminus of the coagulation factor, two to
four CTPs attached to the carboxy terminus of the coagulation
factor, two to five CTPs attached to the carboxy terminus of the
coagulation factor, two to six CTPs attached to the carboxy
terminus of the coagulation factor, two to seven CTPs attached to
the carboxy terminus of the coagulation factor, two to eight CTPs
attached to the carboxy terminus of the coagulation factor, two to
nine CTPs attached to the carboxy terminus of the coagulation
factor, two to ten CTPs attached to the carboxy terminus of the
coagulation factor.
[0206] In one embodiment, disclosed herein is a polypeptide
consisting of a coagulation factor and three CTPs attached to the
carboxy terminus of the coagulation factor, three to four CTPs
attached to the carboxy terminus of the coagulation factor, three
to five CTPs attached to the carboxy terminus of the coagulation
factor, three to six CTPs attached to the carboxy terminus of the
coagulation factor, three to seven CTPs attached to the carboxy
terminus of the coagulation factor, three to eight CTPs attached to
the carboxy terminus of the coagulation factor, three to nine CTPs
attached to the carboxy terminus of the coagulation factor, or
three to ten CTPs attached to the carboxy terminus of the
coagulation factor.
[0207] In one embodiment, disclosed herein is a polypeptide
consisting of a coagulation factor and four CTPs attached to the
carboxy terminus of the coagulation factor, four to five CTPs
attached to the carboxy terminus of the coagulation factor, four to
six CTPs attached to the carboxy terminus of the coagulation
factor, four to seven CTPs attached to the carboxy terminus of the
coagulation factor, four to eight CTPs attached to the carboxy
terminus of the coagulation factor, four to nine CTPs attached to
the carboxy terminus of the coagulation factor, or four to ten CTPs
attached to the carboxy terminus of the coagulation factor.
[0208] In one embodiment, disclosed herein is a polypeptide
consisting of a coagulation factor and five CTPs attached to the
carboxy terminus of the coagulation factor, five to six CTPs
attached to the carboxy terminus of the coagulation factor, five to
seven CTPs attached to the carboxy terminus of the coagulation
factor, five to eight CTPs attached to the carboxy terminus of the
coagulation factor, five to nine CTPs attached to the carboxy
terminus of the coagulation factor, or five to ten CTPs attached to
the carboxy terminus of the coagulation factor.
[0209] In one embodiment, disclosed herein is a polypeptide
consisting essentially of a coagulation factor and two CTPs
attached to the carboxy terminus of the coagulation factor, two to
three CTPs attached to the carboxy terminus of the coagulation
factor, two to four CTPs attached to the carboxy terminus of the
coagulation factor, two to five CTPs attached to the carboxy
terminus of the coagulation factor, two to six CTPs attached to the
carboxy terminus of the coagulation factor, two to seven CTPs
attached to the carboxy terminus of the coagulation factor, two to
eight CTPs attached to the carboxy terminus of the coagulation
factor, two to nine CTPs attached to the carboxy terminus of the
coagulation factor, or two to ten CTPs attached to the carboxy
terminus of the coagulation factor.
[0210] In one embodiment, disclosed herein is a polypeptide
consisting essentially of a coagulation factor and three CTPs
attached to the carboxy terminus of the coagulation factor, three
to four CTPs attached to the carboxy terminus of the coagulation
factor, three to five CTPs attached to the carboxy terminus of the
coagulation factor, three to six CTPs attached to the carboxy
terminus of the coagulation factor, three to seven CTPs attached to
the carboxy terminus of the coagulation factor, three to eight CTPs
attached to the carboxy terminus of the coagulation factor, three
to nine CTPs attached to the carboxy terminus of the coagulation
factor, or three to ten CTPs attached to the carboxy terminus of
the coagulation factor.
[0211] In one embodiment, disclosed herein is a polypeptide
consisting essentially of a coagulation factor and four CTPs
attached to the carboxy terminus of the coagulation factor, four to
five CTPs attached to the carboxy terminus of the coagulation
factor, four to six CTPs attached to the carboxy terminus of the
coagulation factor, four to seven CTPs attached to the carboxy
terminus of the coagulation factor, four to eight CTPs attached to
the carboxy terminus of the coagulation factor, four to nine CTPs
attached to the carboxy terminus of the coagulation factor, or four
to ten CTPs attached to the carboxy terminus of the coagulation
factor.
[0212] In one embodiment, disclosed herein is a polypeptide
consisting essentially of a coagulation factor and five CTPs
attached to the carboxy terminus of the coagulation factor, five to
six CTPs attached to the carboxy terminus of the coagulation
factor, five to seven CTPs attached to the carboxy terminus of the
coagulation factor, five to eight CTPs attached to the carboxy
terminus of the coagulation factor, five to nine CTPs attached to
the carboxy terminus of the coagulation factor, or five to ten CTPs
attached to the carboxy terminus of the coagulation factor.
[0213] In another embodiment, disclosed herein is a polypeptide
comprising, consisting essentially of, or consisting of a
coagulation factor having no CTPs on its amino terminus. In another
embodiment, disclosed herein is a polypeptide comprising,
consisting essentially of, or consisting of a coagulation factor
lacking a CTP on its amino terminus. In another embodiment,
disclosed herein is a polypeptide comprising, consisting
essentially of, or consisting of a coagulation factor having at
least one CTP on its carboxy terminus and no CTPs on its amino
terminus. In another embodiment, disclosed herein is a polypeptide
comprising, consisting essentially of, or consisting of a
coagulation factor having the number of CTPs on its carboxy
terminus as described herein and no CTPs on its amino terminus.
[0214] In another embodiment, disclosed herein is a composition
comprising an expression vector comprising a polynucleotide
encoding a CTP-modified polypeptide consisting of a Factor IX (FIX)
polypeptide and three CTPs attached to the carboxy terminus of said
FIX polypeptide.
[0215] In another embodiment, disclosed herein is a polynucleotide
encoding a CTP-modified polypeptide consisting of an activated
Factor Vila (FVIIa) polypeptide and three CTPs attached to the
carboxy terminus of said FVIIa polypeptide.
[0216] In one embodiment, disclosed herein is a recombinant
coagulation factor as described herein. In one embodiment,
disclosed herein is an engineered coagulation factor as described
herein. A skilled artisan would appreciate that the term
"engineered coagulation factor" may encompass a CTP-modified
coagulation factor.
[0217] In one embodiment, the CTPs that are attached to the carboxy
terminus of the coagulation factor are attached in tandem to the
carboxy terminus.
[0218] In one embodiment, an engineered coagulation factor as
described herein has equivalent or improved biological activity
compared to the non-CTP-modified coagulation factor. In another
embodiment, an engineered coagulation factor as described herein
has equivalent or improved pharmacological measurements compared to
the non-CTP-modified coagulation factor. In another embodiment, an
engineered coagulation factor as described herein has equivalent or
improved pharmacokinetics compared to the non-CTP-modified
coagulation factor. In another embodiment, an engineered
coagulation factor as described herein has equivalent or improved
pharmacodynamics compared to the non-CTP-modified coagulation
factor.
[0219] In one embodiment, disclosed herein is a method of
preventing or treating a clotting or coagulation disorder. In
another embodiment, disclosed herein is a method of preventing or
treating hemophilia in a subject comprising administering a
CTP-modified coagulation factor disclosed herein. In another
embodiment, disclosed herein is a method of preventing and treating
hemophilia in a subject comprising administering a CTP-modified
coagulation factor disclosed herein. In another embodiment,
disclosed herein is a method of treating hemophilia in a subject
comprising administering a CTP-modified Factor VII disclosed
herein.
[0220] In another embodiment, disclosed herein is a method of
preventing or treating hemophilia in a subject comprising
administering a CTP-modified coagulation factor as set forth in SEQ
ID NO: 111. In another embodiment, disclosed herein is a method of
preventing and treating hemophilia in a subject comprising
administering a CTP-modified coagulation factor as set forth in SEQ
ID NO: 111. In another embodiment, disclosed herein is a method of
preventing or treating hemophilia in a subject comprising
administering a CTP-modified coagulation factor as set forth in SEQ
ID NO: 113. In another embodiment, disclosed herein is a method of
preventing and treating hemophilia in a subject comprising
administering a CTP-modified coagulation factor as set forth in SEQ
ID NO: 113.
[0221] In another embodiment, disclosed herein is a method of
treating hemophilia in a subject comprising administering a
CTP-modified Factor IX disclosed herein. In one embodiment,
hemophilia is hemophilia B. In one embodiment, hemophilia B is
known as factor IX deficiency or Christmas disease. In one
embodiment, the hemophilia is severe hemophilia, which in one
embodiment, describes hemophilia in which the coagulation factor
levels are 0-1%. In another embodiment, the hemophilia is moderate
hemophilia, which in one embodiment, describes hemophilia in which
the coagulation factor levels are 1-5%. In another embodiment, the
hemophilia is mild hemophilia, which in one embodiment, describes
hemophilia in which the coagulation factor levels are 5-50%.
[0222] In another embodiment, disclosed herein is a method of
preventing or treating a clotting or coagulation disorder in a
subject comprising administering a CTP-modified Factor IX (FIX)
polypeptide comprising a FIX polypeptide and three chorionic CTPs
attached to the carboxy terminus of said FIX polypeptide to said
subject, thereby preventing or treating a clotting or coagulation
disorder in said subject. In another embodiment, disclosed herein
is a method of preventing or treating a clotting or coagulation
disorder in a subject comprising administering an activated
CTP-modified Factor VII (FVIIa) polypeptide comprising an FVIIa
polypeptide and three chorionic CTPs attached to the carboxy
terminus of said FVIIa polypeptide to said subject, thereby
preventing or treating a clotting or coagulation disorder in said
subject.
[0223] In another embodiment, disclosed herein is a method of
preventing or treating hemophilia in a subject comprising
administering a CTP-modified Factor IX (FIX) polypeptide comprising
a FIX polypeptide and three chorionic CTPs attached to the carboxy
terminus of said FIX polypeptide to said subject, thereby
preventing or treating hemophilia in said subject. In another
embodiment, disclosed herein is a method of preventing or treating
hemophilia in a subject comprising administering an activated
CTP-modified Factor VIIa (FVIIa) polypeptide comprising a FVIIa
polypeptide and three chorionic CTPs attached to the carboxy
terminus of said FVIIa polypeptide to said subject, thereby
preventing or treating hemophilia in said subject.
[0224] In another embodiment, disclosed herein is a method of
treating hemophilia in a subject comprising administering one or
more CTP-modified coagulation factors as described herein to said
subject. Thus, in one embodiment, disclosed herein is a method of
treating hemophilia in a subject comprising administering a
CTP-modified Factor IX (FIX) polypeptide comprising a FIX
polypeptide and three chorionic CTPs attached to the carboxy
terminus of said FIX polypeptide and an activated CTP-modified
Factor VIIa (FVIIa) polypeptide comprising an activated FVII
(FVIIa) polypeptide and three chorionic CTPs attached to the
carboxy terminus of said FVIIa polypeptide to said subject, thereby
treating hemophilia in said subject. In one embodiment, the
CTP-modified FIX and the activated CTP-modified FVIIa are
administered in the same composition at the same time. In another
embodiment, the CTP-modified FIX and the activated CTP-modified
FVIIa are administered in separate compositions at the same time.
In another embodiment, the CTP-modified FIX and the activated
CTP-modified FVIIa are administered in separate compositions at
separate times.
[0225] In another embodiment, disclosed herein is a method of
preventing or treating hemophilia in a subject comprising
administering a CTP-modified Factor IX (FIX) or a CTP-modified
Factor VII polypeptide comprising a FIX or a FVII polypeptide and
three chorionic CTPs attached to the carboxy terminus of said FIX
or said FVII polypeptide to said subject, thereby preventing or
treating hemophilia in said subject. In another embodiment,
disclosed herein is a method of preventing or treating hemophilia
in a subject comprising administering a CTP-modified Factor IX
(FIX) or a CTP-modified Factor VII polypeptide comprising a FIX or
a FVII polypeptide and four chorionic CTPs attached to the carboxy
terminus of said FIX or said FVII polypeptide, five chorionic CTPs
attached to the carboxy terminus of said FIX or said FVII
polypeptide, three to five chorionic CTPs attached to the carboxy
terminus of said FIX or said FVII, three chorionic CTPs attached to
the carboxy terminus of said FIX and said FVII, or three to five
chorionic CTPs attached to the carboxy terminus of said FIX and
said FVII polypeptide to said subject, thereby preventing or
treating hemophilia in said subject.
[0226] In another embodiment, disclosed herein is a method of
preventing or treating hemophilia in a subject comprising
subcutaneously or intravenously administering a CTP-modified Factor
IX (FIX) or a CTP-modified Factor VII polypeptide comprising a FIX
or a FVII polypeptide and three chorionic CTPs attached to the
carboxy terminus of said FIX or said FVII polypeptide to said
subject, thereby preventing or treating hemophilia in said subject.
In another embodiment, disclosed herein is a method of preventing
or treating hemophilia in a subject comprising subcutaneously or
intravenously administering a CTP-modified Factor IX (FIX) or a
CTP-modified Factor VII polypeptide comprising a FIX or a FVII
polypeptide and four chorionic CTPs attached to the carboxy
terminus of said FIX or said FVII, five chorionic CTPs attached to
the carboxy terminus of said FIX or said FVII polypeptide, three to
five chorionic CTPs attached to the carboxy terminus of said FIX or
said FVII polypeptide, three chorionic CTPs attached to the carboxy
terminus of said FIX and said FVII polypeptide, or three to five
chorionic CTPs attached to the carboxy terminus of said FIX and
said FVII polypeptide to said subject, thereby preventing or
treating hemophilia in said subject.
[0227] In some embodiments, disclosed herein is a method of
preventing or treating a hemophilia in a subject, the method
comprising the step of administering to the subject a CTP-modified
coagulation factor, comprising three to five chorionic CTPs
attached to the carboxy terminus of said coagulation factor
polypeptide, wherein the sequence of said CTP-modified coagulation
factor is selected from the group consisting of SEQ ID NO: 38, 40,
42, 111, or 113 thereby preventing hemophilia in said subject.
[0228] In some embodiments, disclosed herein is a method of
preventing or treating a hemophilia in a subject, the method
comprising the step of subcutaneously administering to the subject
a CTP-modified Factor VII, comprising three to five chorionic CTPs
attached to the carboxy terminus of said FVII polypeptide, wherein
the sequence of said CTP-modified FVII is selected from the group
consisting of SEQ ID NO: 38, 40, 42, 111, or 113 thereby preventing
hemophilia in said subject.
[0229] In other embodiments, the engineered coagulation factor is
for the treatment of hemophilia B patients. In one embodiment,
coagulation Factor IX comprising 3 CTPs in tandem in its carboxy
terminus is for the treatment of hemophilia B patients. In another
embodiment, coagulation Factor IX comprising 4 CTPs in tandem in
its carboxy terminus, 5 CTPs in tandem in its carboxy, 2 CTPs in
tandem in its carboxy terminus, or 1 CTP repeat in its carboxy
terminus is for the treatment of hemophilia B patients. In other
embodiments, the engineered coagulation factor can reduce the
number of infusions required for a patient, reduce the required
doses for a patient, or a combination thereof.
[0230] In one embodiment, coagulation Factor IX comprising 3 CTPs
in tandem in its carboxy terminus exhibits an improved PK profile
while maintaining its coagulation activity vs. FIX-CTP-CTP harvest,
FIX-CTP harvest or rhFIX. In one embodiment, the elimination
half-life of rFIX-CTP3 is 2.5- to 4-fold longer than rFIX in rats
and in FIX-deficient mice. In one embodiment, the administration of
rFIX-CTP3 significantly prolonged the procoagulatory effect in
FIX-deficient mice for at least 76 hr after dosing. In one
embodiment, the administration of rFIX-CTP3 produced a higher
activity peak than rFIX in FIX-deficient mice. In another
embodiment, coagulation Factor IX comprising 2 CTPs in tandem in
its carboxy terminus exhibits an improved PK profile while
maintaining its coagulation activity vs. FIX-CTP harvest or rhFIX.
In another embodiment, coagulation Factor IX comprising 2 CTPs in
tandem in its carboxy terminus exhibits 3-fold increase in
half-life and 4.5-fold higher AUC compared to rhFIX.
[0231] In another embodiment, subcutaneous (SC) administration
results in higher bioavailability of CTP-modified FVII as compared
to recombinant FVII. In another embodiment, half-life is longer and
bioavailability (AUC SC/AUC IV) is higher following FVIIa-CTP3 and
5 SC administration when compared to SC administration of
NovoSeven.RTM.. In another embodiment, subcutaneously injected
MOD-5014 and MOD-5019 shows improved mice survival in comparison to
recombinant FVII (NovoSeven.RTM.) (see Example 8 below).
[0232] In another embodiment, a signal peptide is attached to the
amino terminus of the CTP-modified coagulation factor, as described
in U.S. Pat. No. 7,553,940, which is incorporated by reference
herein in its entirety. In another embodiment, a signal peptide is
cleaved from the amino terminus of the CTP-modified coagulation
factor disclosed herein.
[0233] A skilled artisan would appreciate that the term engineered
coagulation factor may encompass the amino acid sequence of a
matured coagulation factor lacking a signal peptide. In one
embodiment, the mature form of an engineered coagulation factor
does not include a signal peptide. In another embodiment, the
mature form of a CTP-modified coagulation factor does not include a
signal peptide. In other embodiments, the term engineered
coagulation factor comprises the amino acid sequence of the
coagulation factor including its signal sequence or signal
peptide.
[0234] In another embodiment, an engineered coagulation factor
comprising at least one CTP as described herein has enhanced in
vivo biological activity compared the same coagulation factor
without at least one CTP. In one embodiment, the enhanced
biological activity stems from the longer half-life of the
engineered coagulation factor while maintaining at least some
biological activity. In another embodiment, the enhanced biological
activity stems from enhanced biological activity resulting from the
CTP modification. In another embodiment, the enhanced biological
activity stems from both a longer half-life and from enhanced
functionality of the CTP-modified coagulation factor.
[0235] In some embodiments, at least one CTP sequence at the
carboxy terminal end of the coagulation factor provides enhanced
protection against degradation of a coagulation factor. In some
embodiments, at least one CTP sequence at the carboxy terminal end
of the coagulation factor provides enhanced protection against
clearance. In some embodiments, at least one CTP sequence at the
carboxy terminal end of the coagulation factor provides prolonged
clearance time. In some embodiments, at least one CTP sequence at
the carboxy terminal end of the coagulation factor enhances its
Cmax. In some embodiments, at least one CTP sequence at the carboxy
terminal end of the coagulation factor enhances its Tmax. In some
embodiments, at least one CTP sequence at the carboxy terminal end
of the coagulation factor prolongs its T1/2.
[0236] In another embodiment, a conjugated coagulation factor
disclosed herein is used in the same manner as an unmodified
conjugated coagulation factor. In another embodiment, a conjugated
coagulation factor disclosed herein has an increased circulating
half-life and plasma residence time, decreased clearance, and
increased clinical activity in vivo. In another embodiment, due to
the improved properties of the conjugated coagulation factor as
described herein, this conjugate is administered less frequently
than the unmodified form of the same coagulation factor.
[0237] In another embodiment, decreased frequency of administration
will result in improved treatment strategy, which in one
embodiment, will lead to improved patient compliance leading to
improved treatment outcomes, as well as improved patient quality of
life. In another embodiment, compared to conventional conjugates of
coagulation factors, it has been found that conjugates having the
molecular weight and linker structure of the conjugates disclosed
herein have an improved potency, improved stability, elevated AUC
levels, and enhanced circulating half-life.
[0238] In another embodiment, disclosed herein is a pharmaceutical
composition comprising a CTP-modified Factor IX (FIX) polypeptide
consisting of a FIX polypeptide and three CTPs attached to the
carboxy terminus of said CTP-modified FIX polypeptide. In another
embodiment, disclosed herein is a pharmaceutical composition
comprising a CTP-modified Factor IX (FIX) polypeptide consisting of
a FIX polypeptide and three CTPs attached to the carboxy terminus
of said CTP-modified FIX polypeptide, as set forth in SEQ ID NO:
113.
[0239] In another embodiment, disclosed herein is a pharmaceutical
composition comprising an activated CTP-modified Factor VIIa
(FVIIa) polypeptide consisting of a FVIIa polypeptide and five CTPs
attached to the carboxy terminus of said FVIIa. In another
embodiment, disclosed herein is a pharmaceutical composition
comprising an activated CTP-modified Factor Vita (FVIIa)
polypeptide consisting of a FVIIa polypeptide and three CTPs
attached to the carboxy terminus of said FVIIa. In another
embodiment, disclosed herein is a pharmaceutical composition
comprising an activated CTP-modified Factor VIIa (FVIIa)
polypeptide consisting of a FVIIa polypeptide and three CTPs
attached to the carboxy terminus, as set forth in SEQ ID NO:
111.
[0240] In another embodiment, disclosed herein is a composition
comprising a conjugated coagulation factor as described herein. In
another embodiment, disclosed herein is a pharmaceutical
composition comprising the conjugated coagulation factor as
described herein. In another embodiment, disclosed herein is a
pharmaceutical composition comprising a therapeutically effective
amount of the conjugated coagulation factor as described herein. In
one embodiment, a therapeutically effective amount of a conjugated
coagulation factor is determined according to factors such as the
specific condition being treated, the condition of the patient
being treated, as well as the other ingredients in the
composition.
[0241] In another embodiment, a conjugated coagulation factor as
described herein is useful in the treatment of subjects afflicted
with Hemophilia. In another embodiment, a conjugated coagulation
factor as described herein is useful in the prophylactic therapy of
Hemophilia thus reducing the risk of bleeding and associated
complications. In another embodiment, a conjugated coagulation
factor as described herein is useful in the treatment of subjects
afflicted with Hemophilia while reducing the risk of developing
inhibitory antibodies to exogenously administered coagulation
factors. In another embodiment, a conjugated coagulation factor as
described herein is useful in the treatment of subjects afflicted
with Hemophilia thus inducing homeostasis.
[0242] In one embodiment, a CTP-modified coagulation factor
disclosed herein has therapeutic uses. In another embodiment, a
CTP-modified coagulation factor disclosed herein has prophylactic
uses.
[0243] In another embodiment, a conjugated coagulation factor as
described herein is useful in the treatment of subjects
experiencing excessive bleeding or bruising or having a prolonged
Prothrombin Time (PT) or Partial Thromboplastin Time (PTT). In
another embodiment, a conjugated coagulation factor as described
herein is useful in the treatment of subjects having an acquired
condition that is causing bleeding, such as vitamin K deficiency or
liver disease. In another embodiment, a conjugated coagulation
factor as described herein is useful in the treatment of subjects
having deficiencies in coagulation factors that are acquired (due
to other diseases) or inherited, mild or severe, permanent or
temporary. In another embodiment, a conjugated coagulation factor
as described herein is useful in the treatment of subjects
afflicted with hemophilia A. In another embodiment, a conjugated
coagulation factor as described herein is useful in the treatment
of subjects afflicted with hemophilia B. In another embodiment, a
conjugated coagulation factor as described herein is useful in the
treatment of subjects having acquired deficiencies due to chronic
diseases, such as liver disease or cancer; to an acute condition
such as disseminated intravascular coagulation (DIC), which uses up
clotting factors at a rapid rate; or to a deficiency in vitamin K
or treatment with a vitamin K antagonist like warfarin (the
production of factors II, VII, IX, and X require vitamin K). In
another embodiment, a conjugated coagulation factor as described
herein is useful in the treatment of subjects afflicted with a
disease in which causes clotting imbalances such as but not limited
to: a liver disease, uremia, a cancer, a bone marrow disorder, an
exposure to snake venom, a vitamin K deficiency, an anticoagulation
therapy, an accidental ingestion of the anticoagulant warfarin,
multiple blood transfusions (stored units of blood lose some of
their clotting factors), or a combination thereof. In another
embodiment, disclosed herein is a method of treating deep vein
thrombosis in a subject comprising administering a CTP-modified
coagulation factor disclosed herein. In another embodiment,
disclosed herein is a method of preventing uncontrolled bleeding in
a subject with hemophilia comprising administering a CTP-modified
coagulation factor disclosed herein. In another embodiment,
disclosed herein is a method of preventing bleeding episodes in a
subject with hemophilia comprising administering a CTP-modified
coagulation factor disclosed herein. In another embodiment,
disclosed herein is a method of controlling bleeding episodes in a
subject with hemophilia B (congenital factor IX deficiency).
[0244] In another embodiment, the compositions and methods
disclosed herein are for the treatment of bleeding episodes in
hemophilia A or B patients with inhibitors to FVIII or FIX and in
patients with acquired hemophilia; prevention of bleeding in
surgical interventions or invasive procedures in hemophilia A or B
patients with inhibitors to FVIII or FIX and in patients with
acquired hemophilia; treatment of bleeding episodes in patients
with congenital FVII deficiency and prevention of bleeding in
surgical interventions or invasive procedures in patients with
congenital FVII deficiency. In another embodiment, the compositions
and methods disclosed herein are for the treatment or prevention of
muscle bleeds. In another embodiment, the compositions and methods
disclosed herein are for the treatment or prevention of joint
bleeds. In another embodiment, the compositions and methods
disclosed herein manufacture therapeutic or prophylactic treatment
of epistaxis and gum bleeding, mucous membrane bleeding, bleeding
into the central nervous system. In another embodiment, the
compositions and methods disclosed herein manufacture therapeutic
or prophylactic treatment of gastrointestinal or cerebral bleeding.
In another embodiment, the compositions and methods disclosed
herein manufacture therapeutic or prophylactic treatment of low
frequency mild bleeds. In another embodiment, the compositions and
methods disclosed herein manufacture therapeutic or prophylactic
treatment of low frequency moderate bleeds. In another embodiment,
the compositions and methods disclosed herein manufacture
therapeutic or prophylactic treatment of high frequency mild
bleeds. In another embodiment, the compositions and methods
disclosed herein manufacture therapeutic or prophylactic treatment
of high frequency moderate bleeds.
[0245] In one embodiment, the compositions and methods disclosed
herein manufacture therapeutic or prophylactic treatment of
asymptomatic hemophilia. In another embodiment, the compositions
and methods disclosed herein manufacture therapeutic or
prophylactic treatment of mild to moderate hemophilia. In another
embodiment, the compositions and methods disclosed herein
manufacture therapeutic or prophylactic treatment of severe
hemophilia.
[0246] In one embodiment, the compositions and methods disclosed
herein manufacture therapeutic or prophylactic treatment of
hemorrhage, which in one embodiment, is uncontrollable hemorrhage,
and, in another embodiment, intracerebral hemorrhage. In another
embodiment, the compositions and methods disclosed herein
manufacture therapeutic or prophylactic treatment of neonatal
coagulopathies; severe hepatic disease; high-risk surgical
procedures; traumatic blood loss; bone marrow transplantation;
thrombocytopenias and platelet function disorders; urgent reversal
of oral anticoagulation; congenital deficiencies of factors V, VII,
X, and XI; or von Willebrand disease, in one embodiment, von
Willebrand disease with inhibitors to von Willebrand factor.
[0247] In one embodiment, a CTP-modified coagulation factor
disclosed herein is for the treatment of hemophilia or a related
disease as described herein in a subject.
[0248] In another embodiment, a [(CTP)n>1-coagulation factor] as
described herein comprises a full length coagulation factor or an
active fragment thereof connected via a peptide bond on its carboxy
terminus to at least one CTP unit with no CTPs on its amino
terminus. In another embodiment, a [(CTP)n>1-coagulation factor]
as described herein comprises a coagulation factor or an active
fragment thereof connected via a peptide bond to at least one CTP
unit which is connected to an additional CTP unit via a peptide
bond with no CTPs on its amino terminus. In another embodiment, one
nucleic acid molecule encodes an engineered coagulation factor
comprising at least one CTP attached to its C-terminus and no CTPs
on its amino terminus.
[0249] In another embodiment, disclosed herein is an expression
vector comprising a polynucleotide molecule as described herein. In
another embodiment, disclosed herein is a n expression vector
comprising a polynucleotide encoding a CTP-modified polypeptide
consisting of a Factor IX (FIX) polypeptide and three CTPs attached
to the carboxy terminus of said FIX polypeptide. In another
embodiment, disclosed herein is a n expression vector comprising a
polynucleotide encoding a CTP-modified polypeptide consisting of a
Factor VIIa (FVIIa) polypeptide and three CTPs attached to the
carboxy terminus of said FVIIa polypeptide.
[0250] In another embodiment, disclosed herein is a cell comprising
the expression vector as described herein. In another embodiment,
disclosed herein is a cell comprising an expression vector
comprising a polynucleotide encoding a CTP-modified polypeptide
consisting of a Factor IX (FIX) polypeptide and three CTPs attached
to the carboxy terminus of said FIX polypeptide. In another
embodiment, disclosed herein is a cell comprising an expression
vector comprising a polynucleotide encoding a CTP-modified
polypeptide consisting of a Factor VIIa (FVIIa) polypeptide and
three CTPs attached to the carboxy terminus of said FVIIa
polypeptide.
[0251] In another embodiment, disclosed herein is a composition
comprising the expression vector as described herein. In another
embodiment, disclosed herein is a composition comprising an
expression vector comprising a polynucleotide encoding a
CTP-modified polypeptide consisting of a Factor IX (FIX)
polypeptide and three CTPs attached to the carboxy terminus of said
FIX polypeptide. In another embodiment, disclosed herein is a
composition comprising an expression vector comprising a
polynucleotide encoding a CTP-modified polypeptide consisting of a
Factor VIIa (FVIIa) polypeptide and three CTPs attached to the
carboxy terminus of said FVIIa polypeptide.
[0252] In another embodiment, disclosed herein is a composition
comprising the cell as described herein. In another embodiment, the
cell is a eukaryotic cell. In another embodiment, the cell is a
prokaryotic cell.
[0253] In another embodiment, disclosed herein is a method of
producing a CTP-modified coagulation factor, comprising the step of
attaching one to ten chorionic CTPs to the carboxy terminus of said
coagulation factor, thereby producing a CTP-modified coagulation
factor. In another embodiment, disclosed herein is a method of
producing a CTP-modified coagulation factor, comprising the step of
attaching one to ten polynucleotide sequences encoding a chorionic
gonadotropin carboxy terminal peptide (CTP) to the carboxy terminus
of a polynucleotide sequence encoding said coagulation factor,
thereby producing a CTP-modified coagulation factor. In another
embodiment, disclosed herein is a method of producing a
CTP-modified Factor IX (FIX) polypeptide, comprising the step of
attaching three chorionic CTPs to the carboxy terminus of said FIX
polypeptide, thereby producing a CTP-modified FIX polypeptide. In
another embodiment, disclosed herein is a method of producing a
CTP-modified Factor VIIa (FVIIa) polypeptide, comprising the step
of attaching three chorionic CTPs to the carboxy terminus of said
FVIIa polypeptide, thereby producing a CTP-modified FVIIa
polypeptide.
[0254] In another embodiment, the engineered coagulation factors
disclosed herein are synthesized using a polynucleotide molecule
encoding a polypeptide disclosed herein. In some embodiments, the
polynucleotide molecule encoding the engineered coagulation factors
disclosed herein 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 an
engineered coagulation factor disclosed herein. In some
embodiments, the cis-regulatory sequence is suitable for directing
tissue-specific expression of the engineered coagulation factors
disclosed herein. In some embodiments, the cis-regulatory sequence
is suitable for directing inducible expression of the engineered
coagulation factors disclosed herein.
[0255] In one embodiment, disclosed herein are long-acting dual
GLP-1/glucagon receptor agonists and methods of producing and using
same. In another embodiment, long-acting GLP-1/glucagon receptor
agonists comprise a carboxy terminal peptide (CTP, also referred to
as CTP unit). In another embodiment, long-acting polypeptides which
comprise a GLP-1/glucagon receptor agonist further comprise a
carboxy terminal peptide (CTP) of human Chorionic Gonadotropin
(hCG). In another embodiment, CTP acts as a protectant against the
degradation of a GLP-1/glucagon receptor agonist. In another
embodiment, CTP extends the Cmax of a GLP-1/glucagon receptor
agonist. In another embodiment, CTP extends the Tmax of a
GLP-1/glucagon receptor agonist. In another embodiment, CTP extends
the circulatory half-life of a GLP-1/glucagon receptor agonist. In
some embodiments, CTP enhances the potency of a GLP-1/glucagon
receptor agonist.
[0256] In another embodiment, disclosed herein is a method of
extending the biological half-life of a GLP-1/glucagon receptor
agonist, comprising the step of attaching one to ten CTPs to the
carboxy terminus of the GLP-1/glucagon receptor agonist, thereby
extending the biological half-life of the GLP-1/glucagon receptor
agonist. In another embodiment, disclosed herein is a method of
extending the biological half-life of a GLP-1/glucagon receptor
agonist, comprising the step of attaching one to five CTPs to the
carboxy terminus of the GLP-1/glucagon receptor agonist, thereby
extending the biological half-life of the GLP-1/glucagon receptor
agonist. In another embodiment, disclosed herein is a method for
extending the circulatory half-life of a GLP-1/glucagon receptor
agonist. In another embodiment, disclosed herein is a method for
increasing the half-life of a GLP-1/glucagon receptor agonist. In
another embodiment, disclosed herein is a method for extending the
half-life of a GLP-1/glucagon receptor agonist.
[0257] In one embodiment, disclosed herein is a CTP-modified
polypeptide comprising a dual GLP-1/Glucagon receptor agonist and
at least one chorionic gonadotropin carboxy terminal peptide (CTP)
attached to the amino terminus or carboxy terminus of said
agonist.
[0258] In another embodiment, the agonist is a protein, a
polypeptide, or a peptide. In another embodiment, the peptide is an
oxyntomodulin (OXM).
[0259] In another embodiment, the CTP-modified polypeptide
comprises a peptide that comprises fewer than 50 amino acids and at
least one chorionic gonadotropin carboxy terminal peptide, attached
to an amino or a carboxy terminus of the peptide. In another
embodiment, the peptide is OXM.
[0260] Oxyntomodulin peptide is useful for the treatment of
metabolic disorders such as diabetes and obesity. However, due to
the short half-life of the peptide and its low stability in-vivo,
repeated daily administrations of supraphysiological doses are
required in order to achieve pharmacological effect in humans. As
demonstrated herein below (see Examples), all OXM-CTP variants
disclosed herein demonstrated superior pharmacokinetic profile in
rats as compared to native OXM, with a substantial increase in the
exposure and elongated half-life. Surprisingly, the OXM-CTP-CTP
variant demonstrated superior PK parameters as compared to the
CTP-OXM-CTP-CTP variant which is fused to 3 copies of CTP, one in
the N-terminus and two in tandem in the C-terminus.
[0261] Fusion of 2 and 3 CTPs to the C-terminus of OXM resulted in
similar fold of increase compared to the native peptide half-life
(21.6 and 21 respectively), following SC administration (see
Examples below). Therefore, it was expected that fusion of four and
five CTP to the C-terminus of OXM would not significantly elongate
the half-life above 20-fold. However, a surprisingly marked
increase in OXM half-life (i.e. 50-fold) was demonstrated for the
OXM-4CTP variants and the OXM-5CTP variant with 30-fold increase in
the exposure as reflected by the area under the curve (AUC)
parameter (see Examples herein).
[0262] Hence, in one embodiment, disclosed herein is a CTP-modified
polypeptide comprising an OXM peptide and at least one chorionic
gonadotropin carboxy terminal peptide (CTP) attached to the amino
terminus or carboxy terminus of the oxyntomodulin peptide.
[0263] In one embodiment, a CTP-modified OXM as described herein
comprises a full length OXM or an active fragment thereof connected
via a peptide bond on its amino or carboxy terminus to at least one
CTP unit with no CTPs on its amino terminus. In another embodiment,
a CTP-modified OXM as described herein comprises a full length OXM
or an active fragment thereof connected via a peptide bond on its
carboxy terminus to at least one CTP unit with no CTPs on its amino
terminus. In another embodiment, a CTP-modified OXM as described
herein comprises a full length OXM or an active fragment thereof
connected via a peptide bond on its amino terminus to at least one
CTP unit with no CTPs on its carboxy terminus. In another
embodiment, disclosed herein is a nucleic acid molecule encoding an
engineered OXM as described herein above, which in one embodiment,
comprises at least one CTP attached to its carboxy terminus or its
amino terminus.
[0264] In another embodiment, the CTP-modified polypeptide
comprises a peptide that comprises fewer than 50 amino acids and at
least one chorionic gonadotropin carboxy terminal peptide, attached
to an amino or a carboxy terminus of the peptide. In one
embodiment, the CTP-modified polypeptide disclosed herein comprises
a peptide that comprises fewer than 40 amino acids and at least one
chorionic gonadotropin carboxy terminal peptide, attached to an
amino or a carboxy terminus of the peptide. In another embodiment,
the CTP-modified polypeptide disclosed herein comprises a peptide
that comprises fewer than 30, 20, or 10 amino acids. In one
embodiment, the peptide comprising fewer than 50 amino acids is a
dual GLP-1/Glucagon receptor agonist. In another embodiment, the
peptide comprising fewer than 50 amino acids is OXM.
[0265] In another embodiment, OXM comprises the following amino
acid (AA) sequence: HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID
NO: 57). In another embodiment, OXM consists of the amino acid
sequence of SEQ ID NO: 57.
[0266] In another embodiment, OXM is human OXM or any mammal OXM. A
skilled artisan would appreciate that OXM may also be termed
glucagon-37 or bioactive enteroglucagon. In another embodiment, OXM
is a dual GLP-1/Glucagon receptor agonist. A skilled artisan would
appreciate that the term OXM may encompass a biologically active
fragment of OXM. In another embodiment, biologically active OXM
extends from amino acid 30 to amino acid 37 of SEQ ID NO: 57. In
another embodiment, biologically active OXM extends from amino acid
19 to amino acid 37 of SEQ ID NO: 57. In another embodiment, OXM
disclosed herein corresponds to an octapeptide from which the two
C-terminal amino acids are deleted. In another embodiment, OXM
disclosed herein corresponds to any fragment of SEQ ID NO: 57 which
retains OXM activity as described herein.
[0267] In one embodiment, two chorionic gonadotropin carboxy
terminal peptides are attached to OXM, one CTP on the carboxy
terminus and one CTP on the amino terminus of the OXM peptide. In
another embodiment, two chorionic gonadotropin carboxy terminal
peptides are attached to OXM on the carboxy terminus of the OXM
peptide. In another embodiment, two chorionic gonadotropin carboxy
terminal peptides are attached to OXM, both on the amino terminus
of the OXM peptide. In another embodiment, three chorionic
gonadotropin carboxy terminal peptides are attached to OXM, one CTP
on the amino terminus and two CTPs on the carboxy terminus of the
OXM peptide. In another embodiment, three chorionic gonadotropin
carboxy terminal peptides are attached to the carboxy terminus of
the OXM peptide. In another embodiment, four chorionic gonadotropin
carboxy terminal peptides are attached to the carboxy terminus of
the OXM peptide. In another embodiment, five chorionic gonadotropin
carboxy terminal peptides are attached to the carboxy terminus of
the OXM peptide. In another embodiment, 1-10 CTP are attached to
the amino or carboxy terminus of OXM. In another embodiment, 1-10
CTPs are attached to the amino terminus of OXM. In another
embodiment, 1-10 CTPs are attached to the carboxy terminus of
OXM.
[0268] In another embodiment, disclosed herein is a method of
producing a CTP-modified polypeptide comprising an OXM peptide and
at least one CTP attached to the amino terminus or carboxy terminus
of the OXM peptide, the method comprising the step of attaching at
least one chorionic gonadotropin carboxy terminal peptide attached
to the amino terminus or carboxy terminus of the OXM peptide.
[0269] In one embodiment, disclosed herein is a method of extending
the biological half-life of a peptide comprising fewer than 50
amino acids, comprising the step of attaching at least one
chorionic gonadotropin carboxy terminal peptides to an amino or a
carboxy terminus of the agonist, thereby improving the biological
half-life of the agonist.
[0270] In one embodiment, disclosed herein is a method of extending
the biological half-life of a dual GLP-1/Glucagon receptor agonist,
comprising the step of attaching at least one chorionic
gonadotropin carboxy terminal peptides to an amino or a carboxy
terminus of the agonist, thereby improving the biological half-life
of the agonist.
[0271] In another embodiment, disclosed herein is a method of
extending the biological half-life of OXM, comprising the step of
attaching at least one CTP to an amino or a carboxy terminus of the
OXM, thereby improving the biological half-life of OXM.
[0272] In one embodiment, disclosed herein is a method of improving
the AUC of a peptide comprising fewer than 50 amino acids,
comprising the step of attaching at least one CTP to a carboxy
terminus of the agonist, thereby improving the AUC of the
agonist.
[0273] In one embodiment, disclosed herein is a method of improving
the AUC of a dual GLP-1/Glucagon receptor agonist, comprising the
step of attaching at least one CTP to a carboxy terminus of the
agonist, thereby improving the AUC of the agonist.
[0274] In another embodiment, disclosed herein is a method of
improving the AUC of OXM, comprising the step of attaching at least
one CTP to a carboxy terminus of the OXM, thereby improving the AUC
of OXM.
[0275] In one embodiment, the term OXM further includes a homologue
of a known OXM. In one embodiment, the homologue is a functional
homologue. A skilled artisan would appreciate that the term
"functional" may encompass the ability of an OXM homologue, peptide
or protein disclosed herein to suppress appetite. In addition, the
skilled artisan would appreciate the ability an OXM homologue,
peptide or protein disclosed herein has to extend another protein's
or peptide's biological half-life. A skilled artisan would
appreciate that the biological half-life (T1/2) of a OXM protein,
peptide or homologue disclosed would encompass the time it takes
for half of the amount of the protein, peptide or homologue to be
degraded or to not be present in a biological medium in a subject.
In another embodiment, the biological medium is serum, cerebospinal
fluid, tissue, mucosa, and the like.
[0276] In one embodiment, OXM binds to a receptor and mediates
appetite suppression. In another embodiment, the receptor is a dual
GLP-1/Glucagon receptor. In another embodiment, the receptor is a
GLP-1 receptor. In another embodiment, the receptor is a glucagon
receptor. In yet another embodiment, the receptor is any receptor
known in the art to bind to OXM.
[0277] In another embodiment, disclosed herein is a homologue of an
OXM. A skilled artisan would appreciate that an OXM homologue may
encompass a peptide homologue of the peptide of SEQ ID NO: 57.
[0278] In another embodiment, disclosed herein is a homologue of an
OXM having an appetite suppressing activity. In another embodiment,
disclosed herein is a homologue of an OXM having functional
binding. In another embodiment, disclosed herein is homologues of
an OXM as described herein having an appetite suppression and
activity. In another embodiment, disclosed herein is homologues of
an OXM as described herein having functional binding. In another
embodiment, homologues comprise polypeptides which are at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 87%, at least 89%,
at least 91%, at least 93%, at least 95%, at least 96%, at least
97%, at least 98%, or at least 99% homologous to an OXM as
determined using BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters.
[0279] In one embodiment, disclosed herein is a pharmaceutical
composition comprising the CTP-modified polypeptide disclosed
herein.
[0280] In one embodiment, disclosed herein is a polypeptide
comprising a GLP-1/Glucagon receptor agonist and two CTPs attached
to the carboxy terminus of the GLP-1/Glucagon receptor agonist. In
another embodiment, disclosed herein is a polypeptide comprising a
GLP-1/Glucagon receptor agonist and two to three CTPs, two to four,
two to five CTPs, two to six CTPs, two to seven CTPs, two to eight
CTPs, two to nine CTPs, or two to ten CTPs attached to the carboxy
terminus of the GLP-1/Glucagon receptor agonist.
[0281] In one embodiment, disclosed herein is a polypeptide
comprising an OXM and two CTPs attached to the carboxy terminus of
the OXM. In another embodiment, disclosed herein is a polypeptide
comprising an OXM and two to three CTPs, two to four CTPs, two to
five CTPs, two to six CTPs, two to seven CTPs, two to eight CTPs,
two to nine CTPs, or two to ten CTPs attached to the carboxy
terminus of the OXM.
[0282] In one embodiment, disclosed herein is a polypeptide
comprising a GLP-1/Glucagon receptor agonist and three CTPs
attached to the carboxy terminus of the GLP-1/Glucagon receptor
agonist. In another embodiment, disclosed herein is a polypeptide
comprising a GLP-1/Glucagon receptor agonist and three to four
CTPs, three to five CTPs, three to six CTPs, three to seven CTPs,
three to eight CTPs, three to nine CTPs, or three to ten CTPs
attached to the carboxy terminus of the GLP-1/Glucagon receptor
agonist.
[0283] In one embodiment, disclosed herein is a polypeptide
comprising an OXM and three CTPs attached to the carboxy terminus
of the OXM. In another embodiment, disclosed herein is a
polypeptide comprising an OXM and three to four CTPs, three to five
CTPs, three to six CTPs, three to seven CTPs, three to eight CTPs,
three to nine CTPs, or three to ten CTPs attached to the carboxy
terminus of the OXM.
[0284] In one embodiment, disclosed herein is a polypeptide
comprising a GLP-1/Glucagon receptor agonist and four CTPs attached
to the carboxy terminus of the GLP-1/Glucagon receptor agonist. In
another embodiment, disclosed herein is a polypeptide comprising a
GLP-1/Glucagon receptor agonist and four to five CTPs, four to six,
four to seven CTPs, four to eight CTPs, four to nine CTPs, or four
to ten CTPs attached to the carboxy terminus of the GLP-1/Glucagon
receptor agonist.
[0285] In one embodiment, disclosed herein is a polypeptide
comprising an OXM and four CTPs attached to the carboxy terminus of
the OXM. In another embodiment, disclosed herein is a polypeptide
comprising an OXM and four to five CTPs, four to six CTPs, four to
seven CTPs, four to eight CTPs, four to nine CTPs, or four to ten
CTPs attached to the carboxy terminus of the OXM.
[0286] In one embodiment, disclosed herein is a polypeptide
comprising a GLP-1/Glucagon receptor agonist and five CTPs attached
to the carboxy terminus of the GLP-1/Glucagon receptor agonist. In
another embodiment, disclosed herein is a polypeptide comprising a
GLP-1/Glucagon receptor agonist and five to six CTPs, five to
seven, five to eight CTPs, five to nine CTPs, or five to ten CTPs
attached to the carboxy terminus of the GLP-1/Glucagon receptor
agonist.
[0287] In one embodiment, disclosed herein is a polypeptide
comprising an OXM and five CTPs attached to the carboxy terminus of
the OXM. In another embodiment, disclosed herein is a polypeptide
comprising an OXM and five to six CTPs, five to seven CTPs, five to
eight CTPs, five to nine CTPs, or five to ten CTPs attached to the
carboxy terminus of the OXM.
[0288] In one embodiment, disclosed herein is a polypeptide
consisting of a GLP-1/Glucagon receptor agonist and two CTPs
attached to the carboxy terminus of the GLP-1/Glucagon receptor
agonist. In another embodiment, disclosed herein is a polypeptide
consisting of a GLP-1/Glucagon receptor agonist and two to three
CTPs, two to four, two to five CTPs, two to six CTPs, two to seven
CTPs, two to eight CTPs, two to nine CTPs, or two to ten CTPs
attached to the carboxy terminus of the GLP-1/Glucagon receptor
agonist.
[0289] In one embodiment, disclosed herein is a polypeptide
consisting of an OXM and two CTPs attached to the carboxy terminus
of the OXM. In another embodiment, disclosed herein is a
polypeptide consisting of an OXM and two to three CTPs, two to four
CTPs, two to five CTPs, two to six CTPs, two to seven CTPs, two to
eight CTPs attached to the carboxy terminus of the OXM. In another
embodiment, disclosed herein is a polypeptide consisting of an OXM
and two to nine CTPs attached to the carboxy terminus of the OXM.
In another embodiment, disclosed herein is a polypeptide consisting
of an OXM and two to ten CTPs attached to the carboxy terminus of
the OXM.
[0290] In one embodiment, disclosed herein is a polypeptide
consisting of a GLP-1/Glucagon receptor agonist and three CTPs
attached to the carboxy terminus of the GLP-1/Glucagon receptor
agonist. In another embodiment, disclosed herein is a polypeptide
consisting of a GLP-1/Glucagon receptor agonist and three to four
CTPs, three to five CTPs, three to six CTPs, three to seven CTPs,
three to eight CTPs, three to nine CTPs, or three to ten CTPs
attached to the carboxy terminus of the GLP-1/Glucagon receptor
agonist.
[0291] In one embodiment, disclosed herein is a polypeptide
consisting of an OXM and three CTPs attached to the carboxy
terminus of the OXM. In another embodiment, disclosed herein is a
polypeptide consisting of an OXM and three to four CTPs, three to
five CTPs, three to six CTPs, three to seven CTPs, three to eight
CTPs, three to nine CTPs, or three to ten CTPs attached to the
carboxy terminus of the OXM.
[0292] In one embodiment, disclosed herein is a polypeptide
consisting of a GLP-1/Glucagon receptor agonist and four CTPs
attached to the carboxy terminus of the GLP-1/Glucagon receptor
agonist. In another embodiment, disclosed herein is a polypeptide
consisting of a GLP-1/Glucagon receptor agonist and four to five
CTPs, four to six CTPs, four to seven CTPs, four to eight CTPs,
four to nine CTPs, or four to ten CTPs attached to the carboxy
terminus of the GLP-1/Glucagon receptor agonist.
[0293] In one embodiment, disclosed herein is a polypeptide
consisting of an OXM and four CTPs attached to the carboxy terminus
of the OXM. In another embodiment, disclosed herein is a
polypeptide consisting of an OXM and four to five CTPs, four to six
CTPs, four to seven CTPs, four to eight CTPs, four to nine CTPs, or
four to ten CTPs attached to the carboxy terminus of the OXM.
[0294] In one embodiment, disclosed herein is a polypeptide
consisting of a GLP-1/Glucagon receptor agonist and five CTPs
attached to the carboxy terminus of the GLP-1/Glucagon receptor
agonist. In another embodiment, disclosed herein is a polypeptide
consisting of a GLP-1/Glucagon receptor agonist and five to six
CTPs, five to seven CTPs, five to eight CTPs, five to nine CTPs, or
five to ten CTPs attached to the carboxy terminus of the
GLP-1/Glucagon receptor agonist.
[0295] In one embodiment, disclosed herein is a polypeptide
consisting of an OXM and five CTPs attached to the carboxy terminus
of the OXM. In another embodiment, disclosed herein is a
polypeptide consisting of an OXM and five to six CTPs, five to
seven CTPs, five to eight CTPs, five to nine CTPs, or five to ten
CTPs attached to the carboxy terminus of the OXM.
[0296] In one embodiment, disclosed herein is a polypeptide
consisting essentially of a GLP-1/Glucagon receptor agonist and two
CTPs attached to the carboxy terminus of the GLP-1/Glucagon
receptor agonist. In another embodiment, disclosed herein is a
polypeptide consisting essentially of a GLP-1/Glucagon receptor
agonist and two to three CTPs, two to four CTPs, two to five CTPs,
two to six CTPs, two to seven CTPs, two to eight CTPs, two to nine
CTPs, or two to ten CTPs attached to the carboxy terminus of the
GLP-1/Glucagon receptor agonist.
[0297] In one embodiment, disclosed herein is a polypeptide
consisting essentially of an OXM and two CTPs attached to the
carboxy terminus of the OXM. In another embodiment, disclosed
herein is a polypeptide consisting essentially of an OXM and two to
three CTPs, two to four CTPs, two to five CTPs, two to six CTPs,
two to seven CTPs, two to eight CTPs, two to nine CTPs, or two to
ten CTPs attached to the carboxy terminus of the OXM.
[0298] In one embodiment, disclosed herein is a polypeptide
consisting essentially of a GLP-1/Glucagon receptor agonist and
three CTPs attached to the carboxy terminus of the GLP-1/Glucagon
receptor agonist. In another embodiment, disclosed herein is a
polypeptide consisting essentially of a GLP-1/Glucagon receptor
agonist and three to four CTPs, three to five CTPs, three to six
CTPs, three to seven CTPs, three to eight CTPs, three to nine CTPs,
or three to ten CTPs attached to the carboxy terminus of the
GLP-1/Glucagon receptor agonist.
[0299] In one embodiment, disclosed herein is a polypeptide
consisting essentially of an OXM and three CTPs attached to the
carboxy terminus of the OXM. In another embodiment, disclosed
herein is a polypeptide consisting essentially of an OXM and three
to four CTPs, three to five CTPs, three to six CTPs, three to seven
CTPs, three to eight CTPs, three to nine CTPs, three to ten CTPs
attached to the carboxy terminus of the OXM.
[0300] In one embodiment, disclosed herein is a polypeptide
consisting essentially of a GLP-1/Glucagon receptor agonist and
four CTPs attached to the carboxy terminus of the GLP-1/Glucagon
receptor agonist. In another embodiment, disclosed herein is a
polypeptide consisting essentially of a GLP-1/Glucagon receptor
agonist and four to five CTPs, four to six CTPs, four to seven
CTPs, four to eight CTPs, four to nine CTPs, or four to ten CTPs
attached to the carboxy terminus of the GLP-1/Glucagon receptor
agonist.
[0301] In one embodiment, disclosed herein is a polypeptide
consisting essentially of an OXM and four CTPs attached to the
carboxy terminus of the OXM. In another embodiment, disclosed
herein is a polypeptide consisting essentially of an OXM and four
to five CTPs, four to six CTPs, four to seven CTPs, four to eight
CTPs, four to nine CTPs, or four to ten CTPs attached to the
carboxy terminus of the OXM.
[0302] In one embodiment, disclosed herein is a polypeptide
consisting essentially of a GLP-1/Glucagon receptor agonist and
five CTPs attached to the carboxy terminus of the GLP-1/Glucagon
receptor agonist. In another embodiment, disclosed herein is a
polypeptide consisting essentially of a GLP-1/Glucagon receptor
agonist and five to six CTPs, five to seven CTPs, five to eight,
five to nine CTPs, or five to ten CTPs attached to the carboxy
terminus of the GLP-1/Glucagon receptor agonist.
[0303] In one embodiment, disclosed herein is a polypeptide
consisting essentially of an OXM and five CTPs attached to the
carboxy terminus of the OXM. In another embodiment, disclosed
herein is a polypeptide consisting essentially of an OXM and five
to six CTPs, five to seven CTPs, five to eight CTPs, five to nine
CTPs, or five to ten CTPs attached to the carboxy terminus of the
OXM.
[0304] In another embodiment, disclosed herein is a polypeptide
comprising, consisting essentially of, or consisting of a
GLP-1/Glucagon receptor agonist having no CTPs on its amino
terminus. In another embodiment, disclosed herein is a polypeptide
comprising, consisting essentially of, or consisting of a
GLP-1/Glucagon receptor agonist lacking a CTP on its amino
terminus. In another embodiment, disclosed herein is a polypeptide
comprising, consisting essentially of, or consisting of a
GLP-1/Glucagon receptor agonist having at least one CTP on its
carboxy terminus and no CTPs on its amino terminus. In another
embodiment, disclosed herein is a polypeptide comprising,
consisting essentially of, or consisting of a GLP-1/Glucagon
receptor agonist having the number of CTPs on its carboxy terminus
as described herein and no CTPs on its amino terminus.
[0305] In another embodiment, disclosed herein is a polypeptide
comprising, consisting essentially of, or consisting of an OXM
having no CTPs on its amino terminus. In another embodiment,
disclosed herein is a polypeptide comprising, consisting
essentially of, or consisting of an OXM lacking a CTP on its amino
terminus. In another embodiment, disclosed herein is a polypeptide
comprising, consisting essentially of, or consisting of an OXM
having at least one CTP on its carboxy terminus and no CTPs on its
amino terminus. In another embodiment, disclosed herein is a
polypeptide comprising, consisting essentially of, or consisting of
an OXM having the number of CTPs on its carboxy terminus as
described herein and no CTPs on its amino terminus.
[0306] In one embodiment, the amino acid sequence of the
CTP-modified polypeptide comprises SEQ ID NO: 58, 59, 60, 61, 62,
63, or 64, further disclosed herein. In another embodiment, the
amino acid sequence of the CTP-modified polypeptide is selected
from the group consisting of: SEQ ID NO: 58, 59, 60, 61, 62, 63, or
64, further disclosed herein.
[0307] In one embodiment, following expression a mature
CTP-modified OXM polypeptide is secreted, the signal peptide having
been cleaved from the precursor protein resulting in a mature
protein. For example, in the precursor CTP-modified OXM polypeptide
set forth in SEQ ID NO: 62, amino acids 1-26: M A T G S R T S L L L
A F G L L C L P W L Q E G S A (SEQ ID NO: 115) represent the signal
peptide of the precursor CTP-modified OXM polypeptide, and amino
acids H S Q G T F T S D Y S K Y L D S R R A Q D F V Q W L M N T K R
N R N N I A S S S S K A P P P S L P S P S R L P G P S D T P I L P Q
S S S S K A P P P S L P S P S R L P G P S D T P I L P Q S S S S K A
P P P S L P S P S R L P G P S D T P I L P Q (SEQ ID NO: 114)
represent the mature engineered CTP-modified OXM polypeptide
lacking the signal peptide. In one embodiment, the amino acid
sequence of CTP-modified OXM without the signal peptide is set
forth in SEQ ID NO: 114. In another embodiment, the signal peptide
of CTP-modified OXM is set forth in SEQ ID NO: 115.
[0308] In another embodiment, disclosed herein is a polynucleotide
encoding a polypeptide as described herein. In another embodiment,
the polynucleotide comprises SEQ ID NO: 65, 66, 67, 68, 69, 70 or
71, further disclosed herein. In another embodiment, the
polynucleotide is selected from the group consisting of SEQ ID NO:
65, 66, 67, 68, 69, 70 or 71, further disclosed herein.
[0309] In one embodiment, disclosed herein is a method of reducing
cholesterol in a subject, comprising the step of attaching at least
one CTP to an amino or a carboxy terminus of the OXM, thereby
reducing cholesterol in a subject.
[0310] In another embodiment, disclosed herein is a method of
reducing glycerol in a subject, comprising the step of attaching at
least one CTP to an amino or a carboxy terminus of the OXM, thereby
reducing glycerol in a subject.
[0311] In one embodiment, disclosed herein is a long-acting OXM. In
one embodiment, a long-acting OXM disclosed herein maintains the
biological activity of unmodified OXM. In another embodiment, the
long-acting OXM disclosed herein comprises OXM biological activity.
In another embodiment, the biological activity of a long-acting OXM
disclosed herein comprises reducing digestive secretions. In
another embodiment, the biological activity of a long-acting OXM
disclosed herein comprises reducing and delaying gastric emptying.
In another embodiment, the biological activity of a long-acting OXM
disclosed herein comprises the inhibition of the fed motility
pattern in the small intestine. In another embodiment, the
biological activity of a long-acting OXM disclosed herein comprises
the inhibition of acid secretion stimulated by pentagastrin. In
another embodiment, the biological activity of a long-acting OXM
disclosed herein comprises an increase of gastric somatostatin
release. In another embodiment, the biological activity of a
long-acting OXM disclosed herein comprises potentiating the effects
of peptide YY. In another embodiment, the biological activity of a
long-acting OXM disclosed herein comprises the inhibition of
ghrelin release. In another embodiment, the biological activity of
a long-acting OXM disclosed herein comprises the stimulation of
aminopyrine accumulation and cAMP production. In another
embodiment, the biological activity of a long-acting OXM disclosed
herein comprises binding the GLP-1 receptor. In another embodiment,
the biological activity of a long-acting OXM disclosed herein
comprises stimulating H+ production by activating adenylate
cyclase. In another embodiment, the biological activity of a
long-acting OXM disclosed herein comprises inhibiting
histamine-stimulated gastric acid secretion. In another embodiment,
the biological activity of a long-acting OXM disclosed herein
comprises inhibiting food intake. In another embodiment, the
biological activity of a long-acting OXM disclosed herein comprises
stimulating insulin release. In another embodiment, the biological
activity of a long-acting OXM disclosed herein comprises inhibiting
exocrine pancreatic secretion.
[0312] In another embodiment, the biological activity of a
long-acting OXM disclosed herein comprises inhibiting pancreatic
secretion through a vagal neural indirect mechanism. In another
embodiment, the biological activity of a long-acting OXM disclosed
herein comprises reducing hydromineral transport through the small
intestine. In another embodiment, the biological activity of a
long-acting OXM disclosed herein comprises stimulating glucose
uptake. In another embodiment, the biological activity of a
long-acting OXM disclosed herein comprises controlling/stimulating
somatostatin secretion. In another embodiment, the biological
activity of a long-acting OXM disclosed herein comprises reduction
in both food intake and body weight gain. In another embodiment,
the biological activity of a long-acting OXM disclosed herein
comprises reduction in adiposity. In another embodiment, the
biological activity of a long-acting OXM disclosed herein comprises
appetite suppression. In another embodiment, the biological
activity of a long-acting OXM disclosed herein comprises induction
of anorexia. In another embodiment, the biological activity of a
long-acting OXM disclosed herein comprises reducing body weight in
overweight and obese subjects. In another embodiment, the
biological activity of a long-acting OXM disclosed herein comprises
inducing changes in the levels of the adipose hormones leptin and
adiponectin. In another embodiment, the biological activity of a
long-acting OXM disclosed herein comprises increasing energy
expenditure in addition to decreasing energy intake in overweight
and obese subjects.
[0313] Thus, in one embodiment, disclosed herein is a method of
effecting any of the above-mentioned biological activities of OXM
by administering a CTP-modified OXM disclosed herein. In another
embodiment, disclosed herein is a method for reducing food intake,
reducing body weight, or both in a subject, comprising the step of
administering to the subject a CTP-modified OXM peptide. In another
embodiment, the subject has diabetes. In another embodiment, the
subject is overweight. In another embodiment, the subject is
obese.
[0314] A skilled artisan would appreciate that the terms "reducing,
reduction, lowering, etc." when used in relation to the methods
disclosed herein may encompass 100% reduction from a previously
measured or determined level or from a normal level. In another
embodiment, the reduction is by 89-99%, is by 79-88%, is by 69-78%,
is by 59-68%, is by 49-58%, is by 39-48%, is by 29-38%, is by
19-28%, is by 9-18%, is by 5-8%, or is by 1-4% from a previously
determined level.
[0315] The in-vivo biological activity of OXM-CTP variants was
assessed in two animal models, IPGTT in mice, which evaluate the
ability of OXM to induce glucose tolerance following glucose
administration, and food intake inhibition in lean rats, which
assess the ability of OXM to inhibit animal's food consumption. It
was demonstrated that OXM-CTP variants: CTP-OXM-CTP-CTP,
OXM-CTP-CTP, OXM-CTP-CTP-CTP, OXM-CTP-CTP-CTP-CTP and
OXM-CTP-CTP-CTP-CTP-CTP induced glucose tolerance as reflected by
reduction of 20-30% of blood glucose AUC compared to vehicle group
(see Example 4, herein). These results indicate that the biological
activity of OXM-CTP variants was not inhibited in-vivo due to
potential steric interference of CTP fusion to the binding of OXM
to its receptor. The marked reduction in glucose induced by these
variants correlated with their improved PK profiles. Therefore, in
one embodiment, disclosed herein is a method of inducing glucose
tolerance in a subject, the method comprising the step of
administering to the subject an effective dose of a composition
comprising a CTP-modified polypeptide comprising an OXM peptide and
at least one CTP attached to the amino terminus or caboxy terminus
of the OXM peptide, thereby inducing glucose tolerance in a
subject.
[0316] In another embodiment, disclosed herein is a method of
inducing glucose tolerance in a subject, the method comprising the
step of administering to the subject an effective dose of a
composition comprising a CTP-modified polypeptide comprising a
GLP-1/Glucagon receptor agonist and at least one CTP attached to
the amino terminus or caboxy terminus of the GLP-1/Glucagon
receptor agonist peptide, thereby inducing glucose tolerance in a
subject.
[0317] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a dual GLP-1/Glucagon receptor
agonist and at least one CTP attached to the amino terminus or
caboxy terminus of said agonist for inducing glucose tolerance in a
subject. In one embodiment, said dual GLP-1/Glucagon receptor
agonist is OXM.
[0318] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a dual GLP-1/Glucagon receptor
agonist and at least one CTP attached to the amino terminus or
caboxy terminus of said agonist in the preparation of a medicament
for inducing glucose tolerance in a subject. In one embodiment,
said dual GLP-1/Glucagon receptor agonist is OXM.
[0319] In one embodiment, disclosed herein is a method of
increasing insulin sensitivity in a subject, the method comprising
the step of administering to the subject an effective dose of a
composition comprising a CTP-modified polypeptide comprising a
GLP-1/Glucagon receptor agonist and at least one CTP attached to
the amino terminus or caboxy terminus of the GLP-1/Glucagon
receptor agonist peptide, thereby increasing insulin sensitivity in
a subject.
[0320] In one embodiment, disclosed herein is a method of reducing
insulin resistance in a subject, the method comprising the step of
administering to the subject an effective dose of a composition
comprising a CTP-modified polypeptide comprising a GLP-1/Glucagon
receptor agonist and at least one CTP attached to the amino
terminus or caboxy terminus of the GLP-1/Glucagon receptor agonist
peptide, thereby reducing insulin resistance in a subject.
[0321] In one embodiment, disclosed herein is a method of
increasing insulin sensitivity and reducing insulin resistance in a
subject, the method comprising the step of administering to the
subject an effective dose of a composition comprising a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one CTP attached to the amino terminus or
caboxy terminus of the GLP-1/Glucagon receptor agonist peptide,
thereby increasing insulin sensitivity and reducing insulin
resistance in a subject.
[0322] The ability of OXM-CTP variants OXM-3CTP, OXM-4CTP and
OXM-5CTP to inhibit food intake was assessed. Native OXM inhibited
food intake only in the first hour following administration,
however, all variants manifested substantial prolonged improved
inhibition in food intake compared to OXM. The cumulative food
inhibition of OXM-5CTP was surprisingly sustained for at least 141
hrs emphasizing the elongated half-life of this variant (see
Examples herein).
[0323] Hence, in one embodiment, disclosed herein is a method of
inducing food intake inhibition in a subject, the method comprising
the step of administering to the subject an effective dose of a
composition comprising a CTP-modified polypeptide comprising an OXM
(OXM) peptide and at least one CTP attached to the amino terminus
or carboxy terminus of the OXM peptide, thereby inducing food
intake inhibition in the subject.
[0324] In another embodiment, disclosed herein is a method of
inducing food intake inhibition in a subject, the method comprising
the step of administering to the subject an effective dose of a
composition comprising a CTP-modified polypeptide comprising a
GLP-1/Glucagon receptor agonist and at least one CTP attached to
the amino terminus or carboxy terminus of the GLP-1/Glucagon
receptor agonist peptide, thereby inducing food intake inhibition
in the subject.
[0325] In another embodiment, disclosed herein is a method of
preventing, reducing or suppressing food intake in a subject, the
method comprising the step of administering to the subject an
effective dose of a composition comprising CTP-modified polypeptide
comprising a GLP-1/Glucagon receptor agonist and at least one CTP
attached to the amino terminus or carboxy terminus of the
GLP-1/Glucagon receptor agonist peptide, thereby preventing
reducing or suppressing food intake by a subject. In another
embodiment, preventing, reducing or suppressing food intake by a
subject reduces the chances of the subject developing undesired
weight gain. In another embodiment, preventing reducing or
suppressing food intake in a subject reduces the chances of the
subject developing obesity.
[0326] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one CTP attached to the amino terminus or
carboxy terminus of said agonist for preventing undesired weight
gain by a subject. In one embodiment, said GLP-1/Glucagon receptor
agonist is OXM.
[0327] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one CTP attached to the amino terminus or
carboxy terminus of said agonist in the preparation of a medicament
for preventing undesired weight gain by a subject. In one
embodiment, said GLP-1/Glucagon receptor agonist is OXM.
[0328] In another embodiment, disclosed herein is a method of
preventing, reducing or suppressing food intake in a subject, the
method comprising the step of administering to the subject an
effective dose of a composition comprising CTP-modified polypeptide
comprising an OXM (OXM) peptide and at least one CTP attached to
the amino terminus or carboxy terminus of the OXM peptide, thereby
preventing reducing or suppressing food intake by a subject. In
another embodiment, preventing, reducing or suppressing food intake
by a subject reduces the chances of the subject developing
undesired weight gain. In another embodiment, preventing reducing
or suppressing food intake in a subject reduces the chances of the
subject developing obesity.
[0329] In another embodiment, disclosed herein is a method of
preventing undesired weight gain by a subject, the method
comprising the step of administering to the subject an effective
dose of a composition comprising CTP-modified polypeptide
comprising a GLP-1/Glucagon receptor agonist and at least one CTP
attached to the amino terminus or carboxy terminus of the
GLP-1/Glucagon receptor agonist peptide, thereby preventing weight
gain in a subject. In another embodiment, the weight gain leads to
or results in obesity of the subject. In another embodiment, the
risk of gaining weight gain is due to a psychological condition, or
due to a genetic predisposposition to gain weight by the subject.
In another embodiment, the psychological condition is depression,
anxiety or post-traumatic stress disorder (PTSD).
[0330] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one CTP attached to the amino terminus or
carboxy terminus of said agonist for preventing undesired weight
gain by a subject. In one embodiment, said GLP-1/Glucagon receptor
agonist is OXM.
[0331] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one CTP attached to the amino terminus or
carboxy terminus of said agonist in the preparation of a medicament
for preventing undesired weight gain by a subject. In one
embodiment, said GLP-1/Glucagon receptor agonist is OXM.
[0332] In another embodiment, disclosed herein is a method of
preventing undesired weight gain by a subject, the method
comprising the step of administering to the subject an effective
dose of a composition comprising CTP-modified polypeptide
comprising an OXM peptide and at least one CTP attached to the
amino terminus or carboxy terminus of the OXM peptide, thereby
preventing weight gain in a subject. In another embodiment, the
weight gain leads to or results in obesity of the subject. In
another embodiment, the risk of gaining weight gain is due to a
psychological condition, or due to a genetic predisposposition to
gain weight by the subject. In another embodiment, the
psychological condition is depression, anxiety or post-traumatic
stress disorder (PTSD).
[0333] In another embodiment, disclosed herein is a method of
treating obesity in a subject, the method comprising administering
to the subject an effective dose of a composition comprising a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist peptide and at least one chorionic gonadotropin carboxy
terminal peptide attached to the amino terminus or carboxy terminus
of the GLP-1/Glucagon receptor agonist peptide, thereby treating
obesity in the subject. In another embodiment, the subject is
genetically predisposed to being obese. In another embodiment, the
method of treating obesity results in a reduction of body weight in
a subject. In another embodiment, the reduction in body weight is
due to body fat reduction.
[0334] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one chorionic gonadotropin carboxy terminal
peptide attached to the amino terminus or carboxy terminus of said
agonist for treating obesity in a subject. In one embodiment, said
GLP-1/Glucagon receptor agonist is OXM.
[0335] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one chorionic gonadotropin carboxy terminal
peptide attached to the amino terminus or carboxy terminus of said
agonist in the preparation of a medicament for treating obesity in
a subject. In one embodiment, said GLP-1/Glucagon receptor agonist
is OXM.
[0336] In another embodiment, disclosed herein is a method of
treating obesity in a subject, the method comprising administering
to the subject an effective dose of a composition comprising a
CTP-modified polypeptide comprising an OXM peptide and at least one
chorionic gonadotropin carboxy terminal peptide attached to the
amino terminus or carboxy terminus of the OXM peptide, thereby
treating obesity in the subject. In another embodiment, the subject
is genetically predisposed to being obese.
[0337] In one embodiment, disclosed herein is a method of treating
type II diabetes in a subject, the method comprising administering
to the subject an effective dose of a composition comprising a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist peptide and at least one chorionic gonadotropin carboxy
terminal peptide attached to the amino terminus or carboxy terminus
of the GLP-1/Glucagon receptor agonist peptide, thereby treating
type II diabetes in the subject.
[0338] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one chorionic gonadotropin carboxy terminal
peptide attached to the amino terminus or carboxy terminus of said
agonist for treating type II diabetes in a subject. In one
embodiment, said GLP-1/Glucagon receptor agonist is OXM.
[0339] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one chorionic gonadotropin carboxy terminal
peptide attached to the amino terminus or carboxy terminus of said
agonist in the preparation of a medicament for treating type II
diabetes in a subject. In one embodiment, said GLP-1/Glucagon
receptor agonist is OXM.
[0340] In one embodiment, disclosed herein is a method of treating
type II diabetes in a subject, the method comprising administering
to the subject an effective dose of a composition comprising a
CTP-modified polypeptide comprising an OXM peptide and at least one
chorionic gonadotropin carboxy terminal peptide attached to the
amino terminus or carboxy terminus of the OXM peptide, thereby
treating type II diabetes in the subject.
[0341] In another embodiment, disclosed herein is a method of
treating a metabolic disorder in a subject, the method comprising
administering to the subject an effective dose of a composition
comprising a CTP-modified polypeptide comprising a GLP-1/Glucagon
receptor agonist and at least one chorionic gonadotropin carboxy
terminal peptide attached to the amino terminus or carboxy terminus
of the GLP-1/Glucagon receptor agonist peptide, thereby treating a
metabolic disorder in the subject.
[0342] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one chorionic gonadotropin carboxy terminal
peptide attached to the amino terminus or carboxy terminus of said
agonist for treating a metabolic disorder in a subject. In one
embodiment, said GLP-1/Glucagon receptor agonist is OXM.
[0343] In another embodiment, disclosed herein is a use of a
CTP-modified polypeptide comprising a GLP-1/Glucagon receptor
agonist and at least one chorionic gonadotropin carboxy terminal
peptide attached to the amino terminus or carboxy terminus of said
agonist in the preparation of a medicament for treating a metabolic
disorder in a subject. In one embodiment, said GLP-1/Glucagon
receptor agonist is OXM.
[0344] In another embodiment, disclosed herein is a method of
treating a metabolic disorder in a subject, the method comprising
administering to the subject an effective dose of a composition
comprising a CTP-modified polypeptide comprising an OXM peptide and
at least one chorionic gonadotropin carboxy terminal peptide
attached to the amino terminus or carboxy terminus of the OXM
peptide, thereby treating a metabolic disorder in the subject.
[0345] In another embodiment, the metabolic disorder is diabetic
ketoacidosis, or diabetes mellitus or any glucose-related metabolic
disorder known in the art. In another embodiment, the metabolic
disorder results from a lack of insulin and an overabundance of
glucose in a subject.
[0346] In another embodiment, the engineered GLP-1/Glucagon
receptor agonist variants disclosed herein are synthesized using a
polynucleotide molecule encoding a polypeptide disclosed herein. In
another embodiment, the polynucleotide molecule encoding the
engineered GLP-1/Glucagon receptor agonist disclosed herein is
ligated into an expression vector, comprising a transcriptional
control of a cis-regulatory sequence (e.g., promoter sequence). In
another embodiment, the cis-regulatory sequence is suitable for
directing constitutive expression of an engineered GLP-1/Glucagon
receptor agonist disclosed herein. In another embodiment, the
cis-regulatory sequence is suitable for directing tissue-specific
expression of the engineered GLP-1/Glucagon receptor agonist
peptides disclosed herein. In another embodiment, the
cis-regulatory sequence is suitable for directing inducible
expression of the engineered GLP-1/Glucagon receptor agonist
variants disclosed herein.
[0347] In another embodiment, disclosed herein is a cell comprising
the expression vector as described herein. In another embodiment,
disclosed herein is a cell comprising an expression vector
comprising a polynucleotide encoding a CTP-modified polypeptide
comprising or consisting of a GLP-1/Glucagon receptor agonist and
at least one CTPs attached to the amino or carboxy terminus of the
agonist. In another embodiment, disclosed herein is a cell
comprising an expression vector comprising a polynucleotide
encoding a CTP-modified polypeptide consisting of a GLP-1/Glucagon
receptor agonist and at least one CTPs attached to the amino or
carboxy terminus of the agonist.
[0348] In another embodiment, disclosed herein is a composition
comprising the expression vector as described herein. In another
embodiment, disclosed herein is a composition comprising an
expression vector comprising a polynucleotide encoding a
CTP-modified polypeptide comprising or consisting of a
GLP-1/Glucagon receptor agonist and at least one CTPs attached to
the amino or carboxy terminus of the agonist. In another
embodiment, disclosed herein is a cell comprising an expression
vector comprising a polynucleotide encoding a CTP-modified
polypeptide consisting of the agonist and at least one CTPs
attached to the amino or carboxy terminus of the agonist. In
another embodiment, the agonist is a polypeptide, or a peptide. In
another embodiment, the peptide is OXM.
[0349] In another embodiment, disclosed herein is a composition
comprising the cell as described herein. In another embodiment, the
cell is a eukaryotic cell. In another embodiment, the cell is a
prokaryotic cell.
[0350] In one embodiment, disclosed herein is a dual GLP-1/Glucagon
receptor agonist. In another embodiment, disclosed herein is a
recombinant OXM as described hereinabove. In one embodiment,
disclosed herein is a n engineered OXM as described hereinabove. In
one embodiment, the engineered OXM as described hereinabove is
referred to as a CTP-modified OXM.
[0351] In one embodiment, the CTPs that are attached to the carboxy
terminus of the OXM are attached in tandem to the carboxy terminus.
In one embodiment, the CTPs that are attached to the amino terminus
of the OXM are attached in tandem to the amino terminus.
[0352] In another embodiment, disclosed herein is a pharmaceutical
composition comprising a CTP-modified polypeptide consisting of a
GLP-1/Glucagon receptor agonist and three CTPs attached to the
carboxy terminus of the OXM.
[0353] In another embodiment, disclosed herein is a pharmaceutical
composition comprising a CTP-modified polypeptide consisting of an
OXM and three CTPs attached to the carboxy terminus of the OXM.
[0354] In one embodiment, an engineered GLP-1/Glucagon receptor
agonist as described herein has equivalent or improved biological
activity compared to the non-CTP-modified GLP-1/Glucagon receptor
agonist. In another embodiment, an engineered GLP-1/Glucagon
receptor agonist as disclosed herein has equivalent or improved
pharmacological measurements compared to the non-CTP-modified
GLP-1/Glucagon receptor agonist. In another embodiment, an
engineered GLP-1/Glucagon receptor agonist as disclosed herein has
equivalent or improved pharmacokinetics compared to the
non-CTP-modified GLP-1/Glucagon receptor agonist. In another
embodiment, an engineered GLP-1/Glucagon receptor agonist as
disclosed herein has equivalent or improved pharmacodynamics
compared to the non-CTP-modified GLP-1/Glucagon receptor
agonist.
[0355] A skilled artisan would appreciate that while particular
embodiments of CTP-modified polypeptides have been disclosed above,
a polypeptide or peptide of interest may encompass any polypeptide
or peptide of interest having a functional activity. In another
embodiment the polypeptide sequence-of-interest is insulin, is
enkephalin, is an ACTH, is a glucagon, is an insulin-like growth
factor, is an epidermal growth factor, is an acidic or basic
fibroblast growth factor, is a platelet-derived growth factor, is a
granulocyte-CSF, is a macrophage-CSF, is an IL-2, is an IL-3, is a
tumor necrosis factor, is an LHRH, is an LHRH analog, is a
somatostatin, is a growth hormone releasing factor, is an
endorphin, is an alveolar surfactant protein, is a natriuretic
factor, is an adhesin, is an angiostatin, is an endostatin, is a
cytokine, is a receptor peptide, is a receptor binding ligand, is
an antibody, is an antibody fragment, or is a peptide or a protein
including any modified form.
[0356] In another embodiment, the peptide disclosed herein
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, or
monoclonal antibodies.
[0357] In another embodiment, the methods disclosed herein
manufacture 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.
[0358] In another embodiment, the methods disclosed herein
manufacture 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.
[0359] In another embodiment, the methods disclosed herein
manufacture 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.
[0360] In another embodiment, the methods disclosed herein
manufacture 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.
[0361] In another embodiment, the methods disclosed herein
manufacture 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.
[0362] In another embodiment, the methods disclosed herein
manufacture immune globulin is a CMV immune globulin. In another
embodiment, the methods disclosed herein manufacture
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.
[0363] In another embodiment, the methods disclosed herein
manufacture 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.
[0364] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein manufacture 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.
[0365] In another embodiment, the methods disclosed herein
manufacture 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.
[0366] In another embodiment, the methods disclosed herein
manufacture 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.
[0367] In another embodiment, the methods disclosed herein
manufacture 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.
[0368] In another embodiment, the methods disclosed herein
manufacture 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.
[0369] In another embodiment, the methods disclosed herein
manufacture 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.
[0370] In another embodiment, the methods disclosed herein
manufacture 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.
[0371] In another embodiment, the methods disclosed herein
manufacture 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.
[0372] In another embodiment, the methods disclosed herein
manufacture 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.
[0373] In another embodiment, the methods disclosed herein
manufacture 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.
[0374] In another embodiment, the methods disclosed herein
manufacture 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 disclosed herein manufacture 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.
[0375] In another embodiment, the methods disclosed herein
manufacture 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.
[0376] In another embodiment, the methods disclosed herein
manufacture 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.
[0377] In another embodiment, the methods disclosed herein
manufacture 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.
[0378] In another embodiment, the methods disclosed herein
manufacture 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.
[0379] In another embodiment, the methods disclosed herein
manufacture 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.
[0380] In another embodiment, the methods disclosed herein
manufacture 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.
[0381] In another embodiment, the methods disclosed herein
manufacture 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.
[0382] In another embodiment, the methods disclosed herein
manufacture 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.
[0383] In some embodiments, the CTP sequences modification is
advantageous in permitting lower dosages to be used. 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 disclosed
hereinunder.
[0384] 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 U-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.
[0385] In some embodiments, natural aromatic amino acids of the
polypeptide such as Trp, Tyr and Phe, are 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 disclosed
herein include one or more modified amino acid or one or more
non-amino acid monomers (e.g. fatty acid, complex carbohydrates
etc).
[0386] 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.
[0387] In some embodiments, the polypeptides disclosed herein are
utilized in therapeutics which require the polypeptides to be in a
soluble form. In some embodiments, the polypeptides disclosed
herein 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.
[0388] In some embodiments, the polypeptides disclosed herein is
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.
[0389] In some embodiments, the polypeptides disclosed herein 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.
[0390] 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.
[0391] In some embodiments, recombinant protein techniques are used
to generate the polypeptides disclosed herein. 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 disclosed
herein. 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.
[0392] In one embodiment, a polypeptide disclosed herein is
synthesized using a polynucleotide encoding a polypeptide disclosed
herein. In some embodiments, the polynucleotide encoding a
polypeptide disclosed herein 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 disclosed herein. In some embodiments, the
cis-regulatory sequence is suitable for directing tissue specific
expression of the polypeptide disclosed herein. In some
embodiments, the cis-regulatory sequence is suitable for directing
inducible expression of the polypeptide disclosed herein.
[0393] In some embodiments, the polypeptides of interest disclosed
herein 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 cytokines
characteristics, cyclic forms of the cytokines can also be
utilized.
[0394] In some embodiments, the polypeptide of interests disclosed
herein is 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 of
interests are 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.
[0395] In some embodiments, solid phase polypeptide of interests
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.
[0396] In some embodiments, recombinant protein techniques are used
to generate the polypeptide of interests disclosed herein. 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 interests
disclosed herein. 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.
[0397] In another embodiment, polypeptides of interests disclosed
herein are synthesized using a polynucleotide encoding a
polypeptide disclosed herein. In some embodiments, the
polynucleotide encoding polypeptide of interests disclosed herein
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 interests
disclosed herein. In some embodiments, the cis-regulatory sequence
is suitable for directing tissue specific expression of the
polypeptide of interests disclosed herein. In some embodiments, the
cis-regulatory sequence is suitable for directing inducible
expression of the polypeptide of interests disclosed herein.
[0398] In some embodiment, tissue-specific promoters suitable for
use with a nucleotide sequence disclosed herein 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 a nucleotide sequence disclosed herein
include for example the tetracycline-inducible promoter (Srour, M.
A., et al., 2003. Thromb. Haemost. 90: 398-405).
[0399] A skilled artisan would appreciate that the phrase "a
polynucleotide" may encompass 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).
[0400] A skilled artisan would appreciate that the term
"complementary polynucleotide sequence" may encompass 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.
[0401] A skilled artisan would appreciate that the term "genomic
polynucleotide sequence" may encompass a sequence derived
(isolated) from a chromosome and thus it represents a contiguous
portion of a chromosome.
[0402] A skilled artisan would appreciate that the term "composite
polynucleotide sequence" may encompass 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 disclosed herein, 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.
[0403] In some embodiments, polynucleotides disclosed herein are
prepared using PCR techniques or any other technique known to one
skilled in the art. In some embodiments, the procedure involves the
ligation of two different DNA sequences (See, for example, "Current
Protocols in Molecular Biology", eds. Ausubel et al., John Wiley
& Sons, 1992).
[0404] In one embodiment, the polypeptides disclosed herein can be
provided to the individual per se. In one embodiment, the
polypeptides disclosed herein can be provided to the individual as
part of a pharmaceutical composition where it is mixed with a
pharmaceutically acceptable carrier.
[0405] In one embodiment, the oral dosage form comprises predefined
release profile. In one embodiment, the oral dosage form disclosed
herein comprises an extended release tablets, capsules, lozenges or
chewable tablets. In one embodiment, the oral dosage form disclosed
herein comprises a slow release tablets, capsules, lozenges or
chewable tablets. In one embodiment, the oral dosage form disclosed
herein 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.
[0406] 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%.
[0407] In some embodiments, compositions for use in the methods
disclosed herein comprise solutions or emulsions, which in some
embodiments are aqueous solutions or emulsions comprising a safe
and effective amount of the compounds disclosed herein 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.
[0408] 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.
[0409] 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 disclosed herein 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.
[0410] In one embodiment, pharmaceutical compositions disclosed
herein 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.
[0411] In one embodiment, pharmaceutical compositions for use in
accordance with a nucleotide sequence disclosed herein 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.
[0412] In one embodiment, injectables, disclosed herein are
formulated in aqueous solutions. In one embodiment, injectables,
disclosed herein 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.
[0413] 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.
[0414] 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.
[0415] 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.
[0416] 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).
[0417] 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).
[0418] 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.
[0419] In one embodiment, the preparation disclosed herein is
formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0420] In some embodiments, pharmaceutical compositions suitable
for use in context disclosed herein 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.
[0421] In one embodiment, determination of a therapeutically
effective amount is well within the capability of those skilled in
the art.
[0422] 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.
[0423] 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.
[0424] 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-HCI., 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.
[0425] 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.
[0426] The compositions also include incorporation of the active
material into or onto particulate preparations of polymeric
compounds such as polylactic acid, polglycolic 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.
[0427] Also comprehended herein 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.
[0428] 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.
[0429] 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.
[0430] 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].
[0431] 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.
[0432] 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.
[0433] In one embodiment, compositions including the preparation
disclosed herein formulated in a compatible pharmaceutical carrier
are also be prepared, placed in an appropriate container, and
labeled for treatment of an indicated condition.
[0434] In one embodiment, compositions disclosed herein 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.
[0435] In one embodiment, it will be appreciated that the
polypeptides disclosed herein 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.
[0436] 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 comprising a polypeptide of interest
even more stable while in a body or more capable of penetrating
into cells.
Manufacturing
[0437] In one embodiment, disclosed herein is a method of
manufacturing a human chorionic gonadotropin peptide (CTP)-modified
polypeptide of interest polypeptide, the method comprising the
steps of: (a) stably transfecting a predetermined number of cells
with an expression vector comprising a coding portion encoding said
CTP-modified polypeptide of interest, wherein said transfected cell
expresses and optionally secretes said CTP-modified polypeptide of
interest; (b) obtaining cell clones that overexpress said
CTP-modified polypeptide of interest; (c) expanding said clones in
solution to a predetermined scale; (d) harvesting said solution
containing said clones; (e) filtering said solution containing said
clones to obtain a clarified harvest solution; and, (f) purifying
said clarified harvest solution to obtain a purified protein
solution having a desired concentration of a CTP-modified
polypeptide of interest, thereby manufacturing a human chorionic
gonadotropin peptide (CTP)-modified polypeptide of interest
polypeptide. In another embodiment, the CTP-modified polypeptide of
interest is secreted. In another embodiment, the CTP-modified
polypeptide of interest is not secreted. In the manufacturing of
CTP-modified polypeptide of interest transfection is an early step.
Once the final high expressing clone is selected, each production
includes thawing of a working cell bank (WCB), expansion, harvest
and purification. (See Example 12, steps 1-7 describing clone
expansion through harvest; steps 8-16 describing purification)
[0438] In one embodiment, polynucleotides disclosed herein are
inserted into expression vectors (i.e., a nucleic acid construct)
to enable expression of the recombinant polypeptide. In one
embodiment, the expression vector disclosed herein includes
additional sequences which render this vector suitable for
replication and integration in prokaryotes. In one embodiment, the
expression vector disclosed herein includes additional sequences
which render this vector suitable for replication and integration
in eukaryotes. In one embodiment, the expression vector disclosed
herein 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, enhancer) and
transcription and translation terminators (e.g., polyadenylation
signals).
[0439] In one embodiment, a method of manufacture of a CTP-modified
polypeptide, for example, a CTP-modified polypeptide of interest,
comprises a step comprising the use of an expression vector,
wherein said expression vector comprises a promoter, a coding
sequence for a CTP-modified polypeptide, and a polyadenylation
sequence. In another embodiment, the polypeptide is polypeptide of
interest. In another embodiment the polyadenylation sequence is a
simian virus (SV) 40 polyadenylation sequence.
[0440] In one embodiment, a variety of prokaryotic or eukaryotic
cells can be used as host-expression systems to express the
polypeptides disclosed herein. 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.
[0441] In one embodiment, non-bacterial expression systems are used
(e g mammalian expression systems such as CHO cells or cells
derived from CHO cells) to express the polypeptide disclosed
herein. In one embodiment, the expression vector used to express
polynucleotides disclosed herein in mammalian cells is pCI-DHFR
vector comprising a CMV promoter and a neomycin resistance
gene.
[0442] In one embodiment, the expression vector disclosed herein
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.
[0443] 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.
[0444] In some embodiments, expression vectors containing
regulatory elements from eukaryotic viruses such as retroviruses
are used by methods disclosed herein. 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.
[0445] In one embodiment, various methods can be used to introduce
the expression vector encoding the CTP-modified polypeptide of
interest disclosed herein into cells. "Transfection" of eukaryotic
host cells with a polynucleotide or expression vector, resulting in
genetically modified cells or transgenic cells, can be performed by
any method well known in the art and described, e.g., 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 at.
[Biotechniques 4 (6): 504-512, 1986] and include, for example,
stable or transient transfection, and electroporation. In addition,
see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative
selection methods. Transfection methods further include but are not
limited to liposome-mediated transfection, calcium phosphate
co-precipitation, electroporation, polycation (such as
DEAE-dextran)-mediated transfection, protoplast fusion, viral
infections (including recombinant viral infections) and
microinjection. Preferably, the transfection is a stable
transfection. The transfection method that provides optimal
transfection frequency and expression of the heterologous genes
encoding the peptide of interest disclosed herein in the particular
host cell line and type is favored. Suitable methods can be
determined by routine procedures. For stable transfectants the
constructs are either integrated into the host cell's genome or an
artificial chromosome/mini-chromosome or located episomally so as
to be stably maintained within the host cell.
[0446] The practice disclosed herein will employ, unless otherwise
indicated, conventional techniques of cell biology, molecular
biology, cell culture, immunology and the like which are in the
skill of one in the art. These techniques are fully disclosed in
the current literature. See e.g. Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2.sup.nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al.,
Current Protocols in Molecular Biology (1987, updated); Brown ed.,
Essential Molecular Biology, IRL Press (1991); Goeddel ed., Gene
Expression Technology, Academic Press (1991); Bothwell et al. eds.,
Methods for Cloning and Analysis of Eukaryotic Genes, Bartlett
Publ. (1990); Wu et al., eds., Recombinant DNA Methodology,
Academic Press (1989); Kriegler, Gene Transfer and Expression,
Stockton Press (1990); McPherson et al., PCR: A Practical Approach,
IRL Press at Oxford University Press (1991); Gait ed.,
Oligonucleotide Synthesis (1984); Miller & Calos eds., Gene
Transfer Vectors for Mammalian Cells (1987); Butler ed., Mammalian
Cell Biotechnology (1991); Pollard et al., eds, Animal Cell
Culture, Humana Press (1990); Freshney et al., eds., Culture of
Animal Cells, Alan R. Liss (1987); Studzinski, ed., Cell Growth and
Apoptosis, A Practical Approach, IRL Press at Oxford University
Press (1995); Melamed et al., eds., Flow Cytometry and Sorting,
Wiley-Liss (1990); Current Protocols in Cytometry, John Wiley &
Sons, Inc. (updated); Wirth & Hauser, Genetic Engineering of
Animals Cells, in: Biotechnology Vol. 2, Paler ed., VCH, Weinheim
663-744; the series Methods of Enzymology (Academic Press, Inc.),
and Harlow et al., eds., Antibodies: A Laboratory Manual
(1987).
[0447] A heterologous gene of interest encoding the CTP-modified
polypeptide of interest may be introduced into the cell disclosed
herein by various methods, for example by viral transformation,
transfection or microinjection. The heterologous gene of interest
may be introduced into the cell as linear DNA or as part of an
expression vector. A number of eukaryotic expression vectors are
known which allow multiple cloning sites for the insertion of one
or more heterologous genes and their expression. Commercial
suppliers include among others companies such as Stratagene, La
Jolla, Calif., USA; Invitrogen, Carlsbad, Calif., USA; Promega,
Madison, Wis., USA or BD Biosciences Clontech, Palo Alto, Calif.,
USA. The transfection of the cells with a DNA or an expression
vector which code(s) for one or more genes of interest is carried
out by conventional methods as described for example in Sambrook et
al., 1989 or Ausubel et al., 1994. Suitable methods of transfection
include for example liposome-mediated transfection, calcium
phosphate co-precipitation, electroporation, polycation- (e.g. DEAE
dextran)-mediated transfection, protoplast fusion, microinjection
and viral infections. Preferably, stable transfection is carried
out in which the DNA molecules are either integrated into the
genome of the host cell or an artificial chromosome/minichromosome,
or are episomally contained in stable manner in the host cell. The
transfection method which gives the optimum transfection frequency
and expression of one or more heterologous genes of interest in the
host cell in question is preferred.
[0448] 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.
[0449] In one embodiment, it will be appreciated that the
polypeptides disclosed herein 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).
[0450] The heterologous gene of interest is usually functionally
linked to a promoter which enables the transcription of the gene of
interest, and to other regulatory elements which allow
transcription and translation (expression) of the gene of interest
or increase its efficiency.
[0451] A skilled artisan would appreciate that the term "promoter"
may encompass a polynucleotide sequence which enables and controls
transcription of the genes or sequences functionally linked to it.
A promoter contains recognition sequences for binding RNA
polymerase and the initiation site for transcription (transcription
initiation site). In order to express a desired sequence in a
certain cell type or a host cell a suitable functional promoter
must be chosen. The skilled man will be familiar with a variety of
promoters from various sources, including constitutive, inducible
and repressible promoters. They are deposited in databanks such as
GenBank, for example, and may be obtained as separate elements or
elements cloned within polynucleotide sequences from commercial or
individual sources. In inducible promoters the activity of the
promoter may be reduced or increased in response to a signal. One
example of an inducible promoter is the tetracycline (tet)
promoter. This contains tetracycline operator sequences (tetO)
which can be induced by a tetracycline-regulated transactivator
protein (tTA). In the presence of tetracycline the binding of tTA
to tetO is inhibited. Examples of other inducible promoters are the
jun, fos, metallothionein and heat shock promoter (see also
Sambrook, J., Fritsch, E. F. & Maniatis, T., Molecular Cloning:
A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1989; Gossen, M. et al., Curr Opi Biotech 1994, 5,
516-520). Of the promoters which are particularly suitable for high
expression in eukaryotes, there are for example the ubiquitin/S27a
promoter of the hamster (WO 97/15664), SV 40 early promoter,
adenovirus major late promoter, mouse metallothionein-1 promoter,
the long terminal repeat region of Rous Sarcoma Virus and the early
promoter of human Cytomegalovirus. Examples of other heterologous
mammalian promoters are the actin, immunoglobulin or heat shock
promoter(s).
[0452] For example, the promoter may be functionally linked to
enhancer sequences in order to increase the transcriptional
activity. For this, one or more enhancers and/or several copies of
an enhancer sequence may be used, e.g. a CMV or SV40 enhancer. For
example, the promoter may be functionally linked to enhancer
sequences in order to increase the transcriptional activity. For
this, one or more enhancers and/or several copies of an enhancer
sequence may be used, e.g. a CMV or SV40 enhancer.
[0453] A skilled artisan would appreciate that the term enhancer
may encompass a polynucleotide sequence which in the cis location
acts on the activity of a promoter and thus stimulates the
transcription of a gene functionally connected to this promoter.
Unlike promoters the effect of enhancers is independent of position
and orientation and they can therefore be positioned in front of or
behind a transcription unit, within an intron or even within the
coding region. The enhancer may be located both in the immediate
vicinity of the transcription unit and at a considerable distance
from the promoter. It is also possible to have a physical and
functional overlap with the promoter. The skilled artisan will be
aware of a number of enhancers from various sources (and deposited
in databanks such as GenBank, e.g. SV40 enhancers, CMV enhancers,
polyoma enhancers, adenovirus enhancers) which are available as
independent elements or elements cloned within polynucleotide
sequences (e.g. deposited at the ATCC or from commercial and
individual sources). A number of promoter sequences also contain
enhancer sequences such as the frequently used CMV promoter. The
human CMV enhancer is one of the strongest enhancers identified
hitherto. One example of an inducible enhancer is the
metallothionein enhancer, which can be stimulated by
glucocorticoids or heavy metals.
[0454] Basically, the regulatory elements include promoters,
enhancers, termination and polyadenylation signals and other
expression control elements. Both inducible and constitutively
regulatory sequences are known for the various cell types.
"Transcription-regulatory elements" generally comprise a promoter
upstream of the gene sequence to be expressed, transcription
initiation and termination sites and a polyadenylation signal.
[0455] A skilled artisan would appreciate that the term
"transcription initiation site" may encompass a nucleic acid in the
construct which corresponds to the first nucleic acid which is
incorporated in the primary transcript, i.e. the mRNA precursor.
The transcription initiation site may overlap with the promoter
sequences.
[0456] A skilled artisan would appreciate that the term
"transcription termination site" may encompass a nucleotide
sequence which is normally at the 3' end of the gene of interest or
of the gene section which is to be transcribed, and which brings
about the termination of transcription by RNA polymerase.
[0457] A skilled artisan would appreciate that the term
"polyadenylation signal" may encompass a signal sequence which
causes cleavage at a specific site at the 3' end of the eukaryotic
mRNA and posttranscriptional incorporation of a sequence of about
100-200 adenine nucleotides (polyA tail) at the cleaved 3'-end. The
polyadenylation signal comprises the sequence AATAAA about 10-30
nucleotides upstream of the cleavage site and a sequence located
downstream. Various polyadenylation elements are known such as tk
polyA, SV40 late and early polyA or BGH polyA (described for
example in U.S. Pat. No. 5,122,458).
[0458] "Translation regulatory elements" comprise a translation
initiation site (AUG), a stop codon and a polyA signal for each
polypeptide to be expressed. For optimum expression it may be
advisable to remove, add or change 5'- and/or 3'-untranslated
regions of the nucleic acid sequence which is to be expressed, in
order to eliminate any potentially unsuitable additional
translation initiation codons or other sequences which might affect
expression at the transcription or expression level. In order to
promote expression, ribosomal consensus binding sites may
alternatively be inserted immediately upstream of the start codon.
In order to produce a secreted polypeptide the gene of interest
usually contains a signal sequence which codes for a signal
precursor peptide which transports the synthesized polypeptide to
and through the ER membrane. The signal sequence is often but not
always located at the amino terminus of the secreted protein and is
cleaved by signal peptidases after the protein has been filtered
through the ER membrane. The gene sequence will usually but not
necessarily contain its own signal sequence. If the native signal
sequence is not present a heterologous signal sequence may be
introduced in known manner Numerous signal sequences of this kind
are known to the skilled artisan and deposited in sequence
databanks such as GenBank and EMBL.
[0459] A skilled artisan would appreciate that the terms
"polypeptides", "polypeptide" or grammatical equivalents thereof,
may be used interchangeably to encompass amino acid sequences or
proteins and may encompass polymers of amino acids of any length.
This term also includes proteins which have been modified
post-translationally by reactions such as glycosylation,
phosphorylation, acetylation or protein processing. The structure
of the polypeptide may be modified, for example, by substitutions,
deletions or insertions of amino acids and fusion with other
proteins while retaining its biological activity.
[0460] In order to produce one or more gene products of interest in
the cells, the cells may be grown in a serum-free culture medium
and in suspension culture under conditions which allow expression
of the gene of interest. If for example the gene of interest is
under the control of a constitutive promoter, there is no need to
add special inducers. If the expression of the gene of interest is
under the control of an inducible promoter, for example, a
corresponding inducer must be added to the cell culture medium in a
sufficient but non-toxic concentration. The cells can be expanded
as desired by multiple subpassaging and transferred into suitable
cell culture vessels. The gene product(s) is or are produced as
either a cellular, membrane-bound or secretory product.
[0461] In one embodiment, a step of manufacturing a CTP-modified
polypeptide of interest comprises stably transfecting a
predetermined number of cells with an expression vector comprising
a coding portion encoding said CTP-modified polypeptide of
interest. In another embodiment, a step of manufacturing a
CTP-modified polypeptide of interest comprises stably transfecting
cells with an expression vector comprising a coding portion
encoding said CTP-modified polypeptide of interest. In one
embodiment, cells are CHO cells. In another embodiment, cells are
DG44 cells. In another embodiment, cells are any cells known in the
art suitable for expression and secretion of CTP-modified
polypeptide of interest.
[0462] In another embodiment, the transfected cells express
CTP-modified polypeptide of interest. In another embodiment, the
CTP-modified polypeptide of interest being manufactured and
expressed consists of two CTP attached to the carboxy terminus of
said polypeptide of interest, and one chorionic gonadotropin
carboxy terminal peptide attached to the amino terminus of said
polypeptide of interest. In another embodiment, the CTP-modified
polypeptide of interest being manufactured and expressed consists
of one chorionic gonadotropin carboxy terminal peptide attached to
the carboxy terminus of said polypeptide of interest. In other
embodiment, the expression of CTP-modified polypeptide of interest
is at a high expression level. In another embodiment, said
CTP-modified polypeptide of interest is highly glycosylated. In
another embodiment, said CTP-modified polypeptide of interest is
highly sialated. As described above in detail, CTP-modified
polypeptide of interest may have different glycosylation content
and patterns. A CTP-modified polypeptide of interest manufactured
by the methods disclosed herein may include any of the
glycosylation patterns and content as disclosed above. In general,
method of manufacture presented here, provide a CTP-modified
polypeptide of interest having a high glycosylation content and a
high percentage of glycosylation sites glycosylated.
[0463] In one embodiment, a step of manufacturing a CTP-modified
polypeptide of interest comprises obtaining cell clones that
overexpress the CTP-modified polypeptide of interest. In another
embodiment, expression of CTP-modified polypeptide of interest is
optimal. In another embodiment, the level of expression is between
30-1500 mg/L. In another embodiment, the level of expression is at
least 30 mg/L. In another embodiment, the level of expression is at
least 40 mg/L. In another embodiment, the level of expression is at
least 50 mg/L. In another embodiment, the level of expression is at
least 60 mg/L. In another embodiment, the level of expression is at
least 70 mg/L. In another embodiment, the level of expression is
between 50-70 mg/L. In another embodiment, the level of expression
is at least 200 mg/L. In another embodiment, the level of
expression is at least 300 mg/L. In another embodiment, the level
of expression is at least 400 mg/L. In another embodiment, the
level of expression is at least 500 mg/L. In another embodiment,
the level of expression is at least 600 mg/L. In another
embodiment, the level of expression is at least 700 mg/L. In
another embodiment, the level of expression is at least 800 mg/L.
In another embodiment, the level of expression is at least 900
mg/L. In another embodiment, the level of expression is at least
1000 mg/L. In another embodiment, the level of expression is at
least 1100 mg/L. In another embodiment, the level of expression is
at least 1200 mg/L. In another embodiment, the level of expression
is at least 1300 mg/L. In another embodiment, the level of
expression is at least 1400 mg/L. In another embodiment, the level
of expression is at least 1500 mg/L. In another embodiment, the
clones in step (b) are propagated in medium to form a master cell
bank (MCB) and a working cell bank (WCB). In one embodiment, clones
at step (c) are obtained from a MCB. In another embodiment, clones
at step (c) are obtained from a WCB.
[0464] The product of interest is obtained from the cell culture
medium as a secreted gene product. If a protein or polypeptide is
expressed without a secretion signal, however, the gene product may
also be isolated from cell lysates. In order to obtain a pure
homogeneous product which is substantially free from other
recombinant proteins and host cell proteins, conventional
purification procedures are carried out. First of all, cells and
cell debris are frequently removed from the culture medium or
lysate. The desired gene product can then be freed from
contaminating soluble proteins, polypeptides and nucleic acids,
e.g. by fractionation on immunoaffinity and ion exchange columns,
ethanol precipitation, reversed phase HPLC or chromatography on
Sephadex, hydroxyapatite, silica or cation exchange resins such as
DEAE (see Examples herein). Methods known in the art and which
result in the purification of a heterologous protein expressed by
recombinant host cells are known to the skilled man and described
in the literature, e.g. by Harris et al. (Harris et al., Protein
Purification: A Practical Approach, Pickwood and Hames, eds., IRL
Press, Oxford, 1995) and Scopes (Scopes, R., Protein Purification,
Springer Verlag, 1988). These methods may be employed in whole or
in part in the methods disclosed herein.
[0465] In another embodiment, disclosed herein is a method of
preparing one or more products in mammalian cells under serum-free
conditions, characterized in that (i) mammalian cells contain a
gene of interest which codes for a peptide of interest disclosed
herein; (ii) the mammalian cells are grown under serum-free
conditions which allow replication of the mammalian cells; (iii) in
each case at least one (1) of these mammalian cell(s) are deposited
in a cell culture vessel under serum-free conditions; (iv) the
suitably deposited mammalian cells are replicated under serum-free
conditions; (v) the replicated cells are cultivated under
serum-free conditions in which the gene of interest is expressed;
and (vi) the gene product is then isolated from the cells or
culture supernatant and purified. In another embodiment of this
process the mammalian cell is a transfected mammalian cell into
which the gene of interest has been introduced. Accordingly,
methods disclosed herein also relates to a method of preparing
recombinant gene products, characterized in that before step (i) of
the process described above the mammalian cells are transfected
with a nucleic acid which at least codes for a gene of interest.
Stable transfection of the corresponding mammalian cell is
preferred.
[0466] Examples of serum-free, protein-free or chemically defined
media include for example the commercially obtainable media Ham's
F12 (Sigma, Deisenhofen, DE), RPMI 1640 (Sigma), Dulbecco's
Modified Eagle's medium (DMEM; Sigma), Minimal Essential medium
(MEM; Sigma), Iscove's Modified Dulbecco's medium (IMDM; Sigma),
CDCHO (Invitrogen, Carlsbad, Calif., USA), CHO-S-SFMII
(Invitrogen), serum-free CHO medium (Sigma), CD-PowerCHO2 medium
(Lonza) and protein-free CHO medium (Sigma). Each of these media
can if desired be supplemented with various compounds such as
hormones and/or other growth factors (e.g. insulin, transferrin,
epidermal growth factor, insulin-like growth factor), salts (e.g.
sodium chloride, calcium, magnesium, phosphate), buffers (e.g.
HEPES), nucleosides (e.g. adenosine, thymidine), glutamine, glucose
or other equivalent nutrients, antibiotics and/or trace elements or
commercially available Feed such as Power Feed A (Lonza) If the
replicable cells are recombinant cells which express one or more
selectable markers, one or more suitable selection agents such as
antibiotics may also be added to the medium.
[0467] 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 disclosed herein can also include sequences engineered to
optimize stability, production, purification, yield or activity of
the expressed polypeptide.
[0468] 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. A skilled artisan would appreciate that
the term "an effective medium" may encompass any medium in which a
cell is cultured to produce the recombinant polypeptide disclosed
herein. 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 disclosed herein 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.
[0469] In one embodiment, culture conditions comprised dissolved
oxygen (DO) content at about 20-80%. In another embodiment, DO
content is at about 20-30%. In another embodiment, DO content is at
about 30-40%. In another embodiment, DO content is at about 40-50%.
In another embodiment, DO content is at about 50-60%. In another
embodiment, DO content is at about 60-70%. In another embodiment,
DO content is at about 70-80%.
[0470] In one embodiment, culture conditions comprise pH starting
at one temperature and shifting to another during the manufacture.
In another embodiment, pH starts at about 7.3 and shifts to about
6.7 during bioreactor incubation. In another embodiment, pH starts
at about 7.3, about 7.2 or about 7.1 and shifts to about 6.7, about
6.8, about 6.9 or about 7.0 during bioreactor incubation. Each
possibility represents an embodiment disclosed herein.
[0471] In some embodiments, depending on the vector and host system
used for production, resultant polypeptides disclosed herein either
remain within the recombinant cell, or are secreted into the
medium.
[0472] In one embodiment, following a predetermined time in
culture, recovery of the recombinant polypeptide is effected.
[0473] A skilled artisan would appreciate that the phrase
"recovering the recombinant polypeptide" used herein may encompass
collecting the whole medium containing the polypeptide and can
imply additional steps of separation or purification.
[0474] In one embodiment, polypeptides disclosed herein 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, hydroxyapatite chromatography, chromatofocusing and
differential solubilization.
[0475] In one embodiment, each column can be run under controlled
or non-controlled temperature.
[0476] In one embodiment, to facilitate recovery, the expressed
coding sequence can be engineered to encode the polypeptide
disclosed herein and fused to a 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 Gardena et al., J. Biol. Chem. 265:15854-15859
(1990)].
[0477] In one embodiment, the polypeptide disclosed herein is
retrieved in "substantially pure" form. A skilled artisan would
appreciate that the phrase "substantially pure" may encompass a
purity that allows for the effective use of the protein in the
applications described herein. Such a form may also include highly
glycosylated and highly sialylated forms as also disclosed
herein.
[0478] In one embodiment, a subject disclosed herein, is a human
subject. In another embodiment, the subject is a domesticated
animal. In another embodiment, the subject is a pet. In another
embodiment, the subject is a mammal. In another embodiment, the
subject is a farm animal. In another embodiment, the subject is a
monkey. In another embodiment, the subject is a horse. In another
embodiment, the subject is a cow. In another embodiment, the
subject is a mouse. In another embodiment, the subject is a rat. In
another embodiment, the subject is canine. In another embodiment,
the subject is feline. In another embodiment, the subject is
bovine, ovine, porcine, equine, murine, or cervine. In one
embodiment, the subject is male. In another embodiment, the subject
is female. In one embodiment, the subject is a child, in another
embodiment, an adolescent, in another embodiment, an adult or, in
another embodiment, an elderly subject. In another embodiment, the
subject is a pediatric subject, in another embodiment, a geriatric
subject.
[0479] In one embodiment, the polypeptide disclosed herein is
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.
[0480] In one embodiment, production of a polypeptide of interest
modified by CTPs using recombinant DNA technology is performed.
[0481] 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 a polypeptide of interest modified by
CTPs disclosed herein can be ascertained using various assays.
[0482] In one embodiment, a method of manufacturing CTP-modified
polypeptide of interest comprises a step for obtaining clones that
optimally express said CTP-modified polypeptide of interest from
said WCB, and expanding said clones. In another embodiment, a
method of manufacturing CTP-modified polypeptide of interest
comprises a step for obtaining clones that optimally express said
CTP-modified polypeptide of interest from said MCB, and expanding
said clones. In another embodiment, the cell clones are expanded in
solution through a series of sub-cultivation steps up to production
bioreactor level. In another embodiment, the solution containing
said sub-cultivated clones is seeded in a bioreactor. In another
embodiment, the bioreactor is a disposable bioreactor. In another
embodiment, the bioreactor comprises a stainless steel bioreactor,
a rocking motion bioreactor such as Wave system from GE, a
perfusion bioreactor, or any other bioreactor system known in the
art. In one embodiment, removal of cells from a bioreactor is
accomplished by use of a disposable filter system. If a large scale
manufacture is performed, continuous centrifugation could be used
prior to use of a filtering system.
[0483] In one embodiment, the cell clones are expanded further or
up-scaled by serially culturing said cells in increasing sizes of
the bioreactor until a desired scale is reached. In another
embodiment, a bioreactor is run in a fed-batch mode. In another
embodiment, a bioreactor is run in a batch mode. In another
embodiment, a bioreactor is run in a repeated-batch mode. In
another embodiment, a bioreactor is run in a perfusion mode. Each
possibility described above, is another embodiment.
[0484] Peak viable cell densities differ depending on the type of
bioreactor employed. In one embodiment, the peak viable cell
density of a bioreactor used in methods of manufacturing disclosed
herein is about 0.2.times.10.sup.6-1.4.times.10.sup.6 cells/ml. In
another embodiment, the peak viable cell density of a bioreactor
used in methods of manufacturing disclosed herein is about
0.05.times.10.sup.6-100.times.10.sup.6. In another embodiment, the
peak viable cell density of a bioreactor is about
0.05.times.10.sup.6-0.5.times.10.sup.6. In another embodiment, the
peak viable cell density of a bioreactor is about
0.5.times.10.sup.6-5.times.10.sup.6. In another embodiment, the
peak viable cell density of a bioreactor is about
5.0.times.10.sup.6-50.times.10.sup.6. In another embodiment, the
peak viable cell density of a bioreactor is about
50.times.10.sup.6-100.times.10.sup.6.
[0485] Feed schemes for bioreactor use could be different, e.g.
repeated daily feeding from a certain day, or fixed in several
days, in addition % of feed added could be different from few % up
to even 50% or more. Each possibility is an embodiment disclosed
herein.
[0486] DMSO may be added to a bioreactor at different
concentrations as is known in the art. In one embodiment, 0.1-3%
DMSO is added to a bioreactor during its use. In another
embodiment, 0.1-0.5% DMSO is added. In another embodiment, 0.5-1.0%
DMSO is added. In another embodiment, 1.0-1.5% DMSO is added. In
another embodiment, 1.5-2.0% DMSO is added. In another embodiment,
2.0-2.5% DMSO is added. In another embodiment, 2.5-3.0% DMSO is
added.
[0487] In one embodiment, a method of manufacturing a CTP-modified
polypeptide of interest comprises the step of purifying a clarified
harvest solution in order to obtain a purified protein solution. In
another embodiment, a purified protein solution manufactured using
methods presented herein, comprises at least 5-95% CTP-modified
polypeptide of interest. In another embodiment, a purified protein
solution manufactured using methods presented herein, comprises at
least 5% CTP-modified polypeptide of interest. In another
embodiment, a purified protein solution manufactured using methods
presented herein, comprises at least 10% CTP-modified polypeptide
of interest. In another embodiment, a purified protein solution
manufactured using methods presented herein, comprises at least 20%
CTP-modified polypeptide of interest. In another embodiment, a
purified protein solution manufactured using methods presented In
another embodiment, a purified protein solution manufactured using
methods presented herein, comprises at least 30% CTP-modified
polypeptide of interest., comprises at least 40% CTP-modified
polypeptide of interest. In another embodiment, a purified protein
solution comprises at least 50% CTP-modified polypeptide of
interest. In another embodiment, a purified protein solution
comprises at least 60% CTP-modified polypeptide of interest. In
another embodiment, a purified protein solution comprises at least
70% CTP-modified polypeptide of interest. In another embodiment, a
purified protein solution comprises at least 80% CTP-modified
polypeptide of interest. In another embodiment, a purified protein
solution comprises at least 90% CTP-modified polypeptide of
interest. In another embodiment, a purified protein solution
manufactured using methods presented herein, comprises at least 95%
CTP-modified polypeptide of interest.
[0488] In one embodiment, a clarified harvest is held up to 24
hours at 2-25.degree. C. In another embodiment, the clarified
harvest is stored at least 5.degree. C. for up to one month.
[0489] In one embodiment, the clarified harvest obtained in step
(e) is tested for bioburden, bacterial endotoxin, specific protein
content, residual DNA, viruses, virus-like particles, and/or
Mycoplasma, or any combination thereof.
[0490] In one embodiment, the purification of the clarified harvest
in step (f) is accomplished by sequentially performing the steps
comprising: (g) concentrating, diafiltering and purifying said
clarified harvest solution, wherein said concentration,
diafiltration and purifying is accomplished by hollow fiber
cassette or tangential flow cassette sequentially passing said
clarified harvest solution through an anion exchange column and a
hydrophobic interaction column; (h) obtaining said clarified
harvest obtained following step; (i) and inactivating viruses
present in said clarified harvest by incubating in a solution toxic
to said viruses; (j) obtaining said clarified harvest solution from
(h) and concentrating, diafiltering and purifying said clarified
harvest solution, wherein said concentration, diafiltration and
purification is followed by sequentially passing said clarified
harvest solution through a Hydroxyapatite Mixed-Mode column and a
cation exchange column; (j) obtaining said clarified harvest
solution following step (i) and physically removing said clarified
harvest solution from viruses by nanofiltration; (k) obtaining said
clarified harvest solution following step (j) and concentrating,
diafiltering and purifying said clarified harvest solution to
arrive at a maximally purified clarified harvest containing said a
highly glycosylated form of CTP-modified polypeptides.
[0491] In one embodiment, ultrafiltration and diafiltration to
concentrate and filter a clarified harvest may be performed using a
hollow fiber cartridge, or equivalent TFF based UFDF step. The
cartridge nominal molecular weight cutoff size is 10,000 kDa. In
another embodiment, a membrane cartridge could comply PES/PS/RC
membranes with a cut-off of 3 kDa to 30 kDa.
[0492] In another embodiment, the anion exchange column of step (g)
is a DEAE-Sepharose Fast Flow column. In another embodiment, the
DEAE column purifies the highly glycosylated form of said
CTP-modified polypeptide of interest. In one embodiment, the higher
the glycosylation the better the pharmacodynamics of the
CTP-modified polypeptide of interest. In another embodiment, an
anion exchange column may comprise other anion exchange columns
known in the art, for example a Capto DEAE anion exchange column or
other resins such as Eshmuno Q.
[0493] In one embodiment, the hydrophobic column of step (g) is a
Phenyl Hydrophobic Interaction Chromatography (HIC) column. The
number of cycles of use for phenyl HIC may range between about
1-10. In one embodiment, 1-3 cycles are performed. In another
embodiment, 1-5 cycles are performed. In another embodiment, 1-6
cycles are performed. In another embodiment, 1-7 cycles are
performed. In another embodiment, 1-8 cycles are performed. In
another embodiment, 1-9 cycles are performed. In another
embodiment, 1-10 cycles are performed. In another embodiment,
buffers known in the art are used for washing and elution. In one
embodiment, an elution buffer comprises Ammonium Sulfate with
propylene glycol. In one embodiment, an elution buffer comprises
Ammonium Sulfate with ethylene glycol.
[0494] In one embodiment, a Hydroxyapatite Mixed-Mode column
comprises a ceramic hydroxyapatite Mixed-Mode column (CHT). The
number of cycles of use for CHT may range between about 1-10. In
one embodiment, 1-3 cycles are performed. In another embodiment,
1-5 cycles are performed. In another embodiment, 1-6 cycles are
performed. In another embodiment, 1-7 cycles are performed. In
another embodiment, 1-8 cycles are performed. In another
embodiment, 1-9 cycles are performed. In another embodiment, 1-10
cycles are performed. Elution from a CHT column may be performed
with between about 3-10 column volumes (CV). In one embodiment,
elution is performed with about 3 CV. In another embodiment,
elution is performed with about 4 CV. In another embodiment,
elution is performed with about 5 CV. In another embodiment,
elution is performed with about 6 CV. In another embodiment,
elution is performed with about 7 CV. In another embodiment,
elution is performed with about 8 CV. In another embodiment,
elution is performed with about 9 CV. In another embodiment,
elution is performed with about 10 CV.
[0495] In one embodiment, viruses that could be present in the
clarified harvest due to contamination are inactivated in the
clarified harvest. In another embodiment, the viruses are
inactivated using a 1% Triton-X 100 solution. In another
embodiment, the viruses are inactivated using a 0.2 to 2% Triton-X
100 solution. In another embodiment, the viruses are inactivated
using 0.5% Triton-X 100 solution. In another embodiment, the
viruses are inactivated using a 1-4% Triton-X 100 solution. In
another embodiment, the viruses are inactivated using a 0.2-0.5%
Triton-X 100 solution. In another embodiment, the viruses are
inactivated using a 0.5-1.0% Triton-X 100 solution. In another
embodiment, the viruses are inactivated using a 2% Triton-X 100
solution. In another embodiment, the viruses are inactivated using
a 3% Triton-X 100 solution. In another embodiment, the viruses are
inactivated using a 4% Triton-X 100 solution. In another
embodiment, the viruses are inactivated using a 5-10% Triton-X 100
solution. In another embodiment, viral inactivation in a Triton-X
100 solution is for about 0.5 to 24 hours. In another embodiment,
viral inactivation in a Triton-X solution is for about 0.5 to 1
hours. In another embodiment, viral inactivation in a Triton-X
solution is for about 1 to 2 hours. In another embodiment, viral
inactivation in a Triton-X solution is for about 2 to 3 hours. In
another embodiment, viral inactivation in a Triton-X solution is
for about 3 to 4 hours. In another embodiment, viral inactivation
in a Triton-X solution is for about 4 to 6 hours. In another
embodiment, viral inactivation in a Triton-X solution is for about
6 to 8 hours. In another embodiment, viral inactivation in a
Triton-X solution is for about 8 to 10 hours. In another
embodiment, viral inactivation in a Triton-X solution is for about
10 to 12 hours. In another embodiment, viral inactivation in a
Triton-X solution is for about 12 to 24 hours.
[0496] It will be appreciated by the skilled artisan that other
concentrations or other solutions available in the art and that are
toxic to these viruses, including but not limited to, sodium
cholate and Tween 80 may be used in methods disclosed herein. In
another embodiment, a mixture of Tri-n-butyl phosphate (TNBP) and
Polysorbate 80 (Tween 80) is used to inactivate the virus in step
(h).
[0497] In one embodiment, viruses are physically removed by using
nanofiltration. It will be appreciated by the skilled artisan that
any filter known in the art for removing viruses may be applied in
methods disclosed herein. In another embodiment, nanofiltration is
carried out using a Planova or Planova type filter cartridge (1-60
mm.sup.2). Such methods are followed by confirmation of viral
clearance from the clarified harvest using methods known in the
art.
[0498] In one embodiment, a cation exchange column of step (i)
herein is a SP-Sepharose Fast Flow column. In another embodiment,
the cation exchange column comprises a CM sepharose Capto S. In
another embodiment, the cation exchange column comprises any known
in the art for the purpose herein.
[0499] In one embodiment, the methods disclosed herein achieve at
least a 20% recovery rate of highly glycosylated CTP-modified
polypeptide of interest. In another embodiment, the methods achieve
a recovery rate of at least 5%, at least 10%, at least 15%, 20%, at
least 25%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, at least 99.9%
recovery rate of highly glycosylated CTP-modified polypeptide of
interest.
[0500] In one embodiment, following purification of highly
glycosylated CTP-modified polypeptide of interest, the methods
disclosed herein further comprise characterizing said CTP-modified
polypeptide. In another embodiment, the purity of the CTP-modified
polypeptide of interest is determined. In another embodiment,
glycosylation content is determined. In another embodiment,
glycosylation site occupancy is determined. In one embodiment,
purity, glycosylation content and glycosylation site occupancy are
determined in the manufactured CTP-modified polypeptide of
interest.
[0501] In another embodiment, the cell clones utilized in methods
disclosed herein are stored in a frozen cell bank. In another
embodiment, the cell clones are stored in a lyophilized cell
bank.
[0502] In another embodiment, the cell bank of methods and
compositions disclosed herein is a master cell bank. In another
embodiment, the cell bank is a working cell bank. In another
embodiment, the cell bank is Good Manufacturing Practice (GMP) cell
bank. In another embodiment, the cell bank is intended for
production of clinical-grade material. In another embodiment, the
cell bank conforms to regulatory practices for human use. In
another embodiment, the cell bank is any other type of cell bank
known in the art.
[0503] "Good Manufacturing Practices" are defined, in another
embodiment, by (21 CFR 210-211) of the United States Code of
Federal Regulations. In another embodiment, "Good Manufacturing
Practices" are defined by other standards for production of
clinical-grade material or for human consumption; e.g. standards of
a country other than the United States. Each possibility represents
a separate embodiment disclosed herein.
[0504] In another embodiment, the medium used for propagating cells
contains methotrexate (MXT). In another embodiment, the medium is
methotrexate-free medium. In another embodiment, the concentration
of MXT present in a medium is between about 0.1-2 uM. In another
embodiment, the concentration of MXT present in the medium is about
0.1-0.5 uM. In another embodiment, the concentration of MXT present
in the medium is about 0.5-1.0 uM. In another embodiment, the
concentration of MXT present in the medium is about 1.0-1.5 uM. In
another embodiment, the concentration of MXT present in the medium
is about 1.5-2.0 uM. It will be well appreciated that the term
"medium" may encompass a liquid or gel or powder that is suitable
for growth or culture of the cells comprising the CTP-modified
polypeptide of interest disclosed herein. Such medium may be
alternatively referred to as "growth medium" or "culture medium"
and may include but is not limited to, nutrient media, enriched
media, minimal media, differential media, transport media, or
selective media. In a further aspect, selective medium may be
suitable for selecting a particular group of cells during the
manufacturing process.
[0505] In one embodiment, the purified protein solution contains at
least 5-95% CTP-modified polypeptide of interest. In another
embodiment, the purified protein solution contains at least 5%
CTP-modified polypeptide of interest. In another embodiment, the
purified protein solution contains at least 10% CTP-modified
polypeptide of interest. In another embodiment, the purified
protein solution contains at least 15 CTP-modified polypeptide of
interest. In another embodiment, a purified protein solution
contains at least 20% CTP-modified polypeptide of interest. In
another embodiment, the purified protein solution contains at least
30% CTP-modified polypeptide of interest. In another embodiment,
the purified protein solution contains at least 40% CTP-modified
polypeptide of interest. In another embodiment, the purified
protein solution contains at least 50% CTP-modified polypeptide of
interest. In another embodiment, the purified protein solution
contains at least 60% CTP-modified polypeptide of interest. In
another embodiment, the purified protein solution contains at least
70% CTP-modified polypeptide of interest. In another embodiment,
the purified protein solution contains at least 80% CTP-modified
polypeptide of interest. In another embodiment, the purified
solution contains at least 90-95% CTP-modified polypeptide of
interest. In another embodiment, the purified solution contains
95.1-99.9% CTP-modified polypeptide of interest. In another
embodiment, the purified solution contains 100% CTP-modified
polypeptide of interest.
[0506] In one embodiment, the CTP-modified polypeptide of interest
is highly glycosylated. It will be well appreciated by the skilled
artisan that the term "highly glycosylated" when in reference to a
CTP-modified polypeptide of interest, may encompass a glycosylation
level of about 70-80% of total CTP-modified polypeptide of interest
polypeptide. In another embodiment, highly glycosylated
CTP-modified polypeptide of interest has a glycosylation level of
at least 70%. In another embodiment, highly glycosylated
CTP-modified polypeptide of interest has a glycosylation level of
at least 80%. In another embodiment, the term may encompass a
glycosylation level of about 81-90% of the total CTP-modified
polypeptide of interest polypeptide. In another embodiment, highly
glycosylated CTP-modified polypeptide of interest has a
glycosylation level of at least 90%. In another embodiment, the
term may encompass a glycosylation level of about 91-95% of the
total CTP-modified polypeptide of interest polypeptide. In another
embodiment, the term may encompass a glycosylation level of about
95.1-99% of the total CTP-modified polypeptide of interest
polypeptide. In another embodiment, the term may encompass a
glycosylation level of 100% of the total CTP-modified polypeptide
of interest polypeptide. Highly glycosylated CTP-modified
polypeptide of interest polypeptides may have beneficial properties
in methods of use for a long-acting polypeptide of interest,
supporting reduced frequency of administration. The high
glycosylation levels contribute to the significant increased
hydrodynamic volume of a CTP-modified polypeptide of interest, for
example a CTP-modified polypeptide of interest, as compared to
recombinant polypeptide of interest. This may result in an
elongated circulating time of CTP-modified polypeptide of
interest.
[0507] In one embodiment, the number of O-glycans per CTP is at
least 4-6. In another embodiment, the number of O-glycans per CTP
is between 4-6. In another embodiment, the number of O-glycans per
CTP is at least 4-8. In another embodiment, the number of O-glycans
per CTP is between 4-8. In one embodiment, the number of O-glycans
per CTP is at least 6-8. In one embodiment, the number of O-glycans
per CTP is between 6-8. In another embodiment, the number of
O-glycans per CTP is at least 4. In another embodiment, the number
of O-glycans per CTP is at least 5. In another embodiment, the
number of O-glycans per CTP is at least 6. In another embodiment,
the number of O-glycans per CTP is at least 7. In another
embodiment, the number of O-glycans per CTP is 8.
[0508] In one embodiment, the number of O-glycans per CTP-modified
polypeptide of interest polypeptide having one CTP attached is at
least 4-6. In another embodiment, the number of 0-glycans per
CTP-modified polypeptide of interest polypeptide having one CTP
attached is at least 6-8. In another embodiment, the number of
O-glycans per CTP-modified polypeptide of interest polypeptide
having one CTP attached is at least 4-8. In another embodiment, the
number of 0-glycans per CTP-modified polypeptide of interest
polypeptide having two CTP units attached is at least 8-12. In
another embodiment, the number of O-glycans per CTP-modified
polypeptide of interest polypeptide having two CTP units attached
is at least 12-16. In another embodiment, the number of O-glycans
per CTP-modified polypeptide of interest polypeptide having two CTP
units attached is at least 8-16. In another embodiment, the number
of O-glycans per CTP-modified polypeptide of interest polypeptide
having three CTP units attached is at least 12-18. In another
embodiment, the number of O-glycans per CTP-modified polypeptide of
interest polypeptide having three CTP units attached is at least
18-24. In another embodiment, the number of 0-glycans per
CTP-modified polypeptide of interest polypeptide having three CTP
units attached is at least 12-24. In another embodiment, the number
of O-glycans per CTP-modified polypeptide of interest polypeptide
having four CTP units attached is at least 16-24. In another
embodiment, the number of O-glycans per CTP-modified polypeptide of
interest polypeptide having four CTP units attached is at least
24-32. In another embodiment, the number of O-glycans per
CTP-modified polypeptide of interest polypeptide having four CTP
units attached is at least 16-32. In another embodiment, the number
of O-glycans per CTP-modified polypeptide of interest polypeptide
having five CTP units attached is at least 20-30. In another
embodiment, the number of O-glycans per CTP-modified polypeptide of
interest polypeptide having five CTP units attached is at least
30-40. In another embodiment, the number of O-glycans per
CTP-modified polypeptide of interest polypeptide having five CTP
units attached is at least 20-40. In another embodiment, the number
of O-glycans per CTP-modified polypeptide of interest polypeptide
having six CTP units attached is at least 24-36. In another
embodiment, the number of O-glycans per CTP-modified polypeptide of
interest polypeptide having six CTP units attached is at least
36-48. In another embodiment, the number of O-glycans per
CTP-modified polypeptide of interest polypeptide having six CTP
units attached is at least 24-48. In another embodiment, the number
of O-glycans per CTP-modified polypeptide of interest polypeptide
having seven CTP units attached is at least 28-35. In another
embodiment, the number of O-glycans per CTP-modified polypeptide of
interest polypeptide having seven CTP units attached is at least
42-56. In another embodiment, the number of 0-glycans per
CTP-modified polypeptide of interest polypeptide having seven CTP
units attached is at least 28-56. In another embodiment, the number
of O-glycans per CTP-modified polypeptide of interest polypeptide
having eight CTP units attached is at least 32-48. In another
embodiment, the number of O-glycans per CTP-modified polypeptide of
interest polypeptide having eight CTP units attached is at least
48-64. In another embodiment, the number of O-glycans per
CTP-modified polypeptide of interest polypeptide having eight CTP
units attached is at least 32-64. In another embodiment, the number
of O-glycans per CTP-modified polypeptide of interest polypeptide
having nine CTP units attached is at least 36-54. In another
embodiment, the number of O-glycans per CTP-modified polypeptide of
interest polypeptide having nine CTP units attached is at least
54-72. In another embodiment, the number of O-glycans per
CTP-modified polypeptide of interest polypeptide having nine CTP
units attached is at least 36-72. In another embodiment, the number
of O-glycans per CTP-modified polypeptide of interest polypeptide
having ten CTP units attached is at least 40-60. In another
embodiment, the number of O-glycans per CTP-modified polypeptide of
interest polypeptide having ten CTP units attached is at least
60-80. In another embodiment, the number of O-glycans per
CTP-modified polypeptide of interest polypeptide having five CTP
units attached is at least 40-80.
[0509] In one embodiment, O-glycan occupancy per CTP is at least
70%. In another embodiment, O-glycan occupancy per CTP is at least
80%. In another embodiment, O-glycan occupancy per CTP is at least
90%. In another embodiment, O-glycan occupancy per CTP is 100%.
[0510] In one embodiment, the CTP-modified polypeptide of interest
is highly sialylated. It will be appreciated by the skilled artisan
that the term "highly sialylated" when in reference to a
CTP-modified polypeptide of interest, may encompass a sialylation
level of about 70-80% of total CTP-modified polypeptide of interest
polypeptide. In another embodiment, the term may encompass a
sialylation level of about 80-90% of the total CTP-modified
polypeptide of interest polypeptide. In another embodiment, the
term may encompass a sialylation level of about 90-95% of the total
CTP-modified polypeptide of interest polypeptide. In another
embodiment, the term may encompass a sialylation level of about
95.1-99% of the total CTP-modified polypeptide of interest
polypeptide. In another embodiment, the term may encompass a
sialylation level of 100% of the total CTP-modified polypeptide of
interest polypeptide. In another embodiment, an 0-glycan structure
in a CTP-modified polypeptide of interest comprises a
mono-sialylated core 1.
[0511] In one embodiment, the CTP-modified polypeptide of interest
polypeptide consists of two CTP attached to the carboxy terminus of
said polypeptide of interest, and one chorionic gonadotropin
carboxy terminal peptide attached to the amino terminus of said
polypeptide of interest. In another embodiment, the CTP-modified
polypeptide of interest polypeptide consists of one chorionic
gonadotropin carboxy terminal peptide attached to the carboxy
terminus of said polypeptide of interest.
[0512] In one embodiment, the expression vector comprising a coding
portion encoding said CTP-modified polypeptide of interest also
comprises a promoter, a coding sequence for said CTP-modified
polypeptide, and a polyadenylation sequence. In one embodiment, the
polyadenylation sequence is a simian virus (SV) 40 polyadenylation
sequence.
[0513] In one embodiment, the CTP-modified polypeptide of interest
is expressed at a level of between 30-1500 mg/L. In another
embodiment, the CTP-modified polypeptide of interest is expressed
at a level of at least 30 mg/L. In another embodiment, the
CTP-modified polypeptide of interest is expressed at a level of at
least 40 mg/L. In another embodiment, the CTP-modified polypeptide
of interest is expressed at a level of at least 50 mg/L. In another
embodiment, the CTP-modified polypeptide of interest is expressed
at a level of at least 60 mg/L. In another embodiment, the
CTP-modified polypeptide of interest is expressed at a level of at
least 70 mg/L. In another embodiment, the CTP-modified polypeptide
of interest is expressed at a level of at least 50-70 mg/L. In
another embodiment, the CTP-modified polypeptide of interest is
expressed at a level of at least 80 mg/L. In another embodiment,
the CTP-modified polypeptide of interest is expressed at a level of
at least 90 mg/L. In another embodiment, the CTP-modified
polypeptide of interest is expressed at a level of at least 70-100
mg/L. In another embodiment, the CTP-modified polypeptide of
interest is expressed at a level of at least 100 mg/L. In another
embodiment, the CTP-modified polypeptide of interest is expressed
at a level of at least 200 mg/L. In another embodiment, the
CTP-modified polypeptide of interest is expressed at a level of at
least 100-200 mg/L. In another embodiment, the CTP-modified
polypeptide of interest is expressed at a level of at least 300
mg/L. In another embodiment, the CTP-modified polypeptide of
interest is expressed at a level of at least 200-300 mg/L. In
another embodiment, the CTP-modified polypeptide of interest is
expressed at a level of at least 400 mg/L. In another embodiment,
the CTP-modified polypeptide of interest is expressed at a level of
at least 300-400 mg/L. In another embodiment, the CTP-modified
polypeptide of interest is expressed at a level of at least 500
mg/L. In another embodiment, the CTP-modified polypeptide of
interest is expressed at a level of at least 500-600 mg/L. In
another embodiment, the CTP-modified polypeptide of interest is
expressed at a level of at least 600 mg/L. In another embodiment,
the CTP-modified polypeptide of interest is expressed at a level of
at least 600-700 mg/L. In another embodiment, the CTP-modified
polypeptide of interest is expressed at a level of at least 700
mg/L. In another embodiment, the CTP-modified polypeptide of
interest is expressed at a level of at least 701-800 mg/L. In
another embodiment, the CTP-modified polypeptide of interest is
expressed at a level of at least 800 mg/L. In another embodiment,
the CTP-modified polypeptide of interest is expressed at a level of
at least 801-900 mg/L. In another embodiment, the CTP-modified
polypeptide of interest is expressed at a level of at least 900
mg/L. In another embodiment, the CTP-modified polypeptide of
interest is expressed at a level of at least 901-1000 mg/L. In
another embodiment, the CTP-modified polypeptide of interest is
expressed at a level of at least 1000 mg/L. In another embodiment,
the CTP-modified polypeptide of interest is expressed at a level of
at least 1001-1100 mg/L. In another embodiment, the CTP-modified
polypeptide of interest is expressed at a level of at least 1100
mg/L. In another embodiment, the CTP-modified polypeptide of
interest is expressed at a level of at least 1101-1200 mg/L. In
another embodiment, the CTP-modified polypeptide of interest is
expressed at a level of at least 1200 mg/L. In another embodiment,
the CTP-modified polypeptide of interest is expressed at a level of
at least 1201-1300 mg/L. In another embodiment, the CTP-modified
polypeptide of interest is expressed at a level of at least 1300
mg/L. In another embodiment, the CTP-modified polypeptide of
interest is expressed at a level of at least 1301-1400 mg/L. In
another embodiment, the CTP-modified polypeptide of interest is
expressed at a level of at least 1400 mg/L. In another embodiment,
the CTP-modified polypeptide of interest is expressed at a level of
at least 1401-1500 mg/L. In another embodiment, the CTP-modified
polypeptide of interest is expressed at a level of at least 1500
mg/L.
[0514] It will be appreciated by the skilled artisan that the term
"expression" may encompass transcription and/or translation of a
heterologous nucleic acid sequence within a host cell. The level of
expression of a desired product/protein of interest in a host cell
may be determined on the basis of either the amount of
corresponding mRNA or cDNA that is present in the cell, or the
amount of the desired polypeptide/protein of interest encoded by
the selected sequence as in the present examples. For example, mRNA
transcribed from a selected sequence can be quantitated by Northern
blot hybridization, ribonuclease RNA protection, in situ
hybridization to cellular RNA or by PCR (see Sambrook et al., 1989;
Ausubel et al., 1987 updated) Proteins encoded by a selected
sequence can be quantitated by various methods, e.g. by ELISA, by
Western blotting, by radioimmunoassays, by immunoprecipitation, by
assaying for the biological activity of the protein, by
immunostaining of the protein followed by FACS analysis (see
Sambrook et al., 1989; Ausubel et al., 1987 updated) or by
homogeneous time-resolved fluorescence (HTRF) assays. In one
embodiment, quantitation of the CTP-modified polypeptide of
interest comprises use of a reverse phase high performance liquid
chromatography (RP-HPLC). In another embodiment, the RP-HPLC
comprises a C-18 column. In another embodiment, the RP-HPLC
comprises a C-8 column. In another embodiment, methods disclosed
herein use an RP-HPLC to quantitate a CTP-modified polypeptide of
interest in the harvest (See Example steps 3 to 8). In another
embodiment, methods disclosed herein use an RP-HPLC to quantitate a
CTP-modified polypeptide of interest in the first steps of
purification (See Example steps 9-12.
[0515] In another embodiment, a cell bank, or frozen stock
disclosed herein exhibits viability upon thawing of greater than
90%. In another embodiment, the storage is for an indefinite amount
of time.
[0516] In another embodiment, the storage is for 2 weeks. In
another embodiment, the storage is for 3 weeks. In another
embodiment, the storage is for 1 month. In another embodiment, the
storage is for 2 months. In another embodiment, the storage is for
3 months. In another embodiment, the storage is for 5 months. In
another embodiment, the storage is for 6 months. In another
embodiment, the storage is for 9 months. In another embodiment, the
storage is for 1 year.
[0517] In another embodiment, a cell bank, or frozen stock
disclosed herein is cryopreserved by a method that comprises
growing a culture of the cells in a defined media disclosed herein,
freezing the culture in a solution comprising glycerol, and storing
the cell clones at below -20 degrees Celsius. In another
embodiment, the temperature is about -70 degrees Celsius. In
another embodiment, the temperature is about .sup.-70-.sup.-80
degrees Celsius. In another embodiment, any defined media disclosed
herein may be used in this method. Each defined media represents a
separate embodiment disclosed herein.
[0518] In another embodiment of methods and compositions disclosed
herein, the culture is inoculated from a cell bank. In another
embodiment, the culture is inoculated from a frozen stock. In
another embodiment, the culture is inoculated from a starter
culture. In another embodiment, the culture is inoculated from a
colony. In another embodiment, the culture is inoculated at mid-log
growth phase. In another embodiment, the culture is inoculated at
approximately mid-log growth phase. In another embodiment, the
culture is inoculated at another growth phase.
[0519] In another embodiment of methods and compositions disclosed
herein, the solution used for freezing comprises DMSO in an amount
of 2-20%. In another embodiment, the amount is 2%. In another
embodiment, the amount is 20%. In another embodiment, the amount is
1%. In another embodiment, the amount is 1.5%. In another
embodiment, the amount is 3%. In another embodiment, the amount is
4%. In another embodiment, the amount is 5%. In another embodiment,
the amount is 2%. In another embodiment, the amount is 2%. In
another embodiment, the amount is 7%. In another embodiment, the
amount is 7.5%. In another embodiment, the amount is 9%. In another
embodiment, the amount is 10%. In another embodiment, the amount is
12%. In another embodiment, the amount is 14%. In another
embodiment, the amount is 16%. In another embodiment, the amount is
18%. In another embodiment, the amount is 22%. In another
embodiment, the amount is 25%. In another embodiment, the amount is
30%. In another embodiment, the amount is 35%. In another
embodiment, the amount is 40%.
[0520] In another embodiment, the additive is sucrose. In another
embodiment, the additive is any other colligative additive or
additive with anti-freeze properties that is known in the art. Each
possibility represents a separate embodiment disclosed herein.
[0521] In one embodiment, a freezing solution used in the methods
and for the compositions disclosed herein comprises conditioned
media and DMSO. In one embodiment, a freezing solution used in the
methods and for the compositions disclosed herein comprises about
46.255% conditioned media and 7.5% DMSO.
[0522] In one embodiment, the cell culture is grown by techniques
routine in the art. In another embodiment, a constant pH is
maintained during growth of the cell culture. In another
embodiment, the pH is maintained at about 7.0. In another
embodiment, the pH is about 6. In another embodiment, the pH is
about 6.5. In another embodiment, the pH is about 7.5. In another
embodiment, the pH is about 8. In another embodiment, the pH is
6.5-7.5. In another embodiment, the pH is 6-8. In another
embodiment, the pH is 6-7. In another embodiment, the pH is
7-8.
[0523] In another embodiment, a constant temperature is maintained
during growth of the culture. In another embodiment, the
temperature is maintained at about 37.degree. C. In another
embodiment, the temperature is 37.degree. C. In another embodiment,
the temperature is 25.degree. C. In another embodiment, the
temperature is 27.degree. C. In another embodiment, the temperature
is 28.degree. C. In another embodiment, the temperature is
30.degree. C. In another embodiment, the temperature is 32.degree.
C. In another embodiment, the temperature is 34.degree. C. In
another embodiment, the temperature is 35.degree. C. In another
embodiment, the temperature is 36.degree. C. In another embodiment,
the temperature is 38.degree. C. In another embodiment, the
temperature is 39.degree. C.
[0524] In another embodiment, a constant dissolved oxygen
concentration is maintained during growth of the culture. In
another embodiment, the dissolved oxygen concentration is
maintained at 20% of saturation. In another embodiment, the
concentration is 15% of saturation. In another embodiment, the
concentration is 16% of saturation. In another embodiment, the
concentration is 18% of saturation. In another embodiment, the
concentration is 22% of saturation. In another embodiment, the
concentration is 25% of saturation. In another embodiment, the
concentration is 30% of saturation. In another embodiment, the
concentration is 35% of saturation. In another embodiment, the
concentration is 40% of saturation. In another embodiment, the
concentration is 45% of saturation. In another embodiment, the
concentration is 50% of saturation. In another embodiment, the
concentration is 55% of saturation. In another embodiment, the
concentration is 60% of saturation. In another embodiment, the
concentration is 65% of saturation. In another embodiment, the
concentration is 70% of saturation. In another embodiment, the
concentration is 75% of saturation. In another embodiment, the
concentration is 80% of saturation. In another embodiment, the
concentration is 85% of saturation. In another embodiment, the
concentration is 90% of saturation. In another embodiment, the
concentration is 95% of saturation. In another embodiment, the
concentration is 100% of saturation. In another embodiment, the
concentration is near 100% of saturation.
[0525] In another embodiment of methods and compositions disclosed
herein, the culture is grown in media having a maximum volume of 2
liters (L) per vessel. In another embodiment, the media has a
maximum volume of 200 ml per vessel. In another embodiment, the
media has a maximum volume of 300 ml per vessel. In another
embodiment, the media has a maximum volume of 500 ml per vessel. In
another embodiment, the media has a maximum volume of 750 ml per
vessel. In another embodiment, the media has a maximum volume of 1
L per vessel. In another embodiment, the media has a maximum volume
of 1.5 L per vessel. In another embodiment, the media has a maximum
volume of 2.5 L per vessel. In another embodiment, the media has a
volume of 3 L per vessel. In another embodiment, the media has a
volume of 5 L per vessel. In another embodiment, the media has a
volume of at least 5 L per vessel. In another embodiment, the media
has a volume of at least 10 L per vessel.
[0526] In another embodiment, the media has a minimum volume of 2 L
per vessel. In another embodiment, the media has a minimum volume
of 500 ml per vessel. In another embodiment, the media has a
minimum volume of 750 ml per vessel. In another embodiment, the
media has a minimum volume of 1 L per vessel. In another
embodiment, the media has a minimum volume of 1.5 L per vessel. In
another embodiment, the media has a minimum volume of 2.5 L per
vessel. In another embodiment, the media has a minimum volume of 3
L per vessel. In another embodiment, the media has a minimum volume
of 4 L per vessel. In another embodiment, the media has a minimum
volume of 5 L per vessel. In another embodiment, the media has a
minimum volume of 6 L per vessel. In another embodiment, the media
has a minimum volume of 8 L per vessel. In another embodiment, the
media has a minimum volume of 10 L per vessel.
[0527] In another embodiment, the step of freezing is performed
when the culture has a density of 1.times.10.sup.6 viable cells
(VC)/ml. In another embodiment, the biomass is 1.5.times.10.sup.6
VC/ml. In another embodiment, the biomass is 1.5.times.10.sup.6
VC/ml. In another embodiment, the biomass is 2.times.10.sup.6
VC/ml. In another embodiment, the biomass is 3.times.10.sup.6
VC/ml. In another embodiment, the biomass is 4.times.10.sup.6
VC/ml. In another embodiment, the biomass is 5.times.10.sup.6
VC/ml. In another embodiment, the biomass is 7.times.10.sup.6
VC/ml. In another embodiment, the biomass is 9.times.10.sup.6
VC/ml. In another embodiment, the biomass is 10.times.10.sup.6
VC/ml. In another embodiment, the biomass is 12.times.10.sup.6
VC/ml. In another embodiment, the biomass is 15.times.10.sup.6
VC/ml. In another embodiment, the biomass is 20.times.10.sup.7
VC/ml. In another embodiment, the biomass is 25.times.10.sup.6
VC/ml. In another embodiment, the biomass is 30.times.10.sup.7
VC/ml. In another embodiment, the biomass is 33.times.10.sup.6
VC/ml. In another embodiment, the biomass is 40.times.10.sup.6
VC/ml. In another embodiment, the biomass is 50.times.10.sup.6
VC/ml. In another embodiment, the biomass is more than
50.times.10.sup.6 VC/ml.
[0528] In another embodiment of methods and compositions disclosed
herein, the cell culture is flash-frozen in liquid nitrogen,
followed by storage at the final freezing temperature. In another
embodiment, the culture is frozen in a more gradual manner; e.g. by
placing in a vial of the culture in the final storage temperature.
In another embodiment, the culture is frozen by any other method
known in the art for freezing cell cultures.
[0529] It will be understood by the skilled artisan that the terms
"cell culture" and "tissue culture" may be used interchangeably and
denote the maintenance of cells in vitro, in suspension culture in
a liquid medium or on surface such as glass, plastic or agar
provided with liquid medium. In general, "cell culture"
necessitates a medium that is buffered to maintain a constant
suitable pH. Media used in cell culture are generally formulated to
include an adequate supply of necessary nutrients and can be
osmotically tailored to the particular cells being maintained, with
temperature and gas phase also being controlled within suitable
limits. Cell culture techniques are well known in the art. See,
e.g., Morgan et al. 1993 Animal Cell Culture, BIOS Scientific
Publishers, Oxford, UK; and Adams, R. L. P. 1990 Cell Culture for
Biochemists, Second Edition, Elsevier.
[0530] It will be appreciated by the skilled artisan that the term
"passage" may encompass the act of subculturing a cell population.
A skilled artisan would appreciate that the term "subculture" may
encompass a cell culture established by the inoculation of sterile
medium, which in one embodiment is a fresh sterile medium, with a
sample from a previous culture.
[0531] It will also be appreciated by the skilled artisan that the
term "cell strain" may encompass a population of cells derived from
a primary culture using subcultivation techniques. Thus, a primary
culture can be subcultured into two or more new cultures and the
subculturing repeated at periodic intervals for several months to
maintain the cell strain. Subculturing can be carried out using
established cell culture techniques.
[0532] In one embodiment, passaged cell strains, and immortalized
cell lines can be characterized by their expression of specific
functional markers such as keratins, hormonal and growth factor
receptors and the like.
[0533] In some aspects, cultures may be carried out in serum-free
defined media with added growth factors. In other aspects the media
contains serum with or without added growth factors. Such
modifications may be empirically determined by the skilled artisan
so as to optimize cell proliferation.
[0534] It will be appreciated by a skilled artisan that the term
"cell line" can encompass a population of cells derived from a
single explant which are characterized as having the potential for
unlimited proliferation in vitro. A cell line can be isolated from
a primary culture based on its ability to survive and continue to
grow in culture. Cell lines which have been derived originally from
tumor tissue may have been transformed in vivo, although not all
neoplastic cell populations have the capacity to grow indefinitely
in vitro. Further, cell lines generally retain their differentiated
character through many rounds of division.
[0535] Suitable cell culture substrates are generally a container
that can be sterilized, does not leach toxic factors and does not
distort microscopy images. Thus plates formed from glass and
plastic are suitable substrates herein. Plastic containers may
further be treated to encourage cell attachment using techniques
known in the art (Ramsey et al. 1984 In vitro 20:802). Suitable
tissue culture media generally consist of an isotonic, buffered,
basal nutrient medium which provides an energy source, coupled with
inorganic salts, amino acids, vitamins and various supplements.
Supplements may include serum (e.g., fetal calf serum, or the like)
various antibiotics to prevent contamination or to provide
selective conditions, attachment and growth factors, or the like. A
number of media formulations are known in the art, such as, but not
limited to, minimal essential medium (MEM), Rosewell Park Memorial
Institute (RPMI) 1640 or Dulbecco's modified Eagle's medium (DMEM).
Suitable tissue culture conditions are also known in the art. See,
e.g., Morgan et al. 1993 Animal Cell Culture, BIOS Scientific
Publishers Ltd., Oxford, U K, and Adams, R. L. P. 1990 Cell Culture
for Biochemists, Second Edition, Elsevier. In another embodiment
disclosed herein, methods of manufacture of CTP-modified
polypeptide of interest is a serum-free process. In another
embodiment disclosed herein, methods of manufacture of CTP-modified
polypeptide of interest is an animal derived-free process.
[0536] In another embodiment of methods and compositions disclosed
herein, the storage temperature of the culture is between .sup.-20
and .sup.-80 degrees Celsius (.degree. C.). In another embodiment,
the temperature is significantly below .sup.-20.degree. C. In
another embodiment, the temperature is not warmer than
.sup.-70.degree. C. In another embodiment, the temperature is
.sup.-70.degree. C. In another embodiment, the temperature is about
.sup.-70.degree. C. In another embodiment, the temperature is
.sup.-20.degree. C. In another embodiment, the temperature is about
.sup.-20.degree. C. In another embodiment, the temperature is
.sup.-30.degree. C. In another embodiment, the temperature is
.sup.-40.degree. C. In another embodiment, the temperature is
.sup.-50.degree. C. In another embodiment, the temperature is
.sup.-60.degree. C. In another embodiment, the temperature is
.sup.-80.degree. C. In another embodiment, the temperature is
.sup.-30-.sup.-70.degree. C. In another embodiment, the temperature
is .sup.-40-.sup.-70.degree. C. In another embodiment, the
temperature is .sup.-50-.sup.-70.degree. C. In another embodiment,
the temperature is .sup.-60-.sup.-70.degree. C. In another
embodiment, the temperature is .sup.-30-.sup.-80.degree. C. In
another embodiment, the temperature is .sup.-40-.sup.-80.degree. C.
In another embodiment, the temperature is .sup.-50-.sup.-80.degree.
C. In another embodiment, the temperature is
.sup.-60-.sup.-80.degree. C. In another embodiment, the temperature
is .sup.-70-.sup.-80.degree. C. In another embodiment, the
temperature is colder than .sup.-70.degree. C. In another
embodiment, the temperature is colder than .sup.-80.degree. C.
[0537] In another embodiment, for cryopreservation, the cells are
frozen slowly until they reach a temperature below -70.degree. C.
in medium that includes a cryoprotectant and vials are then
transferred to a liquid-nitrogen freezer to maintain them at
temperatures below -130.degree. C.
[0538] In another embodiment of methods and compositions disclosed
herein, the cryopreservation, or frozen storage, is for a maximum
of 24 hours. In another embodiment, the cryopreservation, or frozen
storage is for maximum of 2 days, is for maximum of 3 days, is for
maximum of 4 days, is for maximum of 1 week, is for maximum of 2
weeks, is for maximum of 3 weeks, is for maximum of 1 month, is for
maximum of 2 months, is for maximum of 3 months, is for maximum of
5 months, is for maximum of 6 months, is for maximum of 9 months,
or is for maximum of 1 year. Each possibility listed above is an
embodiment disclosed herein.
[0539] In another embodiment, the cryopreservation, or frozen
storage is for a minimum of 1 week, is for minimum of 2 weeks, is
for minimum of 3 weeks, is for minimum of 1 month, is for minimum
of 2 months, is for minimum of 3 months, is for minimum of 5
months, is for minimum of 6 months, is for minimum of 9 months, is
for minimum of 1 year, is for minimum of 1.5 years, is for minimum
of 2 years, is for minimum of 3 years, is for minimum of 5 years,
is for minimum of 7 years, is for minimum of 10 years, or is for
longer than 10 years. Each possibility listed above is an
embodiment disclosed herein.
[0540] In another embodiment of methods and compositions disclosed
herein, the cells exhibit growth after thawing following an
extended period of cryopreservation or frozen storage. In another
embodiment, the cells exhibit growth within about 15-22 hours after
inoculating fresh media with cells from the cell bank or starter
culture. In another embodiment, the cells exhibit growth within
about 12-20 hours after inoculating fresh media with cells from the
cell bank or starter culture. In one embodiment, to ensure
viability, genetic stability, and phenotypic stability, cell lines
need to be maintained in the exponential growth phase (via
subculturing on a regular basis).
[0541] An "extended period" of cryopreservation, or frozen storage,
is, in another embodiment, 1 month. In another embodiment, the
period is 2 months. In another embodiment, the period is 3 months.
In another embodiment, the period is 5 months. In another
embodiment, the period is 6 months. In another embodiment, the
period is 9 months. In another embodiment, the period is 1 year. In
another embodiment, the period is 1.5 years. In another embodiment,
the period is 2 years. In another embodiment, the period is 2-7
years. In another embodiment, the period is for at least 7 years.
In another embodiment, the period is for at least 10 years.
[0542] In another embodiment, the cells of the methods and
compositions disclosed herein retain a viability of over 90% after
thawing following cryopreservation. In another embodiment, the
viability upon thawing is close to 100% following the period of
cryopreservation. In another embodiment, the viability upon thawing
is close to 90%. In another embodiment, the viability upon thawing
is at least 90%. In another embodiment, the viability upon thawing
is over 80%.
[0543] In another embodiment, a cell bank, frozen stock, or batch
of vaccine doses disclosed herein is grown in a defined cell
culture media. Such media are known in the art and may include, but
not limited to Dulbecco's Modified Eagle's Medium (DMEM) (ATCC.RTM.
No. 30-2002), Iscove's Modified Dulbecco's Medium (IMDM) (ATCC.RTM.
No. 30-2005), Hybri-Care Medium (ATCC.RTM. No. 46-X), McCoy's 5A
and RPMI-1640 (ATCC.RTM. No. 30-2007), Ham's Nutrient Mixtures
(ATCC.RTM. CCL-61.TM.), PowerCHO.TM. Chemically Defined, Serum-free
CHO Medium (Lonza Cat. No. 12-771Q); or any other media known in
the art. In another embodiment these media may be supplemented in
antibiotics or animal sera, as will be empirically determined by
the skilled artisan.
[0544] In one embodiment, disclosed herein are bioreactors and
methods, which allow the cultivation of mammalian cells in large
scale volumes. Furthermore, and in another embodiment, said
bioreactors and methods, allow the cultivation of mammalian cells
under optimal conditions, even if grown in large scale volumes and
therefore allow a process performance and product quality
independent of the size of the bioreactor. The duration of time of
incubation within the bioreactor can vary, just by changing the
scale and bioreactor system, for example the duration may be
between 8-9, or it may be between 15-16 days. In another
embodiment, the duration of incubation in a bioreactor is about 7
days, about 8 days, about 9 days, about 10 days, about 11 days,
about 12 days, about 13 days, about 14 days, about 15 days, about
16 days, about 17 days, about 18 days, about 19 days, about 20 days
or more. In another embodiment, when a perfusion bioreactor is
used, the duration of incubation may be up to 7-120 days.
[0545] In another embodiment, disclosed herein are large-scale
bioreactors which allow the cultivation of mammalian cells in a
homogenous environment with respect to process parameters such as
pH, dissolved oxygen tension (DOT) and temperature, maintaining a
wen-mixed cell suspension and blending nutrient feeds within the
bioreactor. In another embodiment, the bioreactor is a disposable
bioreactor.
[0546] Methods disclosed herein solves the technical problems
underlying methods disclosed herein by the provision of
bioreactors, bioreactor systems and methods for the cultivation of
eukaryotic cells, especially of mammalian cells, according to the
claims.
[0547] In one embodiment, the bioreactor has a volume of at least
250 liters (L). In another embodiment, the bioreactor has a volume
of at least 500 L. In another embodiment the volume is at least
1000 L, at least 2000 L, at least 5,000 L, at least 10,000 L at
least 12,000 L or at least 15,000 L.
[0548] In another embodiment, the cells are subcultivated in
increasing volumes of bioreactors (see Example 12).
Compositions
[0549] In some embodiment, polypeptide of interest polypeptides
manufactured using the methods disclosed herein 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 one embodiment, the polypeptides disclosed
herein can be provided to the individual per se. In one embodiment,
the polypeptides disclosed herein can be provided to the individual
as part of a pharmaceutical composition where it is mixed with a
pharmaceutically acceptable carrier.
[0550] A skilled artisan would appreciate that the term a
"pharmaceutical composition" may encompass 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.
[0551] The modified peptides disclosed herein can be formulated
into suitable pharmaceutical preparations such as capsules and
injections in admixture with carriers, diluents, etc. known per se,
which can be orally or parenterally administered to mammals (e.g.
cows, horses, pigs, sheep, humans).
[0552] A skilled artisan would appreciate that the term "active
ingredient" may encompass a polypeptide sequence of interest, which
is accountable for the biological effect.
[0553] In some embodiments, any of the compositions disclosed
herein will comprise at least two CTP sequences bound to a protein
of interest, in any form. In one embodiment, disclosed herein are
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 parts 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.
[0554] A skilled artisan would appreciate that the phrases
"physiologically acceptable carrier" and "pharmaceutically
acceptable carrier" which can be used 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).
[0555] A skilled artisan would appreciate that the term "excipient"
may encompass 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.
[0556] 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.
[0557] 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.
[0558] 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.
[0559] In another embodiment, polypeptides of interest disclosed
herein comprising a polypeptide of interest are administered in a
dose of 1-90 micrograms in 0.1-5 ml solution. In another
embodiment, polypeptides disclosed herein comprising a polypeptide
of interest are administered in a dose of 1-50 micrograms in 0.1-5
ml solution. In another embodiment, polypeptides disclosed herein
comprising a polypeptide of interest are administered in a dose of
1-25 micrograms in 0.1-5 ml solution. In another embodiment,
polypeptides disclosed herein comprising a polypeptide of interest
are administered in a dose of 50-90 micrograms in 0.1-5 ml
solution. In another embodiment, polypeptides disclosed herein
comprising a polypeptide of interest are administered in a dose of
10-50 micrograms in 0.1-5 ml solution.
[0560] In another embodiment, polypeptides of interest disclosed
herein comprising a polypeptide of interest are administered in a
dose of 1-90 micrograms in 0.1-5 ml solution by intramuscular (IM)
injection, subcutaneous (SC) injection, or intravenous (IV)
injection once a week. In another embodiment, polypeptides
disclosed herein comprising a polypeptide of interest are
administered in a dose of 1-90 micrograms in 0.1-5 ml solution by
intramuscular (IM) injection, subcutaneous (SC) injection, or
intravenous (IV) injection twice a week. In another embodiment,
polypeptides disclosed herein comprising a polypeptide of interest
are administered in a dose of 1-90 micrograms in 0.1-5 ml solution
by intramuscular (IM) injection, subcutaneous (SC) injection, or
intravenous (IV) injection three times a week. In another
embodiment, polypeptides disclosed herein comprising a polypeptide
of interest are administered in a dose of 1-90 micrograms in 0.1-5
ml solution by intramuscular (IM) injection, subcutaneous (SC)
injection, or intravenous (IV) injection once every two weeks. In
another embodiment, polypeptides disclosed herein comprising a
polypeptide of interest are administered in a dose of 1-90
micrograms in 0.1-5 ml solution by intramuscular (IM) injection,
subcutaneous (SC) injection, or intravenous (IV) injection once
every 17 days. In another embodiment, polypeptides disclosed herein
comprising a polypeptide of interest are administered in a dose of
1-90 micrograms in 0.1-5 ml solution by intramuscular (IM)
injection, subcutaneous (SC) injection, or intravenous (IV)
injection once every 19 days weeks.
[0561] In another embodiment, protein drugs of molecular weight
lower than 50,000 daltons, such as interferons, are in general
short-lived species in vivo, having short circulatory half-lives of
several hours. In another embodiment, the subcutaneous route of
administration in general provides slower release into the
circulation. In another embodiment, the CTP modified polypeptide
disclosed herein prolongs the half-live of protein drugs of
molecular weight lower than 50,000 daltons, such as interferons. In
another embodiment, the CTP modified polypeptide disclosed herein
enable interferons to exert their beneficial effects for a longer
period of time.
[0562] In another embodiment, the immunogenicity of a CTP modified
polypeptide comprising a polypeptide of interest is equal to an
isolated polypeptide of interest. In another embodiment, the
immunogenicity of a CTP modified polypeptide comprising a
polypeptide of interest is comparable to an isolated polypeptide of
interest. In another embodiment, modifying a polypeptide of
interest as described herein with CTP peptides reduces the
immunogenicity of the polypeptide of interest. In another
embodiment, the CTP modified polypeptide comprising a polypeptide
of interest is as active as an isolated polypeptide of interest
protein. In another embodiment, the CTP modified polypeptide
comprising a polypeptide of interest is more active than an
isolated polypeptide of interest. In another embodiment, the CTP
modified polypeptide comprising a polypeptide of interest maximizes
the polypeptide of interest's protective ability against
degradation while minimizing reductions in bioactivity.
[0563] In another embodiment, the polypeptide of interest disclosed
herein can be provided to the individual per se. In one embodiment,
the polypeptide of interest disclosed herein can be provided to the
individual as part of a pharmaceutical composition where it is
mixed with a pharmaceutically acceptable carrier.
[0564] Various embodiments of dosage ranges are contemplated
herein. The dosage of the polypeptide disclosed herein, 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.
[0565] 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.
[0566] Various embodiments of dosage ranges are contemplated
herein. The dosage of the cytokine disclosed herein, in one
embodiment, is in the range of 0.005-100 mg/day. In another
embodiment, the dosage is in the range of 0.005-5 mg/day. In
another embodiment, the dosage is in the range of 0.01-50 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 0.01-5 mg/day.
In another embodiment, the dosage is in the range of 0.001-0.01
mg/day. In another embodiment, the dosage is in the range of
0.001-0.1 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 0.5-50 mg/day. In another embodiment, the dosage is in the range
of 0.2-15 mg/day. In another embodiment, the dosage is in the range
of 0.8-65 mg/day. In another embodiment, the dosage is in the range
of 1-50 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 8-15 mg/day. In another embodiment, the dosage is in a range of
10-20 mg/day. In another embodiment, the dosage is in the range of
20-40 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
12-40 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
50-100 mg/day. In another embodiment, the dosage is in a range of
1-60 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.
[0567] In another embodiment, a polypeptide comprising a
polypeptide of interest and CTP units is formulated in an
intranasal dosage form. In another embodiment, a polypeptide
comprising a polypeptide of interest and CTP units is formulated in
an injectable dosage form. In another embodiment, a polypeptide
comprising a polypeptide of interest and CTP units is administered
to a subject in a dose ranging from 0.0001 mg to 0.6 mg. In another
embodiment, a polypeptide comprising a polypeptide of interest and
CTP units is administered to a subject in a dose ranging from 0.001
mg to 0.005 mg. In another embodiment, a polypeptide comprising a
polypeptide of interest and CTP units is administered to a subject
in a dose ranging from 0.005 mg to 0.01 mg. In another embodiment,
a polypeptide comprising a polypeptide of interest and CTP units is
administered to a subject in a dose ranging from 0.01 mg to 0.3 mg.
In another embodiment, a polypeptide comprising a polypeptide of
interest and CTP units is administered to a subject in a dose in a
dose ranging from 0.2 mg to 0.6 mg.
[0568] In another embodiment, a polypeptide comprising a
polypeptide of interest and CTP units is administered to a subject
in a dose ranging from 1-100 micrograms. In another embodiment, a
polypeptide comprising a polypeptide of interest and CTP units is
administered to a subject in a dose ranging from 10-80 micrograms.
In another embodiment, a polypeptide comprising a polypeptide of
interest and CTP units is administered to a subject in a dose
ranging from 20-60 micrograms. In another embodiment, a polypeptide
comprising a polypeptide of interest and CTP units is administered
to a subject in a dose ranging from 10-50 micrograms. In another
embodiment, a polypeptide comprising a polypeptide of interest and
CTP units is administered to a subject in a dose ranging from 40-80
micrograms. In another embodiment, a polypeptide comprising a
polypeptide of interest and CTP units is administered to a subject
in a dose ranging from 10-30 micrograms. In another embodiment, a
polypeptide comprising a polypeptide of interest and CTP units is
administered to a subject in a dose ranging from 30-60
micrograms.
[0569] In another embodiment, a polypeptide comprising a
polypeptide of interest and CTP units is administered to a subject
in a dose ranging from 0.2 mg to 2 mg. In another embodiment, a
polypeptide comprising a polypeptide of interest and CTP units is
administered to a subject in a dose ranging from 2 mg to 6 mg. In
another embodiment, a polypeptide comprising a polypeptide of
interest and CTP units is administered to a subject in a dose
ranging from 4 mg to 10 mg. In another embodiment, a polypeptide
comprising a polypeptide of interest and CTP units is administered
to a subject in a dose ranging from 5 mg and 15 mg.
[0570] In another embodiment, a polypeptide comprising a
polypeptide of interest and CTP units is injected into the muscle
(intramuscular injection). In another embodiment, a polypeptide
comprising a polypeptide of interest and CTP units is injected
below the skin (subcutaneous injection). In another embodiment, a
polypeptide comprising an IFN protein and CTP units is injected
into the muscle. In another embodiment, a polypeptide comprising an
IFN protein and CTP units is injected below the skin.
[0571] In another embodiment, the methods disclosed herein include
increasing the compliance in the use of polypeptide of interest
therapy, comprising providing to a subject in need thereof, a
polypeptide comprising a polypeptide of interest, one CTP attached
to an amino terminus of the polypeptide of interest, and two CTP
attached to a carboxy terminus of the polypeptide of interest,
thereby increasing compliance in the use of polypeptide of interest
therapy.
[0572] In another embodiment, the methods disclosed herein include
increasing the compliance of patients afflicted with chronic
illnesses that are in need of a polypeptide of interest therapy. In
another embodiment, the methods disclosed herein enable reduction
in the dosing frequency of a polypeptide of interest by modifying
the polypeptide of interest with CTPs as described hereinabove. In
another embodiment, the term compliance comprises adherence. In
another embodiment, the methods disclosed herein include increasing
the compliance of patients in need of a polypeptide of interest
therapy by reducing the frequency of administration of the
polypeptide of interest. In another embodiment, reduction in the
frequency of administration of the polypeptide of interest is
achieved due to the CTP modifications which render the CTP-modified
polypeptide of interest more stable. In another embodiment,
reduction in the frequency of administration of the polypeptide of
interest is achieved as a result of increasing T1/2 of the
polypeptide of interest. In another embodiment, reduction in the
frequency of administration of the polypeptide of interest is
achieved as a result of increasing the clearance time of the
polypeptide of interest. In another embodiment, reduction in the
frequency of administration of the polypeptide of interest is
achieved as a result of increasing the AUC measure of the
polypeptide of interest.
[0573] In another embodiment, a polypeptide comprising a
polypeptide of interest and CTP units is administered to a subject
once a day. In another embodiment, a polypeptide comprising a
polypeptide of interest and CTP units is administered to a subject
once every two days. In another embodiment, a polypeptide
comprising a polypeptide of interest and CTP units is administered
to a subject once every three days. In another embodiment, a
polypeptide comprising a polypeptide of interest and CTP units is
administered to a subject once every four days. In another
embodiment, a polypeptide comprising a polypeptide of interest and
CTP units is administered to a subject once every five days. In
another embodiment, a polypeptide comprising a polypeptide of
interest and CTP units is administered to a subject once every six
days. In another embodiment, a polypeptide comprising a polypeptide
of interest and CTP units is administered to a subject once every
week. In another embodiment, a polypeptide comprising a polypeptide
of interest and CTP units is administered to a subject once every
7-14 days. In another embodiment, a polypeptide comprising a
polypeptide of interest and CTP units is administered to a subject
once every 10-20 days. In another embodiment, a polypeptide
comprising a polypeptide of interest and CTP units is administered
to a subject once every 5-15 days. In another embodiment, a
polypeptide comprising a polypeptide of interest and CTP units is
administered to a subject once every 15-30 days.
[0574] In another embodiment, the dosage is in a range of 50-500
mg/day. In another embodiment, the dosage is in a range of 50-150
mg/day. In another embodiment, the dosage is in a range of 100-200
mg/day. In another embodiment, the dosage is in a range of 150-250
mg/day. In another embodiment, the dosage is in a range of 200-300
mg/day. In another embodiment, the dosage is in a range of 250-400
mg/day. In another embodiment, the dosage is in a range of 300-500
mg/day. In another embodiment, the dosage is in a range of 350-500
mg/day.
[0575] In one embodiment, the dosage is 20 mg/day. In one
embodiment, the dosage is 30 mg/day. In one embodiment, the dosage
is 40 mg/day. In one embodiment, the dosage is 50 mg/day. In one
embodiment, the dosage is 0.01 mg/day. In another embodiment, the
dosage is 0.1 mg/day. In another embodiment, the dosage is 1
mg/day. In another embodiment, the dosage is 0.530 mg/day. In
another embodiment, the dosage is 0.05 mg/day. In another
embodiment, the dosage is 50 mg/day. In another embodiment, the
dosage is 10 mg/day. In another embodiment, the dosage is 20-70
mg/day. In another embodiment, the dosage is 5 mg/day.
[0576] In another embodiment, the dosage is 1-90 mg/day. In another
embodiment, the dosage is 1-90 mg/2 days. In another embodiment,
the dosage is 1-90 mg/3 days. In another embodiment, the dosage is
1-90 mg/4 days. In another embodiment, the dosage is 1-90 mg/5
days. In another embodiment, the dosage is 1-90 mg/6 days. In
another embodiment, the dosage is 1-90 mg/week. In another
embodiment, the dosage is 1-90 mg/9 days. In another embodiment,
the dosage is 1-90 mg/11 days. In another embodiment, the dosage is
1-90 mg/14 days.
[0577] In another embodiment, the polypeptide of interest dosage is
10-50 mg/day. In another embodiment, the dosage is 10-50 mg/2 days.
In another embodiment, the dosage is 10-50 mg/3 days. In another
embodiment, the dosage is 10-50 mg/4 days. In another embodiment,
the dosage is 10-50 micrograms mg/5 days. In another embodiment,
the dosage is 10-50 mg/6 days. In another embodiment, the dosage is
10-50 mg/week. In another embodiment, the dosage is 10-50 mg/9
days. In another embodiment, the dosage is 10-50 mg/11 days. In
another embodiment, the dosage is 10-50 mg/14 days.
[0578] 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
polypeptide of interest disclosed herein, 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 disclosed herein, and can be readily made
by a person skilled in the art.
[0579] In another embodiment, disclosed herein is a method of
reducing the dosing frequency of a polypeptide of interest,
comprising the step of attaching at least one CTP to the
polypeptide of interest as disclosed hereinabove in detail.
[0580] In another embodiment, disclosed herein is a method of
increasing compliance in the use of therapy administering a
polypeptide of interest, comprising providing to a subject in need
thereof, a CTP-modified polypeptide of interest disclosed herein,
wherein said CTP-modified polypeptide is manufactured as disclosed
herein.
[0581] In one embodiment, determination of a therapeutically
effective amount is well within the capability of those skilled in
the art.
[0582] In one embodiment, compositions including the preparation
disclosed herein formulated in a compatible pharmaceutical carrier
are also be prepared, placed in an appropriate container, and
labeled for treatment of an indicated condition.
[0583] In another embodiment, a polypeptide of interest, for
example EPO, IFN, a cytokine, a coagulation factor, FVII, FVIIa,
FIX, dual GLP-1/Glucagon receptor agonist, GLP-1, or OXM --as
described herein, is lyophilized (i.e., freeze-dried) preparation
in combination with complex organic excipients and stabilizers such
as nonionic surface active agents (i.e., surfactants), various
sugars, organic polyols and/or human serum albumin. In another
embodiment, a pharmaceutical composition comprises a lyophilized
polypeptide of interest, for example EPO, IFN, a cytokine, a
coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon receptor
agonist, GLP-1, or OXM--as described herein as described in sterile
water for injection. In another embodiment, a pharmaceutical
composition comprises a lyophilized coagulation factor as described
in sterile PBS for injection. In another embodiment, a
pharmaceutical composition comprises a lyophilized polypeptide of
interest, for example EPO, IFN, a cytokine, a coagulation factor,
FVII, FVIIa, FIX, GLP-1/Glucagon receptor agonist, GLP-1, or
OXM--as described herein as described in sterile 0.9% NaCl for
injection.
[0584] In another embodiment, the pharmaceutical composition
comprises a polypeptide of interest, for example EPO, IFN, a
cytokine, a coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon
receptor agonist, GLP-1, or OXM--as described herein and complex
carriers such as human serum albumin, polyols, sugars, and anionic
surface active stabilizing agents. In another embodiment, the
pharmaceutical composition comprises a coagulation factor as
described herein a polypeptide of interest, for example EPO, IFN, a
cytokine, a coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon
receptor agonist, GLP-1, or OXM--as described herein n and
lactobionic acid and an acetate/glycine buffer. In another
embodiment, the pharmaceutical composition comprises a polypeptide
of interest, for example EPO, IFN, a cytokine, a coagulation
factor, FVII, FVIIa, FIX, GLP-1/Glucagon receptor agonist, GLP-1,
or OXM--as described herein and amino acids, such as arginine or
glutamate that increase the solubility of interferon compositions
in water. In another embodiment, the pharmaceutical composition
comprises a lyophilized polypeptide of interest, for example EPO,
IFN, a cytokine, a coagulation factor, FVII, FVIIa, FIX,
GLP-1/Glucagon receptor agonist, GLP-1, or OXM--as described herein
as described herein and glycine or human serum albumin (HSA), a
buffer (e g. acetate) and an isotonic agent (e.g NaCl). In another
embodiment, the pharmaceutical composition comprises a lyophilized
polypeptide of interest, for example EPO, IFN, a cytokine, a
coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon receptor
agonist, GLP-1, or OXM--as described herein as described herein and
phosphate buffer, glycine and HSA.
[0585] In another embodiment, the pharmaceutical composition
comprising a polypeptide of interest, for example EPO, IFN, a
cytokine, a coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon
receptor agonist, GLP-1, or OXM--as described herein as described
herein is stabilized when placed in buffered solutions having a pH
between about 4 and 7.2. In another embodiment, the pharmaceutical
composition comprising a polypeptide of interest, for example EPO,
IFN, a cytokine, a coagulation factor, FVII, FVIIa, FIX,
GLP-1/Glucagon receptor agonist, GLP-1, or OXM--as described herein
is in a buffered solution having a pH between about 4 and 8.5. In
another embodiment, the pharmaceutical composition comprising a
polypeptide of interest, for example EPO, IFN, a cytokine, a
coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon receptor
agonist, GLP-1, or OXM--as described herein is in a buffered
solution having a pH between about 6 and 7. In another embodiment,
the pharmaceutical composition comprising a polypeptide of
interest, for example EPO, IFN, a cytokine, a coagulation factor,
FVII, FVIIa, FIX, GLP-1/Glucagon receptor agonist, GLP-1, or
OXM--as described herein is in a buffered solution having a pH of
about 6.5. In another embodiment, the pharmaceutical composition
comprising a polypeptide of interest, for example EPO, IFN, a
cytokine, a coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon
receptor agonist, GLP-1, or OXM--as described herein as described
herein is stabilized with an amino acid as a stabilizing agent and
in some cases a salt (if the amino acid does not contain a charged
side chain).
[0586] In another embodiment, the pharmaceutical composition
comprising a polypeptide of interest, for example EPO, IFN, a
cytokine, a coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon
receptor agonist, GLP-1, or OXM--as described herein as described
herein is a liquid composition comprising a stabilizing agent at
between about 0.3% and 5% by weight which is an amino acid.
[0587] In another embodiment, the pharmaceutical composition
comprising a polypeptide of interest, for example EPO, IFN, a
cytokine, a coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon
receptor agonist, GLP-1, or OXM--as described herein as described
herein provides dosing accuracy and product safety. In another
embodiment, the pharmaceutical composition comprising a polypeptide
of interest, for example EPO, IFN, a cytokine, a coagulation
factor, FVII, FVIIa, FIX, GLP-1/Glucagon receptor agonist, GLP-1,
or OXM--as described herein as described herein provides a
biologically active, stable liquid formulation for use in
injectable applications. In another embodiment, the pharmaceutical
composition comprises a non-lyophilized polypeptide of interest,
for example EPO, IFN, a cytokine, a coagulation factor, FVII,
FVIIa, FIX, GLP-1/Glucagon receptor agonist, GLP-1, or OXM--as
described herein as described herein.
[0588] In another embodiment, the pharmaceutical composition
comprising a polypeptide of interest, for example EPO, IFN, a
cytokine, a coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon
receptor agonist, GLP-1, or OXM--as described herein as described
herein provides a liquid formulation permitting storage for a long
period of time in a liquid state facilitating storage and shipping
prior to administration.
[0589] In another embodiment, the pharmaceutical composition
comprising a polypeptide of interest, for example EPO, IFN, a
cytokine, a coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon
receptor agonist, GLP-1, or OXM--as described herein as described
herein comprises solid lipids as matrix material. In another
embodiment, the injectable pharmaceutical composition comprising a
polypeptide of interest, for example EPO, IFN, a cytokine, a
coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon receptor
agonist, GLP-1, or OXM--as described herein as described herein
comprises solid lipids as matrix material. In another embodiment,
the production of lipid microparticles by spray congealing was
described by Speiser (Speiser and al., Pharm. Res. 8 (1991) 47-54)
followed by lipid nanopellets for peroral administration (Speiser
EP 0167825 (1990)). In another embodiment, lipids, which are used,
are well tolerated by the body (e. g. glycerides composed of fatty
acids which are present in the emulsions for parenteral
nutrition).
[0590] In another embodiment, the pharmaceutical composition
comprising a polypeptide of interest, for example EPO, IFN, a
cytokine, a coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon
receptor agonist, GLP-1, or OXM--as described herein as described
herein comprises polymeric microparticles. In another embodiment,
the pharmaceutical composition comprising a polypeptide of
interest, for example EPO, IFN, a cytokine, a coagulation factor,
FVII, FVIIa, FIX, GLP-1/Glucagon receptor agonist, GLP-1, or
OXM--as described herein as described herein comprises
nanoparticles. In another embodiment, the pharmaceutical
composition comprising a polypeptide of interest, for example EPO,
IFN, a cytokine, a coagulation factor, FVII, FVIIa, FIX,
GLP-1/Glucagon receptor agonist, GLP-1, or OXM--as described herein
as described herein comprises liposomes. In another embodiment, the
pharmaceutical composition comprising a polypeptide of interest,
for example EPO, IFN, a cytokine, a coagulation factor, FVII,
FVIIa, FIX, GLP-1/Glucagon receptor agonist, GLP-1, or OXM--as
described herein as described herein comprises lipid emulsion. In
another embodiment, the pharmaceutical composition comprising a
polypeptide of interest, for example EPO, IFN, a cytokine, a
coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon receptor
agonist, GLP-1, or OXM--as described herein as described herein
comprises microspheres. In another embodiment, the pharmaceutical
composition comprising a polypeptide of interest, for example EPO,
IFN, a cytokine, a coagulation factor, FVII, FVIIa, FIX,
GLP-1/Glucagon receptor agonist, GLP-1, or OXM--as described herein
as described herein comprises lipid nanoparticles. In another
embodiment, the pharmaceutical composition comprising a polypeptide
of interest, for example EPO, IFN, a cytokine, a coagulation
factor, FVII, FVIIa, FIX, GLP-1/Glucagon receptor agonist, GLP-1,
or OXM--as described herein as described herein comprises lipid
nanoparticles comprising amphiphilic lipids. In another embodiment,
the pharmaceutical composition comprising a polypeptide of
interest, for example EPO, IFN, a cytokine, a coagulation factor,
FVII, FVIIa, FIX, GLP-1/Glucagon receptor agonist, GLP-1, or
OXM--as described herein as described herein comprises lipid
nanoparticles comprising a drug, a lipid matrix and a surfactant.
In another embodiment, the lipid matrix has a monoglyceride content
which is at least 50% w/w.
[0591] In one embodiment, compositions disclosed herein 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.
[0592] In one embodiment, it will be appreciated that the
polypeptide of interest, for example EPO, IFN, a cytokine, a
coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon receptor
agonist, GLP-1, or OXM--as described hereins disclosed herein 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 avoid
adverse side effects which are associated with combination
therapies.
[0593] In one embodiment, disclosed herein is a cell comprising the
expression vector. In another embodiment, disclosed herein is a
composition comprising the expression vector.
[0594] As is generally known in the art, the modified peptides and
proteins disclosed herein may be coupled to labels, drugs,
targeting agents, carriers, solid supports, and the like, depending
on the desired application. The labeled forms of the modified
biologicals may be used to track their metabolic fate; suitable
labels for this purpose include, especially, radioisotope labels
such as iodine 131, technetium 99, indium 111, and the like. The
labels may also be used to mediate detection of the modified
proteins or peptides in assay systems; in this instance,
radioisotopes may also be used as well as enzyme labels,
fluorescent labels, chromogenic labels, and the like. The use of
such labels is particularly helpful if the peptide or protein is
itself a targeting agent such as an antibody or a receptor
ligand.
[0595] Similar linking techniques, along with others, may be
employed to couple the modified peptides and proteins disclosed
herein to solid supports. When coupled, these modified peptides and
proteins can then be used as affinity reagents for the separation
of desired components with which specific reaction is
exhibited.
[0596] Finally, the modified peptides and proteins disclosed herein
may be used to generate antibodies specifically immunoreactive with
these new compounds. These antibodies are useful in a variety of
diagnostic and therapeutic applications, depending on the nature of
the biological activity of the unmodified peptide or protein. It is
to be understood that disclosed herein is antibodies that are
immunoreactive with CTP-modified polypeptide as described herein.
In one embodiment, such antibodies may be used to distinguish or
identify CTP-modified polypeptides of interest, for example EPO,
IFN, a cytokine, a coagulation factor, FVII, FVIIa, FIX,
GLP-1/Glucagon receptor agonist, GLP-1, or OXM--as described herein
that were administered from endogenous polypeptides of interest,
for example EPO, IFN, a cytokine, a coagulation factor, FVII,
FVIIa, FIX, GLP-1/Glucagon receptor agonist, GLP-1, or OXM--as
described herein, respectively. In another embodiment, the
antibodies may be used to localize administered CTP-modified
polypeptides of interest, for example EPO, IFN, a cytokine, a
coagulation factor, FVII, FVIIa, FIX, GLP-1/Glucagon receptor
agonist, GLP-1, or OXM--as described herein.
[0597] It is to be understood that the compositions and methods
disclosed herein comprising the elements or steps as described
herein may, in another embodiment, consist of those elements or
steps, or in another embodiment, consist essentially of those
elements or steps. A skilled artisan would appreciate that the term
"comprise" may encompass inclusion of the indicated active agent,
such as the CTP-modified polypeptide of interest, as well as
inclusion of other active agents, and pharmaceutically acceptable
carriers, excipients, emollients, stabilizers, etc., as are known
in the pharmaceutical industry. A skilled artisan would appreciate
that the term "consisting essentially of" may encompass a
composition, whose only active ingredient is the indicated active
ingredient, however, other compounds may be included which are for
stabilizing, preserving, etc. the formulation, but are not involved
directly in the therapeutic effect of the indicated active
ingredient. A skilled artisan would appreciate that the term
"consisting essentially of" may encompass components which
facilitate the release of the active ingredient. A skilled artisan
would appreciate that the term "consisting" may encompass a
composition, which contains the active ingredient and a
pharmaceutically acceptable carrier or excipient.
[0598] Additional objects, advantages, and novel features disclosed
herein 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 disclosed herein as delineated hereinabove and as claimed
in the claims section below finds experimental support in the
following examples.
EXAMPLES
Example 1--Generation of EPO Constructs
[0599] Materials and Methods:
[0600] 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:
[0601] 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.
[0602] 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.
[0603] The dhfr gene was ligated into pCI vector to form an
expression vector containing the dhfr gene (pCI-dhfr).
[0604] 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_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 disclosed herein.
[0605] Table 2 hereinbelow summarizes the primer sequences used for
constructing the CTP--containing polypeptides disclosed herein.
TABLE-US-00055 TABLE 2 Restriction site SEQ (underlined Primer ID
in number NO sequence sequence) 1 72 5' AATCTAG XbaI AGGTCATCA
TGGGGGTG C3' 2 73 5' ATTGCGG NotI CCGCGGATCC AGAAGACCTT TATTG3'
17.sup.R 74 5' TAAATAT SspI TGGGGTGTCC GAGGGCCC 3' 10 75 5'
CCAATATT SspI ACCACAAG CCCCACCA CGCCTCA T 3' 11.sup.R 76 5'
TGCGGCCG NotI CGGATCCT TATCTGTC CCCTGTCC TGC 3' 15 77 5' GCCCTGCT
GTCGGAA GC 3' 2R 78 5' ATTGCGGC NotI CGCGGATC CAGAAGAC CTTTATTG 23R
79 5' CTTTGAGG AAGAGGA GCCCAGG ACTGGGA GGC 3' 24 80 5' CCTGGGCT
CCTCTTC CTCAAAG GC 3' 38R 81 5' GCTTCCGA CAGCAG GGC 3'
[0606] 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: 72-81). Then the XbaI-NotI PCR fragment containing Epo-ctp
sequence was ligated into pCI-dhfr expression vector.
[0607] 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.
[0608] 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.
[0609] 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.
[0610] The two fragments were ligated into pCI-dhfr to construct
the p701-4-p42-1 clone.
[0611] 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: 77) and primer 2.sup.R (SEQ ID NO: 78) 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).
[0612] Three sequential PCR reactions were performed. The first
reaction was conducted with primer 1 (SEQ ID NO: 72) and primer
23.sup.R (SEQ ID NO: 79) 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).
[0613] The second reaction was conducted with primer 24 (SEQ ID NO:
80) and primer 11.sup.R (SEQ ID NO: 76) and plasmid DNA of
701-4-p42-1 as a template; as a result of the PCR amplification, a
610 bp product was formed.
[0614] The last reaction was conducted with primers 1 (SEQ ID NO:
72) and 11.sup.R (SEQ ID NO: 76) 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.
[0615] 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.
[0616] 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: 72) and primer 11.sup.R (SEQ ID NO: 76) 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.
[0617] 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: 72) and primer 38.sup.R (SEQ ID NO: 81) and
plasmid DNA of 701-3-p56-6 as a template; as a result of the PCR
amplification, a 400 bp product was formed.
[0618] The second reaction was conducted with primer 15 (SEQ ID NO:
77) and primer 2.sup.R (SEQ ID NO: 78) and plasmid DNA of
701-1-p17-6 as a template; as a result of the PCR amplification, a
390 bp product was formed.
[0619] The last reaction was conducted with primers 1 (SEQ ID NO:
72) and 2.sup.R (SEQ ID NO: 78) 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
[0620] 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 least 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.
[0621] 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.
[0622] 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.
[0623] Western Blotting: Samples were electrophoresed on
nondenaturing 15 SDS-polyacrylamide gels. Gels were allowed to
equilibrate for 10 mM 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 least 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
[0624] 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-00056 TABLE 3 Post Dilution in Mock sup. according Post
Stock to Epo3 Ultra- # # Titer titer filtration Version Clone
IU/ml.sup.1 IU/ml.sup.2 IU/ml.sup.3 Epo0 17 3093 102 335 SEQ ID NO:
8 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.
[0625] All proteins were detected by Western blot as illustrated in
FIG. 2.
Example 3--Generation and Utilization of Coagulation Factor IX
[0626] Cloning and Expression of Recombinant FIX Molecule:
[0627] Factor IX clones were constructed in our eukaryotic
expression vector pCI-neo (Promega, catalog no. E1841). ORF Clone
of Homo sapiens coagulation factor IX was ordered from "OriGene"
(RC219065). Primers were ordered from Sigma-Genosys.
[0628] Construction of 301-1-pCI-Neo-p200-11 (Factor IX-Ctp
x2):
TABLE-US-00057 Primer 101: (SEQ ID NO: 82) 5' GTTTAGTGAACCGTCAGAAT
3' Primer 103R: (SEQ ID NO: 83) 5' TTGAGGAAGATGTTCGTGTA 3'
(contains the SspI site of factor IX)
[0629] A PCR reaction was conducted with primer 101 and primer
103.sup.R and plasmid DNA, cDNA clone of Factor IX (OriGene"
RC219065) as a template; as a result of the PCR amplification, a
.about.1085 bp (per 10) product was formed and purified from the
gel (the fragment containing the amino terminus of Factor IX
sequence).
TABLE-US-00058 Primer 98: (SEQ ID NO: 84) 5' ATTACAGTTGTCGCAGGTGA
3' Primer 99R: (SEQ ID NO: 85) 5' GCTGGAGCTAGTGAGCTTTGTTTTTTCCTT 3'
Primer 100: (SEQ ID NO: 107) 5' GCTCACTAGCTCCAGCAGCAAGGCC 3' Primer
27R: (SEQ ID NO: 86) 5' TTTTCACTGCATTCTAGTTGTGG 3'
[0630] Three PCR reactions were performed. The first reaction was
conducted with primer 98 and primer 99.sup.R and plasmid DNA, cDNA
clone of Factor IX (OriGene, RC219065) as a template; as a result
of the PCR amplification, a .about.540 bp product was formed.
[0631] The second reaction was conducted with primer 100 and primer
27.sup.R and plasmid DNA of 402-2-p72-3 (hGH-CTP-CTP) as a
template; as a result of the PCR amplification, a .about.258 bp
product was formed.
[0632] The last reaction (per 3) was conducted with primers 98 and
27.sup.R and a mixture of the products of the previous two
reactions as a template; as a result of the PCR amplification, a
.about.790 bp product was formed and ligated into TA cloning vector
(Invitrogen, catalog K2000-01). SspI-EcoRI fragment was isolated
(TA 3-3).
[0633] Another PCR reaction was conducted (per 12) with primer 101
and primer 27.sup.R and a mixture of the products of per 10 and
SspI-EcoRI fragment from per 3 as a template; as a result of the
PCR amplification, a .about.1700 bp product was formed (Factor
IX-ctp-ctp) and ligated into TA cloning vector (Invitrogen, catalog
K2000-01) (lig 180).
[0634] A mistake was found in the Factor IX sequence so fragments
were replaced in order to form an insert of Factor IX-ctp-ctp with
the correct DNA sequence.
[0635] TA-pcr 3-3 was digested with SspI and XbaI and the large
fragment was isolated (vector). TA 180-4 was digested with SspI and
XbaI and the small fragment (insert) was isolated and ligated to
the isolated large fragment of TA-per-3-3 digested with SspI and
XbaI. The new plasmid TA-183-2 was digated with Sal I and NotI, and
the Factor IX-CTP-CTP insert was isolated (.about.1575 bp). This
fragment was inserted into eukaryotic expression vector pCI-neo
(digested with Sal I and Not I) to yield the 301-2-p200-11
clone.
[0636] pCI-dhfr-Factor 9-ctpx2 (p223-4) construction: Vector
pCI-dhfr (p6-1) was digested with SmaI and NotI. Factor IX-CTP-CTP
(p200-11) was digested with ASisI F.I. and Nod. The two fragments
were ligated.
[0637] pCI-dhfr Factor 9-ctp x3 (p225-7) construction: Vector
pCI-dhfr OXM-CTPx3 (p216-4) was digested with XbaI and ApaI. Factor
IX-CTP-CTP (223-4) was digested with XbaI and ApaI. The two
fragments were ligated.
[0638] pCI-dhfr Factor 9-ctp x3 T148A (p243-2) construction:
Plasmid p225-7 contained Threonine at position 148, since the more
common version of FIX contains Alanine at this position, Thr was
replaced to Ala using site directed mutagenesis method.
TABLE-US-00059 Primer 75: (SEQ ID NO: 87) ctcccagttcaattacagct
Primer 122r: (SEQ ID NO: 88) ggaaaaactgcctcagcacgggtgagc Primer
123: (SEQ ID NO: 89) gtgctgaggcagtattcctgatgtggactat Primer 124r:
(SEQ ID NO: 90) caacacagtgggcagcag
[0639] Three PCR reactions were performed. The first reaction was
conducted with primer 75 and primer 122r and plasmid DNA p225-7 as
a template; as a result of the PCR amplification, a 692 bp product
was formed and purified from the gel. A second PCR reaction was
conducted with primer 123 and primer 124r and plasmid DNA p225-7 as
a template; as a result of the PCR amplification, a .about.237 bp
product was formed and purified from the gel. The third--overlap
PCR reaction was conducted with primers 75 and 124r, and a mixture
of the products of the previous two reactions as a template; as a
result of the PCR amplification, a .about.910 bp product was
formed. This overlap PCR product was digested with XbaI and NsiI
and re ligated into p225-7 plasmid (digested with XbaI and NsiI) to
yield Factor IX-ctpx3 T148A designated p243-2.
[0640] FIX-4CTP (p259-4) construction: 3.5CTP fragment was isolated
from oxym-4CTP (p254-3) by restriction enzymes ApaI and XbaI.
FIX+0.5CTP fragment was isolated from FIX-3CTP (p243-2) with
restriction enzymes ApaI and XbaI. The two fragments were
ligated.
[0641] FIX-5CTP (p260-18) construction: 4.5CTP fragment was
isolated from oxym-5CTP (255-1) by restriction enzymes ApaI and
XbaI. FIX+0.5CTP fragment was isolated from FIX-3CTP (p243-2) using
enzymes ApaI and XbaI. The two fragments were ligated.
[0642] Dg44 cells were plated in 100 mm tissue culture dishes and
grown to 50-60% confluence. A total of 2 .mu.g (microgram) of FIX
cDNA was used for the transfection of one 100 mm plate using the
FuGene reagent (Roche) in protein-free medium (Invitrogene CD
Dg44). The media was removed 48 hours after transfection and
replaced with a protein-free medium (Invitrogene CD Dg44) without
nucleosides and in the presence of 800 .mu.g/ml of G418 (Neomycin).
After 14 days, the transfected cell population was transferred into
T25 tissue culture flasks, and selection continued for an
additional 10-14 days until the cells began to grow as stable
clones. High expressing clones were selected. Approximately
2.times.10.sup.7 cells were used to inoculate 300 ml of growth
medium in a 1700 cm.sup.2 roller bottle (Corning, Corning N.Y.)
supplemented with 5 ng/ml of Vitamin K3 (menadione sodium
bisulfate; Sigma). The production medium (harvest) was collected
after a rapid decrease in cell viability to about 70%. The
production medium was first clarified and then concentrated
approximately 20-fold and dialyzed with PBS using flow filtration
cassette (10 KDa MWCO; Millipore Corp.).
[0643] Determination of FIX antigen level: FIX-CTP harvest antigen
levels were determined using AssayMax Human FIX ELISA kit
(AssayPro-EF1009-1). The calculated protein concentration is the
average of three different dilutions in two independent runs (Table
4).
TABLE-US-00060 TABLE 4 Calculated protein concentration FIX-CTP
FIX-CTP-CTP FIX Ag level (.mu.g/ml) 41.9 19.2 SD 8.76 3.67 % CV
20.92 19.15
[0644] FIX SDS-PAGE--immune blot: FIX-CTP harvests or purified
rhFIX (American Diagnostics), 100 ng of protein, were loaded on 12%
Tris-Glycine gel using Precision Plus Dual Color Protein Marker
(Bio-Rad). The SDS-PAGE analysis was performed by Western
immunoblot using anti-human FIX polyclonal antibody and anti-human
gamma carboxylation monoclonal antibody (American Diagnostics). As
previously reported, rhFIX migrated at least 55 KDa, while FIX
fused to two CTPs migrated at 75 KDa. Both variants of FIX-CTP
proteins were shown to be gamma carboxylated, an essential
post-translation modification for FIX activity and function.
[0645] Determination of FIX chromogenic activity: A comparative
assessment of the in vitro potency of FIX-CTP harvests versus rhFIX
protein (American Diagnostics) was performed using the commercially
available chromogenic activity test kit, BIOPHEN (Hyphen BioMed
221802). In the presence of thrombin, phospholipids, calcium,
excess amounts of FXIa activates sampled FIX into FIXa. FIXa forms
an enzymatic complex with thrombin, activated FVIII:C (supplied in
an excess amounts), phospholipids, and calcium and activates Factor
X, present in the assay system, into FXa. The activity directly
correlates with the amount of FIX, which is the limiting factor.
The generated FXa is then measured by its specific activity on FXa
chromogenic substrate (pNA). The amount of pNA generated is
directly proportional to FIXa activity. rhFIX and FIX-CTP harvests
were serially diluted, and the potency was assessed by comparing a
dose-response curve of the FIX harvests to a reference preparation
consisting of rhFIX or human plasma. The average EC50 of FIX was 21
ng/ml, while the FIX-(CTP).sub.2 harvest calculated EC50 was 382
ng/ml, and the FIX-CTP harvest calculated EC50 was 1644 ng/ml. An
approximately 15-fold decrease in the enzymatic activity of the
FIX-(CTP).sub.2 harvest was observed.
[0646] FIX Clotting activity (aPTT): The activated partial
thromboplastin time (aPTT) is a measure of the integrity of the
intrinsic and common pathways of the coagulation cascade. The aPTT
is the time, in seconds, for plasma to clot following the addition
of an intrinsic pathway activator, phospholipid and calcium. The
aPTT reagent is called a partial thromboplastin because tissue
factor is not included with the phospholipid as it is with the
protime (PT) reagent. The activator initiates the system and then
the remaining steps of the intrinsic pathway take place in the
presence of phospholipid. Reference aPTT range varies from
laboratory to laboratory, but is usually in the range of 27-34
seconds.
[0647] The principal of the assay was to quantitate the ability of
FIX-CTP harvests to restore the clotting activity of FIX-depleted
human plasma by the addition of rhFIX. 300 .mu.l of FIX-deficient
human plasma was mixed with 100 .mu.l of rhFIX or FIX-CTP harvests
and serially diluted. Following a 60 second incubation at
37.degree. C., thromboplastin, CaCl.sub.2, and phospholipids were
added to the mixture, and clotting time in seconds was determined
(performed by American Medical Laboratories). The potency was
assessed by comparing a dose-response curve of the FIX harvests to
a reference preparation consisting of rhFIX or human plasma. One
unit of FIX activity corresponds to the FIX concentration that
equals the activity of one ml normal human plasma. The presented
aPTT results indicate that FIX-(CTP).sub.2 exhibit a 5.7-fold
reduction in its specific coagulation activity compared to rhFIX
(Table 5). Moreover, the aPTT results together with the chromogenic
activity in vitro assay suggest that FIX-(CTP).sub.2 harvest has an
improved enzymatic activity vs. FIX-CTP harvest (Table 5). An
improved activity of FIX-CTP proteins can be obtained following
optimization of the expression system (i.e. co-transfection with
Furin and optimization of Vitamin K3 medium concentration), which
was strengthened following super-transfection with Furin (data not
shown).
TABLE-US-00061 TABLE 5 FIX clotting activity rhFIX(AD) PTT FIX-CTP
PTT FIX-CTP-CTP PTT (.mu.g/ml) (Sec) (.mu.g/ml) (Sec) (.mu.g/ml)
(Sec) 5 31.3 9 45.2 4 47.5 1.25 35.7 2.25 53.3 1 55.9 0.3125 43
0.5625 64.1 0.25 67 0.078125 52.1 0.140625 76.3 0.0625 77.4
[0648] Pharmacokinetic study: rhFIX (American Diagnostic) and
FIX-CTP harvests were administered in a single intravenous
injection to Sprague-Dawley rats (six rats per substance) at a dose
of 75 .mu.g/kg body weight (Table 6).
TABLE-US-00062 TABLE 6 PK study plan of operation Dose No. of Dose
Level Injected Treated animals/ Dose Level (.mu.g per Vol. Con.
*Time-Points Groups Test Article group Route Gender (.mu.g/kg)
animal) (.mu.l) (.mu.g/ml) (hours post-dose) 1 rFIX 6 IV M 75 15
500 30 0 (Pre-dose) 0.083, 0.5, 1.5, 4, 8, 24, 48, 72. 2 rFIX-CTP 6
IV M 75 15 500 30 0 (Pre-dose) 0.083, 0.5, 1.5, 4, 8, 24, 48, 72. 3
rFIX-CTP- 6 IV M 75 15 1000 15 0 (Pre-dose) CTP 0.083, 0.5, 1.5, 4,
8, 24, 48, 72.
[0649] Blood samples were drawn retro-orbitally from 3 rats
alternately at 0.083, 0.5 1.5, 4, 8, 24, 48, and 72 hours
post-dosing. Plasma was prepared immediately after sampling and
stored at -20.degree. C. until analysis. FIX concentration was
quantitated by FIX ELISA-specific assay (AssayPro). A
pharmacokinetic profile was calculated for each protein and
represents the mean of 3 animals at each time point (data not
shown). The terminal half-lives were calculated using PK solutions
2.0 software. Table 7 summarizes the observed FIX concentrations at
the different sampling time points.
TABLE-US-00063 TABLE 7 Observed FIX concentrations Time FIX-AD
FIX-CTP FIX-CTP-CTP (Hr) (ng/ml) (ng/ml) (ng/ml) 0.083 1506.7
1477.5 1914.8 0.5 1949.8 1150.1 1830.1 1.5 2189.4 1009.0 1264.3 4
733.90 709.33 1000.00 8 319.80 167.20 1234.67 24 BLQ 54.625 230 48
BLQ BLQ 120.9
[0650] The PK profile and summary of the terminal half-lives are
summarized in Table 8. FIX-CTP harvests exhibit an improved
T1/2.beta. values compared to rhFIX (2- and 5-fold increases,
respectively). Since in FIX dosing collection, animal serum
concentrations of FIX at 24 hr were below limit of quantitation
(BLQ), additional PK parameters were not calculated.
TABLE-US-00064 TABLE 8 Summary of PK parameters Terminal Ratio
Product half-life- (hr) (FIX-(CTP).sub.X/rhFIX) rhFIX (American
2.62 -- Diagnostics) FIX-CTP 5.55 2.11 FIX-CTP 12.9 4.92
(FIX-CTP-CTP)
[0651] In this study, a novel approach was described for prolonging
FIX half-life while retaining the therapeutic potency. Adding a CTP
peptide to an active protein has a harmful potential in interfering
with the protein's activity. Therefore, the generation of an active
recombinant FIX-CTP by adding a CTP sequence at the C-terminus of
the FIX is unexpected.
Characterization of an Immunoaffinity Purified FIX-CTP-CTP
[0652] FIX-CTP-CTP Purification
[0653] In order to evaluate a protein at high grade content with
increased activity whose PK profile mimics and can be extrapolated
to a clinical setting, FIX-CTP-CTP is a FIX modified with 2 CTP
units in tandem in its carboxy-terminal FIX-CTP-CTP was purified
using matrix-bound monoclonal antibody against .gamma.
carboxyglutamyl (Gla) residues present in the N-terminal region of
FIX (American Diagnostics Cat. #3570MX). The monoclonal antibody
was bound to Sepharose CL-4B. The FIX-CTP-CTP harvest at a
concentration of 88 .mu.g/ml was dialyzed against 20 mM Tris, 150
Mm NaCl and 10 mM EDTA at PH=7.4. The loading rate was 0.5 ml/min,
elution was performed using 20 Mm Tris-HCl, 350 mM NaCl and 50 mM
CaCl, and the unbound fraction was recycled five times Finally, the
elution fraction was dialyzed with PBS, pulled and
concentrated.
[0654] Determination of FIX antigen level: FIX-CTP harvests,
FIX-(CTP).sub.2 harvests, and FIX-(CTP).sub.2 purified protein
levels were determined using the Human FIX ELISA kit (Affinity
Biologicals; Cat. #FIX-AG RUO). The calculated protein
concentration (.mu.g/ml) is the average of two independent runs
(Data not shown, Table 9).
TABLE-US-00065 TABLE 9 Calculated protein concentration FIX-CTP-CTP
FIX-CTP FIX-CTP-CTP (purified) FIX Ag level 125.78 88.53 172.9
(.mu.g/ml) SD 17.28 21.31 2.63 % CV 13.74 24.08 1.52
[0655] Additionally, FIX-CTP-CTP was quantitated by Bradford assay.
The calculated concentration was 202 .mu.g/ml, which is similar to
the concentration obtained by human FIX ELISA.
[0656] SDS-PAGE blots: FIX-CTP-CTP harvest, unbound fraction and
purified protein, were loaded on a 12% Tris-Glycine gel using
Precision Plus Dual Color Protein Marker (Bio-Rad). The SDS-PAGE
Coomassie analysis was performed by staining the gel with Coommasie
blue reagent (800 ng of protein). A Western immunoblot was
performed with 100 ng of protein, anti-human FIX polyclonal
antibody (Ab), and anti-human gamma carboxylation monoclonal Ab
(American Diagnostics Cat #499 and #3570). The immunoaffinity
purification procedure significantly enriched the FIX-CTP-CTP
portion while reduced impurity (data not shown).
[0657] N-terminal sequencing: FIX-CTP-CTP purified protein was
separated by 12% Tris-Glycine SDS-PAGE and subsequently
electro-blotted to PVDF membrane. The band of interest was cut out
and put on a purified Biobrene treated glass fiber filter. The
N-terminal sequence analysis was carried out by Edmann degradation
using a pulsed liquid protein sequencer equipped with a 140 C HPLC
micro-gradient system. N-terminal sequencing revealed that
FIX-CTP-CTP is a mixture of incomplete and complete pro-peptide
cleaved proteins. Inadequate pro-peptide cleavage was shown to
reduce FIX coagulation activity. By co-transfection with Furin, the
pro-peptide cleavage process can be an improved.
[0658] Determination of FIX chromogenic activity: A comparative
assessment of the in vitro potency of FIX-CTP-CTP purified protein
versus rhFIX (American Diagnostics) and a pool of human normal
plasma was performed using the commercially available chromogenic
activity test kit, BIOPHEN (Hyphen BioMed 221802). In the presence
of thrombin, phospholipids and calcium, excess amounts of FXIa
activates FIX into FIXa. FIXa forms an enzymatic complex with
thrombin (supplied in excess amounts), phospholipids and calcium
activates Factor X, present in the assay system, into FXa. The
activity directly correlates with the amount of FIX, which is the
limiting factor. The generated FXa was measured by its specific
activity on FXa chromogenic substrate (pNA). The amount of pNA
generated was directly proportional to FIXa activity. rhFIX, human
plasma and FIX-CTP-CTP were serially diluted, and potency was
assessed by comparing a dose-response curve (Data not shown). The
average EC50 of rhFIX was 68.74 ng/ml while FIX-CTP-CTP calculated
EC50 was 505 ng/ml. An approximately 7-fold decrease in the
enzymatic activity of FIX-CTP-CTP was observed vs. recombinant FIX
and a 16.5-fold decrease versus normal human pulled plasma. This
reduced activity could be explained by inadequate cleavage of
N-terminal pro-peptide, which was identified by N-terminal
analysis.
[0659] FIX Clotting activity (aPTT): The activated partial
thromboplastin time (aPTT) is a measure of the integrity of the
intrinsic and common pathways of the coagulation cascade. The aPTT
is the time (measured in seconds) it takes plasma to clot following
the addition of an intrinsic pathway activator, phospholipid and
calcium.
[0660] The assay quantitated the ability of the FIX-CTP-CTP protein
to restore the clotting activity of FIX depleted human plasma by
the addition of rhFIX. 300 .mu.l of FIX-deficient human plasma was
mixed with 100 .mu.l of rhFIX, FIX-CTP-CTP (FIX-CTP-CTP (the CTP
are in tandem at the C-terminal)), or normal pool human plasma
which was further diluted. Following a 60 second incubation at
37.degree. C., Tissue Factor (TF), CaCl.sub.2, and phospholipids
were added to the mixture. Clotting time in seconds was determined.
Potency was assessed by comparing a dose-response curve of
FIX-CTP-CTP to a reference preparation of rhFIX or human plasma.
One unit of FIX was defined as the amount of FIX which equals to
the activity of 1 ml human normal plasma.
[0661] The aPTT results indicate that FIX-CTP-CTP coagulation
activity is only 1.4 less then normal pool human plasma and similar
to the rhFIX. The aPTT results together with the chromogenic
activity in vitro assay suggest that FIX-CTP-CTP purification did
not damage its activity.
[0662] Pharmacokinetic activity of FIX-CTP-CTP: Purified
FIX-CTP-CTP, rhFIX (American Diagnostic) and harvests containing
FIX-CTP-CTP and FIX-CTP were administered in a single intravenous
injection to Sprague-Dawley rats (eight rats per substance) in a
dose of 100 .mu.g/kg body weight (Table 10).
TABLE-US-00066 TABLE 10 PK study outline No. of Dose animals/ Dose
Level Injected Treated group/time Level (.mu.g per Vol. Con.
Time-Points Groups Test Article point (.mu.g/kg) animal) (.mu.l)
(.mu.g/ml) (hours post-dose) A rFIX 8 100 20 500 40 0 (Pre-dose)
0.083, 0.5, 1, 2, 4, 7, 10, 24, 48, 72. B rFIX-CTP 8 100 20 500 40
0 (Pre-dose) (harvest) 0.083, 0.5, 1, 2, 4, 7, 10, 24, 48, 72. C
rFIX-CTP-CTP 6 100 20 500 40 0 (Pre-dose) (harvest) 0.083, 0.5, 1,
2, 4, 7, 10, 24, 48, 72. D rFIX-CTP-CTP 4 100 20 500 40 0.083, 0.5
1, 2, 4, (purified) 7, 10, 24, 4, 8, 72.
[0663] Blood samples were drawn retro-orbitally from 4 rats
alternately at 0.083, 0.5, 2, 4, 7 10, 24, 48, and 72 hours
post-dosing. Citrated plasma (0.32%) was prepared immediately after
sampling and stored at -20.degree. C. until analysis. FIX
concentration was quantitated using a human FIX ELISA kit (Affinity
Biologicals). The pharmacokinetic profile was calculated for each
protein as the mean of 4 animals at each time point (FIG. 4). The
terminal half-life was calculated using PK Solutions 2.0 Software.
Table 11 summarizes the observed FIX concentrations at different
sampling time points.
TABLE-US-00067 TABLE 11 Observed FIX concentrations FIX-CTP
FIX-(CTP).sub.2 Purified FIX- Time harvest harvest rhFIX CTP-CTP
(hr) ng/ml ng/ml ng/ml ng/ml 0.085 1038.97 1123.62 325.05 886.48
0.5 939.12 956.80 274.58 670.92 1 791.97 843.85 222.90 674.17 2
304.98 673.31 186.00 503.91 4 315.37 525.50 109.69 337.36 7 171.45
384.36 67.62 257.02 10 50.34 250.73 40.20 158.66 24 10.07 78.50 BLQ
52.13 48 BLQ 23.40 BLQ 18.07
[0664] A summary of the PK parameters are presented in Table
12.
TABLE-US-00068 TABLE 12 Summary of PK parameters T1/2 AUC MRT Vd CL
(hr) ng-hr/ml (hr) ml/Kg Ml/hr/Kg FIX-CTP 4.17 3622 4.5 155.1 27.6
harvest FIX-(CTP).sub.2 10.44 9105.7 12 165.4 10.9 harvest rhFIX
3.72 1416.8 5.4 373.8 70.183 Purified FIX- 11.14 6314.2 12.3 254.5
15.83 CTP-CTP
[0665] The FIX-CTP-CTP harvest demonstrated an improved PK profile
compared to FIX-CTP harvest. Furthermore, purified FIX-CTP-CTP
exhibited a 3-fold increase in T1/2.sub..beta. value and a 4.5-fold
increase in AUC compared to rhFIX.
[0666] The reduced amount of secreted FIX fused to tandem CTP
molecules versus fusion of a single CTP appears to be due to the
addition of an extra CTP and not to reduced detection by ELISA,
because the Bradford-purified FIX-CTP-CTP calculated concentration
was similar to the ELISA-calculated concentration.
[0667] FIX-CTP-CTP clotting activity was similar to pooled human
plasma; however, its in vitro chromogenic activity was
significantly lower when compared to rhFIX or pooled human plasma.
The chromogenic activity assay was reported as a very sensitive
assay compared to the coagulation assay. The reason for reduced
activity of FIX-CTP-CTP may vary. Addition of CTP may decrease the
affinity of FIX to FXIa or reduce post-transcriptional
modifications (e.g. 12-10 GLA residues and pro-peptide cleavage).
N-terminal analysis revealed that the proteolytic cleavage of the
FIX-CTP-CTP pro-peptide was not fully completed prior to secretion.
Since this post-transcriptional modification is crucial for the
normal enzymatic activity of the protein, co-transfection with
Furine-PACE plasmid is favorable and may improve FIX-CTP-CTP
activity.
[0668] Finally, FIX-CTP-CTP comparative PK study in rats
demonstrated that fusion of two tandem CTPs to the C-terminal of
FIX generated a FIX with an extended half-life.
[0669] FIX depleted mouse model: In order to assess the in vivo
activity, FIX knockout mice are obtained, and a breeding colony is
established. 10 .mu.g of either commercial recombinant hFIX
(BeneFIX.RTM.) or rFIX-(CTP).sub.2 (FIX-CTP-CTP) are injected into
the tail vein of an anaesthetized FIX knockout mouse (22-28 g). The
amount of injected protein equals to the required concentration of
FIX in normal plasma (5 .mu.g/ml). Blood samples are taken from the
clipped tail into heparinized capillary tubes at specific time
points. Plasma samples are assessed for FIX levels by ELISA and
efficacy is measured by aPTT coagulation assay.
[0670] Increasing FIX Propeptide cleavage efficacy: CTP peptide
cDNA was fused to the 3' end of human FIX cDNA. The corresponding
rFIX and Furin expressing constructs were co-transfected into Dg44
cells; a human rFIX cDNA was also co-transfected with the Furin
plasmid as a control. Secretion of high level of FIX leads to
secretion of a mixture of pro-factor and a mature factor FIX, due
to limited amount of the Furin protease in the cell.
Co-transfection of a Furin expressing vector with a pro-factor
expressing vector increases the recovery and result in the
secretion of fully processed FIX in to the medium.
[0671] Following FIX-(CTP).sub.2 and Furin co-transfection, stable
clones are generated and harvest is collected for pro-peptide
cleavage evaluation. 100 ng of protein, are loaded on 12%
Tris-Glycine gel using Precision Plus Dual Color Protein Marker
(Bio-Rad). The SDS-PAGE analysis is performed by Western immunoblot
using anti-human FIX polyclonal Ab (American Diagnostics) and
anti-pro-peptide polyclonal antibody. As previously reported, rhFIX
migrated at least 55 KDa, while FIX fused to two CTPs migrated at
75 kDa. Both variants of FIX proteins are shown to undergo a
proper, full pro-peptide cleavage.
[0672] To determine whether proper pro-peptide cleavage improves
FIX-(CTP).sub.2 enzymatic activity, a comparative assessment of
chromogenic and coagulation activity of FIX-(CTP).sub.2 harvest co
transfecated with Furin is performed. A significant improvement in
FIX-(CTP).sub.2 specific activity is observed, which is similar to
rhFIX.
[0673] In conclusion, the results described herein suggest that
FIX-CTP-CTP can be used efficiently for treating Hemophilia B
patients. FIX fused to CTP constructs benefit from improved in vivo
pharmacologic performance that overcomes the drawback in certain in
vitro measures. This proposed treatment is advantageous over
previous treatments as the rate of infusions and the amount of
required doses are reduced.
[0674] It is important to notice that when an albumin-fused
molecule strategy was used to improve the FIX half-life, the
recombinant FIX became inactive. The present novel approach lead to
the design and purification of a novel recombinant FIX-fused
protein that presents an improved long-lasting activity. Since mere
size modifications did not improve the pharmacokinetics of injected
FIX, the finding that CTP fused to FIX facilitates pharmacokinetic
parameters was unexpected. The presence of highly glycosylated
peptide-sialic acid residues stabilized the protein and protected
it from interactions with vascular receptors without abrogating key
determinants of FIX function.
[0675] FIX-CTP has a similar therapeutic efficacy to rFIX in
hemophilia B patients and required less frequent dosing. A single
injection of FIX-CTP is sufficient to control bleeding episodes and
reduce the number of injections that are needed during surgical
intervention in hemophilia B patients.
[0676] The CTP technology was utilized for the development of a
long-acting FIX. Specifically, extending the half-life of
recombinant rFIX molecule was performed by fusion of at least one
human CTP to FIX. The recombinant FIX-CTP was expressed in
mammalian cells and characterized in vitro and in vivo. It was
demonstrated that the in vitro activity of rFIX-CTP was comparable
to rFIX. Pharmacokinetics and efficacy studies in rats demonstrated
improved properties of the rFIX-CTP. The results of this study
demonstrate that it is feasible to develop a half-life extended
rFIX molecule having similar haemostatic properties to the wild
type enzyme.
Example 4--Comparative Assessment of Purified FIX-CTP.sub.3 vs.
FIX-CTP.sub.4 and FIX-CTP.sub.5
[0677] 2.1 Study Objective
[0678] A comparative assessment of the pharmacokinetic parameters
of FIX-CTP.sub.4 and FIX-CTP.sub.5 versus FIX-CTP.sub.3 following a
partial purification process.
[0679] 2.2 Production of FIX-CTP4 and FIX-CTP.sub.5 Harvests
[0680] FIX cDNA (OriGene RC219065) fused at the C-terminal to four
or five tandem CTP sequences was expressed in Dg44 cells using
Excellgene expression system in the presence of 10 ng/L of vitamin
K3 (Sigma, Mennadion). The harvests were collected (300 ml),
filtered and frozen.
[0681] 2.3 Production of FIX-CTP3 Harvest
[0682] FIX-CTP.sub.3 was expressed in-house in CHO cells using
pCI-DHFR vector, clone 196, BR-9 in the presence of 25 ng/L of
vitamin K3 (Sigma). The harvests were collected and filtered.
[0683] All FIX-CTP samples (3, 4 and 5 CTP) were purified only by
Jacalin column because of a lack of material.
[0684] 2.4 Determination of FIX Antigen Level
[0685] FIX antigen level was determined using Human FIX ELISA kit
(Affinity Biologicals; Cat. #FIX-AG RUO). The calculated protein
concentration is the average of four independent runs.
FIX-CTP.sub.3 concentration was slightly higher as compared to the
two additional versions (Table 13).
TABLE-US-00069 TABLE 13 FIX antigen level 3 CTP 4 CTP 5 CTP Final
Final Final Jacalin40 Jacalin40 Jacalin40 Av. (ng/ml) 1016.69
4644.11 1686.82 SD 225.41 925.63 160.07 % CV 22.17 19.93 9.49
[0686] 2.5 FIX-CTP Coomassie Stain and Immune-Blot
[0687] FIX-CTP.sub.3, FIX-CTP.sub.4, and FIX-CTP.sub.5 harvests
were loaded on 12% Tris-Glycine gel using Precision Plus Dual Color
Protein Marker (Bio-Rad). The SDS-PAGE analysis was performed by
Western immuno-blot using anti-CTP polyclonal Ab (Adar Biotech
Production) or anti-Gla Ab (American Diagnostica).
[0688] As previously reported, FIX fused to three CTPs migrated at
80 kDa while FIX fused to four or five CTPs migrated at 85 KDa or
90 KDa, respectively. As expected, FIX-CTP.sub.4 and FIX-CTP.sub.5
harvests from Excellgene showed very low levels of gamma
carboxylation compared to FIX-CTP.sub.3 harvest, which was produced
at Prolor (FIG. 5).
[0689] After a purification process utilizing Jacalin column
(immunoaffinity purification of glycosylated proteins),
FIX-CTP.sub.3, FIX-CTP.sub.4, and FIX-CTP.sub.5 were loaded on 12%
Tris-Glycine gel using Precision Plus Dual Color Protein Marker
(Bio-Rad). The SDS-PAGE was stained by Coomassie blue Dye for
samples detection. All variants showed much cleaner band profiles
(FIG. 6), suggesting an improved purity.
[0690] 2.6 Determination of FIX Chromogenic Activity
[0691] A comparative assessment of the in vitro potency of fully
purified (HA column) FIX-CTP3, FIX-CTP4, and FIX-CTP.sub.5 versus
human pool normal plasma was performed using a commercially
available chromogenic activity test kit, BIOPHEN (Hyphen BioMed
221802). All samples were serially diluted, and the potency was
assessed by comparing a dose-response curve to a reference
preparation of normal human plasma. The reduced chromogenic
activity of FIX-CTP.sub.4 and FIX-CTP.sub.5 (FIG. 7) as compared to
plasma can be a consequence of improper post-transcriptional
modifications of FIX proteins, e.g. inappropriate gamma
carboxylation and pro-peptide cleavage or, alternatively, due to
the addition of CTP cassettes. The fluctuation in the FIX-CTP.sub.4
and FIX-CTP.sub.5 activity (Table 14) might be caused by
inappropriate quantitation capabilities of the FIX ELISA due to CTP
masking of the antigen site.
TABLE-US-00070 TABLE 14 Sample/plasma EC50 ratio Sample
Sample/plasma EC50 ratio Plasma 1 3 CTP Final HA 2 4 CTP Final HA
5.35 5 CTP Final HA 2.73
[0692] 2.7 Pharmacokinetic Study
[0693] Jacalin-purified FIX-CTP3, FIX-CTP4, and FIX-CTP5 (Group A,
B and C, respectively) were administered in a single intravenous
injection to Sprague-Dawley rats (six rats per treatment group) at
a dose of 250 .mu.g/kg body weight. Blood samples were drawn
retro-orbitally from 3 rats alternately at 0.083, 0.5 2, 5, 8, 24,
48, 72 and 96 hours post-dosing (Table 15). Citrated plasma (0.38%)
was prepared immediately after sampling and stored at -20.degree.
C. until analysis.
TABLE-US-00071 TABLE 15 PK study plan of operation Dose No. of
Level Injected Treatment animals/ Dose (.mu.g per Vol. Conc.
Time-Points Group Treatment group Route animal) (.mu.l) (.mu.g/ml)
(hr post-dose) A FIX-CTP*3 6 IV 50 200 250 0.083, 0.5, 2, 5,
Jacalin 40 8, 24, 48, 72, 96 B FIX-CTP*4 6 IV 50 200 250 0.083,
0.5, 2, 5, Jacalin 40 8, 24, 48, 72, 96 C FIX-CTP*5 6 IV 50 200 250
0.083, 0.5, 2, 5, Jacalin 40 8, 24, 48, 72, 96
[0694] FIX concentration in plasma samples were quantified using
human FIX ELISA kits (Affinity Biologicals). The pharmacokinetic
profile was calculated and is the mean of 3 animals at each time
point. Terminal half-lives were calculated using PK Solutions 2.0
Software. Table 16 below summarizes the calculated FIX
concentrations at the different sampling time points.
TABLE-US-00072 TABLE 16 Calculated FIX concentrations Time Av. 3
CTP SD Av. 4 CTP SD Av. 5 CTP SD (hr) ng/ml 3 CTP ng/ml 4 CTP ng/ml
5 CTP 0.083 1087.82 72.39 904.54 21.06 1097.23 82.24 0.5 774.18
86.31 736.82 66.93 998.79 70.43 2 562.23 3.70 627.09 32.47 747.85
14.02 5 357.44 8.63 431.23 29.41 576.49 27.36 8 239.20 7.82 327.46
30.26 394.96 36.48 24 77.08 4.26 107.38 5.18 142.42 16.13 48 27.73
2.02 39.83 1.85 53.66 3.33 72 12.55 1.48 21.53 1.55 23.54 3.32 96
6.66 1.23 10.63 0.13 18.54 3.39
[0695] The PK profile and a summary of the PK parameters are
presented in Table 17 below and in FIG. 8. A full PK analysis
profile at all time points suggested that addition of 4 or 5 CTP
cassettes to FIX did not increase its half-life as compared to
FIX-CTP.sub.3. The AUC following FIX-CTP.sub.5 administration
increased by 1.4- to 1.6-fold versus FIX-CTP.sub.3, which was not
statistically significant.
TABLE-US-00073 TABLE 17 PK profile and a summary of the PK
parameters 24-96 hr 3 CTP 4 CTP 5 CTP Half-life (hr) 20.43 22.02
23.96 AUC (ng-hr/ml) 8218.38 10504.49 13329.41 Vd (ml/kg) 700.76
586.02 494.89 CL (ml/hr/kg) 23.77 18.45 14.32
[0696] Since 96 hr post-dosing samples were shown to have very low
FIX concentrations, which were at the lower limit of quantification
of the assay, the terminal half-life was recalculated providing a
more precise and scientifically appropriate calculation (Table 18).
According to this calculation, even smaller differences were
obtained between the half-life of FIX-CTP.sub.3, FIX-CTP.sub.4, and
FIX-CTP.sub.5.
TABLE-US-00074 TABLE 18 Recalculated terminal half-life 8-72 hr 3
CTP 4 CTP 5 CTP Half-life (hr) 15.38 16.63 16.04
[0697] 2.8 Conclusions:
[0698] In this study, the pharmacokinetic parameters and potential
clotting activity of FIX-CTP.sub.3, FIX-CTP.sub.4, and
FIX-CTP.sub.5 were assessed. Fusion of 4 and 5 CTPs to FIX did not
provide a superior or improved half-life extension, as compared to
FIX-CTP.sub.3, and reduced chromogenic activity was observed. Table
19 below summarizes the percent improvement of half-life for the
different FIX-CTP fused variants (1 to 5 CTPs). Fusion of CTP to
FIX improved its pharmacokinetic behavior, but, unpredictably, this
improvement was limited. Surprisingly, following fusion of 3, 4 or
5 CTPs in tandem to FIX, a similar half-life value was
calculated.
TABLE-US-00075 TABLE 19 Summary of the percent improvement of
half-life T1/2 (8-72 hr) FIX Version % increase rhFIX vs. 1CTP 112
1CTP vs. 2CTP 141 2CTP vs. 3CTP 37 3CTP vs. 4CTP 6 4CTP vs. 5CTP
0
[0699] These data suggest that fusion of 3 CTPs to FIX produces a
maximal improvement in protein half-life, confirming that
FIX-CTP.sub.3 is the optimal variant in terms of half-life,
structure and potential clotting activity for further clinical
development.
Example 5--Generation and Utilization of Coagulation Factor
FVII
[0700] A long-acting version of activated Factor VII (FVIIa)
coagulation factor will be useful for the treatment of patients
with hemophilia A and B. FVIIa-CTP.sub.3 recombinant protein has
the clinical potential to improve the treatment of hemophilia
patients by reducing the frequency of infusions and even by
reducing the drug load, enabling a prophylactic treatment approach
which can significantly improves a patient's quality of life, avoid
spontaneous bleeding episodes and accumulated damage to the joint
and other organs.
[0701] The generation of a recombinant FVIIa-CTP molecule with an
extended half-life based on fusion of FVII to a human CTP is
described herein. The recombinant FVIIa-CTP was expressed in
mammalian cells and characterized in vitro and in vivo. It was
demonstrated that rFVII-CTP activity was comparable to rFVII.
Pharmacokinetic and efficacy studies in rats demonstrated improved
properties of rFVII-CTP. The results of this study demonstrated
that it is feasible to develop a half-life extended rFVIIa molecule
with very similar haemostatic properties to the wild-type
enzyme.
[0702] Cloning and expression of recombinant FVII molecule: Several
Factor VII clones were constructed in our eukaryotic expression
vector (pCI-dhfrr) (FIG. 9). Human MGC verified FL cDNA clone
(IRCM) containing the sequence of Homo sapiens coagulation Factor
VII was ordered from "Open Biosystems" (OB-MHS4426). The following
primers were synthesized by Sigma-Genosys in the following
sequence: Primer 67: 5'CTCGAGGACATGGTCTCCCAGGCCC3' (contains the 5'
end of Factor VII DNA and the restriction site of XhoI) (SEQ ID NO:
91); Primer 68.sup.R: 5' TCTAGAATAGGTATTTTTCCACATG3' (contains the
restriction site of XbaI) (SEQ ID NO: 92); Primer 69: 5'
TCTAGAAAAAAGAAATGCCAGC3' (contains the restriction site of XbaI)
(SEQ ID NO: 93); and Primer 70.sup.R:
5'GCGGCCGCATCCTCAGGGAAATGGGGCTCGCA3' (contains the 3' end of Factor
VII DNA and the restriction site of NotI) (SEQ ID NO: 94).
[0703] Cloning was performed in two sets of PCR reactions. The
first reaction was conducted with primer 67 and primer 68.sup.R
using a cDNA plasmid with the Factor VII sequence (OB-MHS4426) as a
template; as a result of the PCR amplification, a .about.534 bp
product was formed, isolated and ligated into a TA cloning vector
(Invitrogen, Catalog No: K2000-01). A XhoI-XbaI fragment containing
the amino terminus of the Factor VII sequence was isolated. The
second reaction was conducted with primer 69 and primer 70.sup.R
and again, a cDNA plasmid with the Factor VII sequence (OB-MHS4426)
was used as a template. As a result of the PCR amplification, a
.about.813 bp product was formed and ligated into TA cloning vector
(Invitrogen, Catalog No: K2000-01). A XbaI-NotI fragment containing
the carboxy terminus of Factor VII sequence was isolated. The two
fragments were inserted into our eukaryotic expression vector
pCI-dhfr (triple ligation) to yield the 501-0-p136-1 clone.
[0704] Plasmid 501-p136-1 (Factor VII in pCI-dhfr vector) was
digested with restriction enzymes XhoI and KpnI. A fragment of
.about.1186 bp was isolated. A partial Factor VII clone (1180
bp-1322 bp) followed by a CTP sequence, termination sequence and
NotI sequence that was synthesized by GeneArt (0721543) was
digested with restriction enzymes KpnI and Nod. A fragment of
.about.253 bp was isolated. The two fragments were inserted into
our eukaryotic expression vector pCI-dhfr (triple ligation) to
yield the 501-1-p137-2 clone. pCI-dhfr-701-2-p24-2 was digested
with restriction enzymes XhoI and ApaI, and the large fragment
(vector) was isolated.
[0705] pCI-dhfr-501-2-p137-2 (Factor VII-ctp x1) was digested with
restriction enzymes XhoI and ApaI, and a .about.1200 bp insert was
isolated. The vector and insert were ligated to yield 501-2-p139-2.
Dg44 cells were plated in 100 mm tissue culture dishes and grown to
confluence of 50-60%. A total of 2 .mu.g of DNA was used for
transfection of one 100 mm plate using the FuGene reagent (Roche)
in protein-free medium (Invitrogen CD Dg44). The medium was removed
48 hours post-transfection and replaced with a protein-free medium
(Invitrogen CD Dg44) without nucleosides. After 14 days, the
transfected cell population was transferred into T25 tissue culture
flasks, and the selection was continued for 10-14 days until the
cells began to grow well as a stable clone. High-expressing clones
were selected and approximately 2.times.10.sup.7 cells were used to
inoculate 300 ml of growth medium in a 1700 cm.sup.2 roller bottle
(Corning, Corning N.Y.) supplemented with 5 ng/ml of Vitamin K3
(menadione sodium bisulfate; Sigma). The production medium
(harvest) was collected after a rapid decrease in the cell
viability to around 70%. The production medium was first clarified
and then concentrated approximately 20-fold and dialyzed to PBS
using flow filtration cassette (10KDaMWCO; Millipore Corp,
Billerica, Mass.).
[0706] Determination of FVII Antigen Level
[0707] The cDNA coding the CTP peptide was fused to the 3' end of
the cDNA coding human FVII. The corresponding rFVII construct was
transfected into Dg44 cells. As a control, a human rFVII cDNA was
utilized. The production medium (harvest) was collected,
concentrated and the secreted recombinant FVII was further
evaluated. rFVII, rFVII-CTP and rFVII-CTP-CTP antigen levels were
determined by AssayMax Human FVII ELISA kit (AssayPro) (FIG. 10A).
There was no significant difference in the secretion level of
rFVII-CTP and rFVII-(CTP)2 compared to native rFVII.
[0708] SDS-PAGE Blots
[0709] SDS-PAGE analysis was done by loading 50 ng of either
harvest, purified or activated rFVII protein. Samples were loaded
on 12% Tris-Glycine gel using Precision Plus Dual Color Protein
Marker (Bio-Rad). The SDS-PAGE analysis was done by performing a
Western immunoblot using an anti-human FVII monoclonal antibody
(Ab) (R&D systems) or anti-CTP polyclonal antibody generated in
Rabbit.
[0710] The level of rFVII antigen correlated with the detected
protein level in a SDS-PAGE immunoblotted with anti-FVII Ab.
rFVII-CTP migrated as a single band, while the corresponding
molecular weight of the FVII control was approximately 52 KDa (data
not shown). Both proteins reacted with antibodies specific for FVII
on immunoblots. The rFVII-CTP also reacted with antibodies specific
for CTP. rFVII was secreted in its zymogene form with no trace of
activated protein.
[0711] FVII Chromogenic Activity:
[0712] rFVII, rFVII-CTP and rFVII-(CTP).sub.2 harvest activities
were determined using a commercially available chromogenic test kit
(AssaySense Human FVII Chromogenic Activity Assay Kit (AssayPro).
For functional characterization of the rFVII-CTP and its ability to
be further activated (FVIIa), concentrated rFVII-CTP (harvests)
were placed in a commercially available chromogenic test kit that
measure the ability of TF/FVIIa to activate Factor X to Factor Xa
that in the presence of FXa specific substrate releases a
quantitated signal (AssayPro). The addition of the CTP peptide at
the C-terminal of the rFVII protein did not impair the FVII serine
protease activity (FIG. 10B, 10C).
[0713] FVII Clotting Activity:
[0714] Prothrombin time (PT) measures the extrinsic pathway of
coagulation. The PT is the time (measured in seconds) it takes
plasma to clot following the addition of an extrinsic pathway
activator, phospholipid and calcium. It is used to determine the
clotting tendency of blood, specifically in the measure of warfarin
dosage, liver damage, and vitamin K status. The reference range for
prothrombin time is usually around 12-15 seconds. Specifically, the
assay quantitated the ability of FVII-CTP and FVII-(CTP)2 harvest
to restore the clotting activity of FVII-depleted human plasma by
the addition of rhFVII. 300 .mu.l of FVII-deficient human plasma
was mixed with 100 .mu.l of FVII, FVII-CTP and FVII-(CTP).sub.2
harvests at specific concentrations, or normal pool human plasma
and were further diluted. Following a 60 second incubation at
37.degree. C., Tissue Factor (TF), CaCl.sub.2, and phospholipids
were added to the mixture. The clotting time in seconds was
determined. Potency was assessed by comparing a dose-response curve
of FVII-CTP and FVII-(CTP).sub.2 harvests to a reference
preparation consisting of rhFVII or human pool plasma. One unit of
active FVII was defined as the amount of FVII which equals to the
activity of one ml human normal plasma. The PT Clotting activity of
rFVII and rFVII-CTP was measured on a coagulometer (Instrumentation
Laboratory).
[0715] As previously shown, the addition of a CTP peptide at the
C-terminal of the rFVII protein did not damage its serine protease
activity and lead to the initiation and activation of a native
Factor X and Factor IX in human plasma. Following the insertion of
an additional CTP at the C terminal, there was a three-fold
reduction in the serine protease activity (data not shown).
[0716] Pharmacokinetics Study:
[0717] rFVII, rFVII-CTP, and rFVII-(CTP).sub.2 harvests were
administered intravenously to Sprague-Dawley rats (six rats per
substance) with a dose of 100 .mu.g/kg body weight. For all of the
in vivo experiments, the amount of the respective protein was
determined on the basis of FVII ELISA kit. For each FVII test
substance, the injected amount was calculated by taking into
account the differences in the molecular weight of rFVII versus
rFVII-CTP, which leads to a different molar concentration.
[0718] Blood samples were drawn retro-orbitally using an altering
sampling scheme to minimize interference of the sampling procedure
levels to be quantified: from 3 rats at 30 and 90 min and at 2, 6,
and 48 hrs, and from the remaining three rats at 15 and 60 min and
at 1.5, 4, and 24 hrs alternately. Plasma was prepared immediately
after sampling and stored at -20.degree. C. until analysis. FVII
concentration was quantified by FVII ELISA specific assay.
Half-life and AUC were calculated using a linear trapezoidal rule.
Comparison of these clearance parameters revealed that the in vivo
half-life and rFVII-(CTP).sub.2 AUC are significantly higher than
those of rFVII (Table 20).
TABLE-US-00076 TABLE 20 PK study parameters Dose T1/2 AUC.sub.0-t
CL/F MRT Group Route .mu.g/kg min ng/min/mL mL/min/kg min FVII IV
60 4.07 3314.7 6.195 6.2 FYII-CTP IV 60 .beta. = 31353.9 0.287 73.7
51.06 FVII-CTP- IV 60 .beta. = 7626.8 1.18 15.4 CTP 13.66
[0719] Characterization of Recombinant FVIIa-CTP:
[0720] During activation, FVII is cleaved at R152 resulting in
heavy and light chain domains that are held together by a single
disulfide bridge. rFVIIa-(CTP).sub.2 is purified and activated by
an ion exchange column purification process. In order to fully
evaluate rFVIIa-(CTP).sub.2, the protein is loaded on SDS-PAGE
under reducing conditions to commercial FVIIa (NovoSeven.RTM.). The
heavy and the light chain domains are separated and migrate as
separated bands of molecular weights 55 and 25 KDa. Both proteins
react with antibodies specific for FVII, but the heavy chain of the
rFVIIa-CTP specifically reacts with anti-CTP-specific antibodies,
indicating that this band represents the FVII heavy chain fused to
CTP. The light chain reacts specifically with anti-gamma
carboxylase Ab. The FVIIa protein concentration is determined by
FVIIa-specific ELISA kit.
[0721] FVIIa N-Terminal Sequencing:
[0722] rFVII-CTP-CTP in activated or zymogene purified proteins is
separated by SDS-PAGE (on 12% Tris-Glycine) and subsequently
electroblotted to a PVDF membrane. The bands of interest are cut
out and put on a purified Biobrene-treated glass fiber filter. The
N-terminal sequence analysis is carried out by Edmann degradation
using a pulsed liquid protein sequencer equipped with a 140 C HPLC
microgradient system. The identity of the recombinant protein and
proper pro-peptide cleavage is further verified by N-terminal
sequencing.
[0723] FVIIa Clotting Activity:
[0724] In order to evaluate FVII-(CTP).sub.2 coagulation activity,
activated partial thromboplastin time assay (aPTT) is performed.
FVII-deficient plasma sample is substituted with rFVIIa
(NovoSeven.RTM.) or rFVIIa-(CTP).sub.2. 300 .mu.l of FVII deficient
human plasma is mixed with 100 .mu.l of FVIIa or rFVIIa-(CTP).sub.2
at specific concentrations, or normal pooled human plasma which is
further diluted. Following 60 seconds incubation at 37.degree. C.
Tissue Factor (TF), CaCl.sub.2, and phospholipids are added to the
mixture. Clotting time in seconds is determined. Potency is
assessed by comparing a dose-response curve of rFVIIa-(CTP).sub.2
to a reference preparation consisting of rhFVIIa or human pool
normal plasma. One unit of FVIIa is defined as the amount of FVIIa
which equals to the activity of 1 ml human normal plasma. The aPTT
clotting activity of rFVII and rFVIIa-(CTP).sub.2 is measured on a
coagulometer (Instrumentation Laboratory). The aPTT clotting
activity of rFVIIa and rFVIIa-(CTP).sub.2 is similar.
[0725] Pharmacokinetics Studies in Rats:
[0726] In order to characterize the influence of the CTP addition
to the rFVIIa on its longevity potential, a comparative
pharmacokinetic study in rats is performed. NovoSeven.RTM. (rFVIIa)
and rFVIIa-(CTP).sub.2 in TBS are injected IV to 6 SD rats. The
levels of FVIIa over time are detected using a FVIIa ELISA kit. The
half-life and AUC are calculated for each protein. Comparison of
these clearance parameters reveals that the in vivo measures of
half-life, recovery, and AUC of the rFVIIa-(CTP).sub.2 are superior
to those of NovoSeven.RTM..
[0727] FVIIa-CTP In Vivo Efficacy Model (FVIII-Deficient Mouse
Model of Hemophilia):
[0728] In order to assess the in vivo activity model, FVIII
knockout mice are obtained, and a breeding colony is established.
10 .mu.g of either commercial recombinant hFVIIa (NovoSeven.RTM.)
or rFVIIa-(CTP).sub.2 are injected into the tail vein of an
anaesthetized FVIII knockout mouse (22-28 g). The amount of
injected protein equals to the required concentration of FVIII in
normal plasma (5 .mu.g/ml). Blood samples are taken from the
clipped tail into heparinized capillary tubes at specific time
points. Plasma samples are assessed for FVIIa levels by ELISA, and
efficacy is measured by a PTT coagulation assay.
[0729] In this study, a fusion construct of FVII with CTP is
generated. This recombinant protein is the basis for a treatment
that provides a prolonged half-life and retention of therapeutic
potency.
[0730] These results suggest that rFVIIa-(CTP)2 has a similar
therapeutic efficacy to rFVIIa in hemophilia patients. Moreover,
this technology requires less frequent dosing. It appears that a
single injection of rFVIIa-(CTP).sub.2 is sufficient to control
bleeding episodes and reduce the number of injections that are
needed during surgical intervention. This recombinant protein may
be used as a long term prophylactic treatment.
Example 6--Comparative Assessment of Purified FVII-CTP.sub.3,
FVII-CTP.sub.4, and FVII-CTP.sub.5
[0731] 5.1 Study Objective
[0732] Comparative assessment of pharmacokinetic parameters and
clotting activity of FVII-CTP.sub.4 and FVII-CTP.sub.5 versus
FVII-CTP.sub.3.
[0733] 5.2 Production of FVII-CTP4 and FVII-CTP5 Harvests
[0734] FVII cDNA fused at the C-terminal to four or five tandem CTP
sequences was expressed in Dg44 cells using the Excellgene
expressing system in the presence of 20 .mu.g/L of vitamin K3
(Sigma, Mennadion). The harvest was collected (300 ml), filtered
and frozen.
[0735] 5.3 Production of FVII-CTP3 Harvest
[0736] FVII-CTP.sub.3 was expressed in-house in mammalian
expressing system, CHO cells, using pCI-DHFR vector. Stable
transfected pool #71 was grown in shake flasks, in the presence of
25 ng/L of vitamin K3 (Sigma). The harvests were collected and
filtered.
[0737] All FVII-CTP harvests (3, 4 and 5 CTPs) were concentrated
and dialyzed against TBS (50 mM Tris, 150 mM NaCl, pH 7.4) using
Pellicon XL MWCO 10 kDa. Auto-activation of the FVII was induced by
incubation of purified FVII-CTP.sub.3 in the presence of CaCl.sub.2
overnight at 2-8.degree. C.
[0738] 5.4 Determination of FVII Antigen Level
[0739] FVII antigen level was determined using Human FVII ELISA kit
(Zymotest HyPhen) (Table 21). The calculated protein concentration
is the average of two independent runs.
TABLE-US-00077 TABLE 21 FVII antigen level FVII-CTP.sub.3
FVII-CTP.sub.4 FVII-CTP.sub.5 Av. (ng/ml) 224357.3 87884.1 589423
SD 44789.5 3248.7 5309 % CV 20.0 3.7 9
[0740] 5.5 FVII-CTP immune-Blot
[0741] FVII-CTP.sub.3, FVII-CTP.sub.4, and FVII-CTP.sub.5 harvests
were loaded on 12% Tris-Glycine gel (expedeon) using Precision plus
dual color protein marker (Bio-Rad). The SDS-PAGE analysis was
performed by Western immune-blot using anti-CTP polyclonal Ab (Adar
Biotech Production) or anti-Gla Ab (American Diagnostica).
[0742] FVII fused to three, four and five CTP migrated at 80, 90
and 100 kDa, respectively. As expected, FVII-CTP.sub.4 and
FVII-CTP.sub.5 harvests from Excellgene contain low gamma
carboxylation content as compared to FVII-CTP3 harvest which was
produced at Prolor since the production process wasn't optimized
(Data not shown).
[0743] 5.6 Comparative Assessment of FVII In Vitro Potency
[0744] A comparative assessment of the in vitro potency of HA
purified (highly gamma carboxylated fraction) FVII-CTP.sub.3,
FVII-CTP.sub.4, and FVII-CTP.sub.5 versus normal human pool plasma
was performed using a commercially available chromogenic activity
test kit, BIOPHEN (Hyphen BioMed 221304). All samples were serially
diluted, and the potency was assessed by comparing a dose-response
curve to a reference preparation consisting of normal human plasma.
FVII-CTP.sub.3 and FVII-CTP.sub.5 demonstrated chromogenic activity
lower than pooled normal plasma (Data not shown). FVII-CTP.sub.4
demonstrated higher activity as reflected by EC50 ratios, compared
to FII-CTP.sub.3 and FVII-CTP.sub.5 (Table 22).
TABLE-US-00078 TABLE 22 FVII In Vitro Clotting Activity EC50
Sample/plasma Sample (ng/ml) EC50 ratio Plasma 0.05 FVII 3CTP 0.12
2.72 FVII 4CTP 0.03 0.71 FVII 5CTP 0.06 1.35
[0745] 5.7 FVII In Vitro Clotting Activity:
[0746] Factor VII (FVII) activity assay, which was performed in
Sheba Medical Center, the Israel National Coagulation Center, is a
prothrombin (PT)-based assay using immuno-adsorbed plasma deficient
in Factor VII (Siemens). The PT reagent is innovin, and the assay
is performed in the Sysmex.RTM. CA 1500 instrument. FVII normal
range is within 55-145%.
TABLE-US-00079 TABLE 23 FVII In Vitro Chromogenic Activity
Concentration in FVII % of tested sample Concentration Sample
activity (.mu.g/ml) (.mu.g/ml) FVII 3CTP 36 0.5 224.2 18 0.25 6
0.125 FVII 4 CTP 334 0.5 87.9 176 0.25 93 6.25 FVII 5 CTP 38 0.5
58.9 19 0.25 10 0.125
[0747] Since the normal level of circulating FVII in the body is
around 0.5 .mu.g/ml, FVII-CTP.sub.3 and FVII-CTP.sub.5 harvests
exhibit 3-fold reductions in their coagulation activity versus
normal human pool plasma; this result correlates with the obtained
chromogenic activity (Table 23).
[0748] The FVII-CTP.sub.4 harvest exhibits a 3-fold increase in its
potential coagulation activity versus normal human pool plasma as
observed in the chromogenic activity assay (Table 23). The activity
percentage of FVII-CTP.sub.4 is much higher compared to activity
percentage of FVII-CTP.sub.3 and FVII-CTP5. Methodological
limitations of the ELISA method may limit the accuracy of Ag level
calculations of FVII-CTP4.
[0749] 5.8 Pharmacokinetic Study
[0750] Two pharmacokinetic studies were performed in order to
determine the FVII-CTP3, FVII-CTP.sub.4, and FVII-CTP.sub.5
pharmacokinetics (PK) parameters. During the first study,
FVII-CTP.sub.3, FVII-CTP4, and FVII-CTP5 (Group A, B and C,
respectively) were administered in a single intravenous injection
to Sprague Dawley rats (six rats per treatment) in a dose of 250
.mu.g/kg body weight. Blood samples were drawn retro-orbitally from
3 rats alternately at 0.083, 0.5 2, 5, 8, 24, 48, 72 and 96 hours
post-dosing (Table 24). Citrated plasma (0.38%) was prepared
immediately after sampling and stored at -20.degree. C. until
analysis.
TABLE-US-00080 TABLE 24 Pharmacokinetic Study Design - Concentrated
Harvest No. of Dose animals/ Level Injected Treatment group/time
Dose (.mu.g per Vol. Conc. Time-Points Group Test Article point
Route animal) (.mu.l) (.mu.g/ml) (hours post-dose) A FVII-CTP*3 6
IV 50 200 250 0 (Pre-dose) 0.083, 0.5, 2, 5, 8, 24, 48, 72, 96 B
FVII-CTP*4 6 IV 50 200 250 0 (Pre-dose) 0.083, 0.5, 2, 5, 8, 24,
48, 72, 96 C FVII-CTP*5 6 IV 50 200 250 0 (Pre-dose) 0.083, 0.5, 2,
5, 8, 24, 48, 72, 96
[0751] FVII concentration in plasma samples were quantified using
human FVII Elisa kits (Zymutest FVII-Biophen). The pharmacokinetic
profile was calculated and is the mean of 3 animals at each time
point. Terminal half-life values were calculated using PK Solutions
2.0 Software. Table 25 below summarizes the calculated FVII
concentrations at the different sampling time points. The PK
profile (FIGS. 23-24) and a summary of the PK parameters (Table 26)
are also presented below. FVII-CTP.sub.5 demonstrated a superior
profile as compared to FVII-CTP.sub.3 and FVII-CTP.sub.4 (Table
26).
TABLE-US-00081 TABLE 25 First Pharmacokinetic Study - FVII
Concentrations AVE-FVII- AVE-FVII- AVE-FVII- Time 3-CTP 4-CTP 5-CTP
(hr) (ng/ml) SD (ng/ml) SD (ng/ml) SD 0.083 4214 583 3600 427 4888
504 0.5 3386 892 5213 1682 5384 2549 2 1138 219 3603 1338 3082 289
5 1390 374 2726 1127 2480 561 8 333 167 1349 44 2316 633 24 133 12
476 98 788 34 48 38 3 165 24 384 61 72 12 2 91 62 167 31 96 26 1 42
8 93 49
TABLE-US-00082 TABLE 26 Pharmacokinetic Analysis FVII 3-CTP
FVII-4-CTP FVII-5CTP half-life (0.083-8 hr) (hr) 2.5 4.9 6.6
half-life (8-72 hr) (hr) 13.3 16.6 17.7 AUC (ng-hr/ml)(8-72 hr)
18374.6 51224.4 72954.2 Vd (ml/kg)(8-72 hr) 203.7 91.9 67.7
CL(ml/hr/kg) (8-72 hr) 10.6 3.8 2.7
[0752] The addition of four or five CTPs significantly elongated
FVII half-life as compared to 3 CTPs by 2- and 3-fold, respectively
(Table 26). This superiority was more significant in the initial
part of the study (0.083-8 hr), suggesting a potential improved
protein recovery and reduced extra vascular clearance. AUC
following FVII-CTP.sub.4 and FVII-CTP.sub.5 administration
increased by 3- and 4-fold, respectively, versus FVII-CTP.sub.3.
Clearance was also reduced while adding 4 and 5 CTPs to FVII (Table
26).
[0753] As observed in the study, the addition of four and five CTPs
significantly elongated FVII half-life as compared to 3 CTPs, both
in the initial and terminal half-life. The half-life values in the
first and second study are different due to a different analysis
approach which was effected by the dose and study duration,
nevertheless the overall trend was maintained. The AUC following
FVII-CTP.sub.4 and FVII-CTP.sub.5 administration increased by 2.5-
and 7-fold, respectively, versus FVII-CTP.sub.3.
[0754] 5.9 Conclusions:
[0755] In this study, the PK parameters and potential clotting
activity of FVII-CTP.sub.3, FVII-CTP.sub.4, and FVII-CTP.sub.5 were
assessed. Fusion of 4 and 5 CTPs to FVII provided a superior and
improved half-life, exposure and reduced clearance as compared to
FVII-CTP.sub.3 while maintaining a similar chromogenic and in vitro
clotting activity. These results were observed at different
concentrations of protein and were consistent for both harvest and
purified protein. While evaluating the overall effect of fusion of
CTP at the C terminus to FVII, fusion of 1-5 CTPs considerably
increased the half-life and AUC of FVII in a CTP proportional
manner, suggesting that as the CTP portion of the molecule
increases, FVII longevity and stability is significantly improved
while maintaining its initial in vitro clotting activity, as
summarized in Table 27 hereinbelow.
TABLE-US-00083 TABLE 27 T.sub.1/2 percent AUC percent Comparative
assessment increase increase FVII vs. FVII-CTP.sub.2 268 200
FVII-CTP.sub.2 vs. FVII-CTP.sub.3 67 57.8 FVII-CTP.sub.3 vs.
FVII-CTP.sub.4 24 178 FVII-CTP.sub.4 vs. FVII-CTP.sub.5 6 42
[0756] As previously reported, FVII half-life correlates with the
half-life of the activated form of FVII (FVIIa) both in humans and
animals. Therefore, it is anticipated that a similar improvement in
half-life will be obtained for the activated versions following CTP
fusion.
Example 7: Expression and Isolation of IFN-CTP Polypeptides
Materials and Methods
[0757] DNA transfection and clone selection: DG44 cells were
transfected with pCI-DHFR expression vectors containing
IFN.beta.-CTP variants using FuGENE6 Reagent (FuGENE Transfection
Reagent--Roche Cat. 11 815 091 001). 48 hr following transfection,
cells were diluted and seeded at least 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.
[0758] 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.
[0759] 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.
[0760] Western Blotting: Samples were electrophoresed on
nondenaturing 15 SDS-polyacrylamide gels. Gels were allowed to
equilibrate for 10 mM 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 IFN anti-serum (1:1000 titer) overnight
at least 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 mM, dried with a Whatman sheet, and exposed
to X-ray film.
[0761] FIG. 3 indicates that MOD-901X-variants are recognized by
anti IFN-.beta.1a antibodies. The SDS PAGE gel was stained using
Coomassie blue (a) or (B). blotted and stained using monoclonal
anti-IFN-.beta.1a antibodies
Materials and Methods (Examples 8-11)
[0762] Plasmid Construction
[0763] Seven OXM plasmids were constructed in eukaryotic expression
vector (pCI-dhfrr) based on the OXM amino acid sequence (accession
#NP_002045) and the CTP amino acid sequence (accession
#NP_149032).
[0764] A schematic presentation of these plasmids is shown in Table
28. A detailed description of plasmids construction follows.
TABLE-US-00084 TABLE 28 Schematic description of plasmids Plasmid
variant description Nucleic Acid Sequence 1 OXM-CTP-CTP SEQ ID NO:
67 2 CTP-OXM-CTP SEQ ID NO: 65 3 CTP-CTP-OXM SEQ ID NO: 68 4
CTP-OXM-CTP-CTP SEQ ID NO: 66 5 OXM-CTP-CTP-CTP SEQ ID NO: 69 6
OXM-CTP-CTP-CTP-CTP SEQ ID NO: 70 7 OXM-CTP-CTP-CTP-CTP-CTP SEQ ID
NO: 71
[0765] A nucleic acid sequence of the OXM variant CTP-OXM-CTP (0.5C
CTP-OXM-CTP, GenArt, GA #0804377) was synthesized after a codon
usage optimization for DG44 expression system. An XbaI-NotI
fragment containing 0.5C CTP-OXM-CTP sequence was isolated. The
fragment was inserted into the eukaryotic expression vector
pCI-dhfr to yield the 601-0-p142-1 clone.
[0766] Construction of CTP-OXM-CTP
[0767] The following primers were used in order to synthesize
601-6-p149-1 (CTP-OXM-CTP).
TABLE-US-00085 Primer 25 (SEQ ID NO: 95) 5' CTCTAGAGGACATGGCCAC 3'.
Primer 85R (SEQ ID NO: 96) 5' CTGGCTGTGCTGGGGCAGAATGGGTGT 3'.
Primer 86 (SEQ ID NO: 97) 5' CCCCAGCACAGCCAGGG 3'. Primer 74R (SEQ
ID NO: 98) 5' GCGGCCGCATCCAGACCT 3'.
[0768] Three PCR reactions were performed. The first reaction was
conducted with primer 25 and primer 85R and plasmid DNA of
402-3-p81-4 (CTP-hGH-CTP-CTP) as a template; as a result of the PCR
amplification, a .about.181 bp product was formed. The second
reaction was conducted with primer 86 and primer 74R and plasmid
DNA of 601-0-p142-1 (0.5C CTP-OXM-CTP) as a template; as a result
of the PCR amplification, a .about.224 bp product was formed. The
last reaction was conducted with primers 25 and 74R and a mixture
of the products of the previous two reactions as a template; as a
result of the PCR amplification, a .about.391 bp product was formed
and ligated into TA cloning vector (Invitrogen, catalog K2000-01).
An XbaI-NotI fragment containing CTP-OXM-CTP sequence was isolated.
The fragment was inserted into our eukaryotic expression vector
pCI-dhfr to yield 601-6-p149-1 clone.
[0769] The nucleic acid sequence encoding CTP-OXM-CTP is as
follows:
TABLE-US-00086 (SEQ ID NO: 65)
tctagaggacatggccaccggcagcaggaccagcctgct
gctggccttcggcctgctgtgcctgccatggctgcagga
gggcagcgccagctcacttctaaggctccacccccatct
ctgcccagccccagcagactgccgggccccagcgacaca
cccattctgccccagcacagccagggcaccacaccagcg
actacagcaagtacctggacagcagaagggcccaggact
tcgtccagtggctgatgaacaccaagaggaacaggaaca
acatcgcacctctagctccaaggcccctccaccctctct
gcctagcccctctcggctgcctggcccatccgacacacc
aatcctgccacagtgatgaaggtctggatgcggccgc.
[0770] The amino acid sequence encoding CTP-OXM-CTP is as
follows:
TABLE-US-00087 (SEQ ID NO: 58)
MATGSRTSLLLAFGLLCLPWLQEGSASSSSKAPPPSLPSPSRLPGPSDTPI
LPQHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIASSSSKAPPPSL
PSPSRLPGPSDTPILPQ.
[0771] Construction of CTP-OXM-CTP-CTP
[0772] The following primers were used in order to synthesize
601-3-p158-2 (CTP-OXM-CTP-CTP):
TABLE-US-00088 Primer 25 (SEQ ID NO: 99) 5' CTCTAGAGGACATGGCCAC 3'.
(contains the restriction site of XbaI) Primer 87.sup.R (SEQ ID NO:
100) 5' GCTGGAGCTAGCGATGTTGTTCCTGTTCC 3'. (contains the 3' end of
OXM and the 5' end of CTP) Primer 88 (SEQ ID NO: 101) 5'
ACATCGCTAGCTCCAGCAGCAAGGCC 3'. (contains the 3' end of OXM and the
5' end of CTP) Primer 74.sup.R (SEQ ID NO: 102) 5'
GCGGCCGCATCCAGACCT 3'. (contains the restriction site of NotI)
[0773] Three PCR reactions were performed. The first reaction was
conducted with primer 25 and primer 87R and plasmid DNA of
601-6-p149-1 (CTP-OXM-CTP-CTP) as a template; as a result of the
PCR amplification, a .about.290 bp product was formed. The second
reaction was conducted with primer 88 and primer 74R and plasmid
DNA of 402-3-p81-4 (CTP-hGH-CTP-CTP) as a template; as a result of
the PCR amplification, a .about.200 bp product was formed. The last
reaction was conducted with primers 25 and 74R and a mixture of the
products of the previous two reactions as a template; as a result
of the PCR amplification, a .about.450 bp product was formed and
ligated into TA cloning vector (Invitrogen, catalog K2000-01).
XbaI-NotI fragment containing CTP-OXM-CTP-CTP sequence was
isolated. The fragment was inserted in to our eukaryotic expression
vector pCI-dhfr to yield 601-3-p158-2 clone.
[0774] The nucleic acid sequence encoding CTP-OXM-CTP-CTP is as
follows:
TABLE-US-00089 (SEQ ID NO: 66)
tctagaggacatggccaccggcagcaggaccagcctgctgctggccttcgg
cctgctgtgcctgccatggctgcaggagggcagcgccagctcttcttctaa
ggctccacccccatctctgcccagccccagcagactgccgggccccagcga
cacacccattctgccccagcacagccagggcaccttcaccagcgactacag
caagtacctggacagcagaagggcccaggacttcgtccagtggctgatgaa
caccaagaggaacaggaacaacatcgctagctccagcagcaaggcccctcc
cccgagcctgccctccccaagcaggctgcctgggccctccgacacaccaat
cctgccacagagcagctcctctaaggcccctcctccatccctgccatcccc
ctcccggctgcctggcccctctgacacccctatcctgcctcagtgatgaag
gtctggatgcggccgc.
[0775] The amino acid sequence encoding CTP-OXM-CTP-CTP is as
follows:
TABLE-US-00090 (SEQ ID NO: 59)
MATGSRTSLLLAFGLLCLPWLQEGSASSSSKAPPPSLPSPSRLPGPSDTPI
LPQHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIASSSSKAPPPSL
PSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ.
[0776] Construction of OXM-CTP-CTP
[0777] The following primers were used in order to synthesize
601-2-p160-2 (OXM-CTP-CTP):
TABLE-US-00091 Primer 75 (SEQ ID NO: 103) 5' CTCCCAGTTCAATTACAGCT
3'. (contains sequence of pCI-dhfr before XbaI restriction site)
Primer 89.sup.R (SEQ ID NO: 104) 5' GCTGTGAGCGCTGCCCTCCTGCAG 3'.
(contains 5' end of OXM) Primer 90 (SEQ ID NO: 105) 5'
GCGCTCACAGCCAGGGCACCTTC 3'. (contains the 5' end of OXM) Primer
74.sup.R (SEQ ID NO: 106) 5' GCGGCCGCATCCAGACCT 3'. (contains the
restriction site of NotI)
[0778] Three PCR reactions were performed. The first reaction was
conducted with primer 75 and primer 89R and plasmid DNA of
601-0-p142-1 (0.5C CTP-OXM-CTP) as a template; as a result of the
PCR amplification, a .about.175 bp product was formed. The second
reaction was conducted with primer 90 and primer 74R and plasmid
DNA of 601-3-p158-2 (CTP-OXM-CTP-CTP) as a template; as a result of
the PCR amplification, a .about.200 bp product was formed. The last
reaction was conducted with primers 25 and 74R and a mixture of the
products of the previous two reactions as a template; as a result
of the PCR amplification, a .about.391 bp product was formed and
ligated into TA cloning vector (Invitrogen, catalog K2000-01).
XbaI-NotI fragment containing OXM-CTP-CTP sequence was isolated.
The fragment was inserted into our eukaryotic expression vector
pCI-dhfr to yield the 601-2-p160-2 clone.
[0779] The nucleic acid sequence encoding OXM-CTP-CTP is as
follows:
TABLE-US-00092 (SEQ ID NO: 67)
tctagaggacatggccacagggagcaggaccagcctgctgctggctttcgg
cctgctgtgtctgccatggctgcaggagggcagcgctcacagccagggcac
cttcaccagcgactacagcaagtacctggacagcagaagggcccaggactt
cgtccagtggctgatgaacaccaagaggaacaggaacaacatcgctagctc
cagcagcaaggcccctcccccgagcctgccctccccaagcaggctgcctgg
gccctccgacacaccaatcctgccacagagcagctcctctaaggcccctcc
tccatccctgccatccccctcccggctgcctggcccctctgacacccctat
cctgcctcagtgatgaaggtctggatgcggccgc
[0780] The amino acid sequence encoding OXM-CTP-CTP is as
follows:
TABLE-US-00093 (SEQ ID NO: 60)
LEDMATGSRTSLLLAFGLLCLPWLQEGSAHSQGTFTSDYSKYLDSRRAQDF
VQWLMNTKRNRNNIASSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPP
PSLPSPSRLPGPSDTPILPQ
[0781] Construction of CTP-CTP-OXM
[0782] A nucleic acid sequence of OXM variant (CTPx2-OXM, GenArt,
GA #1067101) was synthesized after a codon usage optimization for
DG44 expression system. The fragment was inserted in to Excellgene
eukaryotic expression vector to yield p162 clone.
[0783] The nucleic acid sequence encoding CTPx2-OXM is as
follows:
TABLE-US-00094 (SEQ ID NO: 68)
ctatagggcgaattgaaggaaggccgtcaaggccgcatgagctctctagag
gacatggccaccggcagcaggaccagcctgctgctggccttcggcctgctg
tgcctgccatggctgcaggagggcagcgccagctccagcagcaaggcccct
cccccgagcctgccctccccaagcaggctgcctgggccctccgacacacca
atcctgccacagagcagctcctctaaggcccctcctccatccctgccatcc
ccctcccggctgcctggcccctctgacacccctatcctgcctcagcacagc
cagggcaccttcaccagcgactacagcaagtacctggacagcagaagggcc
caggacttcgtccagtggctgatgaacaccaagaggaacaggaacaacatc
gcttgatgaaggtctggatgcggccgcggtaccctgggcctcatgggcctt
cctttcactgcccgctttccag.
[0784] The amino acid sequence encoding CTPx2-OXM is as
follows:
TABLE-US-00095 (SEQ ID NO: 61) M A T G S R T S L L L A F G L L C L
P W L Q E G S A S S S S K A P P P S L P S P S R L P G P S D T P I L
P Q S S S S K A P P P S L P S P S R L P G P S D T P I L P Q H S Q G
T F T S D Y S K Y L D S R R A Q D F V Q W L M N T K R N R N N I A R
S G C G R G T L G L M G L P F T A R F P.
[0785] Construction of OXM-CTPx3:
[0786] A nucleic acid sequence of OXM variant (OXM-CTPx3, GenArt,
GA #1017864) was synthesized after a codon usage optimization for
DG44 expression system. XbaI-NotI fragment containing OXM-CTPx3
sequence was isolated. The fragment was inserted in to our
eukaryotic expression vector pCI-dhfr to yield
pCI-dhfr-OXM-ctpx3-p216-4 clone.
[0787] The nucleic acid sequence encoding OXM-CTPx3 is as
follows:
TABLE-US-00096 (SEQ ID NO: 69)
tctagactcgagcgatcgccatggccaccggctctaggacctccctgctgc
tggccacggcctgctgtgcctgccctggctgcaggaaggcagcgctcactc
ccagggcaccttcacctccgactactccaagtacctggactctcggagagc
ccaggacttcgtgcagtggctgatgaacaccaagcggaaccggaacaatat
cgcctcctcaagctccaaggcacctccaccttccctgcctagcccttccag
actccctgggcccagtgacacccctatcctgcctcagtccagctccagcaa
ggccccaccccctagcctgccttctccttctcggctgcctggccccagcga
tactccaattctgccccagtcctccagcagtaaggctccccctccatctct
gccatcccccagcagactgccaggcccttctgatacacccatcctcccaca
gtgatgaggatccgcggccgc.
[0788] The amino acid sequence encoding OXM-CTPx3 is as
follows:
TABLE-US-00097 (SEQ ID NO: 62) M A T G S R T S L L L A F G L L C L
P W L Q E G S A H S Q G T F T S D Y S K Y L D S R R A Q D F V Q W L
M N T K R N R N N I A S S S S K A P P P S L P S P S R L P G P S D T
P I L P Q S S S S K A P P P S L P S P S R L P G P S D T P I L P Q S
S S S K A P P P S L P S P S R L P G P S D T P I L P Q.
[0789] Construction of OXM-CTPx4:
[0790] A nucleic acid sequence of OXM variant (OXM-CTPx4, GenArt,
GA #1115769) was synthesized after a codon usage optimization for
DG44 expression system. XbaI-NotI fragment containing OXM-CTPx4
sequence was isolated. The fragment was inserted in to the
eukaryotic expression vector pCI-dhfr to yield
pCI-dhfr-OXM-ctpx4-p254-3 clone.
[0791] The nucleic acid sequence encoding OXM-CTPx4 is as
follows:
TABLE-US-00098 (SEQ ID NO: 70)
tctagactcgagcgatcgccatggctaccggctccagaacctctctgctgc
tggccacggcctgctgtgtctgccttggctgcaagagggcagcgctcattc
ccagggcaccacacctccgactactccaagtacctggactctcgcagagcc
caggacttcgtgcagtggctgatgaacaccaagcggaaccggaacaatatc
gcctcctccagctccaaggcccctcctccatctctgccatcccccagtaga
ctgcctgggccctctgacacccctatcctgcctcagtccagctcctctaag
gccccaccaccttccctgcctagcccttcaagactgccaggccctagcgat
acaccaattctgccccagtcctccagcagcaaggctcccccacctagcctg
ccttctccatcaaggctgcctggcccatccgataccccaattttgcctcag
agcagctctagcaaggcacctccccccagtctgccctctccaagcagactc
cctggcccttcagacactcccattctgccacagtgatgaggatccgcggcc gc
[0792] The amino acid sequence encoding OXM-CTPx4 is as
follows:
TABLE-US-00099 (SEQ ID NO: 63) M A T G S R T S L L L A F G L L C L
P W L Q E G S A H S Q G T F T S D Y S K Y L D S R R A Q D F V Q W L
M N T K R N R N N I A S S S S K A P P P S L P S P S R L P G P S D T
P I L P Q S S S S K A P P P S L P S P S R L P G P S D T P I L P Q S
S S S K A P P P S L P S P S R L P G P S D T P I L P Q S S S S K A P
P P S L P S P S R L P G P S D T P I L P Q.
[0793] Construction of OXM-CTPx5:
[0794] A nucleic acid sequence of OXM variant (OXM-CTPx5, GenArt,
GA #1115770) was synthesized after a codon usage optimization for
DG44 expression system. XbaI-NotI fragment containing OXM-CTPx5
sequence was isolated. The fragment was inserted in to the
eukaryotic expression vector pCI-dhfr to yield
pCI-dhfr-OXM-ctpx5-p255-1 clone.
[0795] The nucleic acid sequence encoding OXM-CTPx5 is as
follows:
TABLE-US-00100 (SEQ ID NO: 71)
ctctagactcgagcgatcgccatggctaccggctccagaacctctctgctg
ctggccttcggcctgctgtgtctgccaggctgcaagagggcagcgctcatt
cccagggcaccttcacctccgactactccaagtacctggactctcgcagag
cacaggacttcgtgcagtggctgatgaacaccaagcggaaccggaacaata
tcgcctcctccagctccaaggcccctcctccatctctgccatcccccagta
gactgcctgggccctctgacacccctatcctgcctcagtccagctcctcta
aggctccaccaccttccctgcctagcccttcaagactgccaggccctagcg
atacaccaattctgccccagtcctccagcagcaaggctcccccacctagcc
tgccttctccatcaaggctgcctggcccatccgataccccaattttgcctc
agagcagctctagcaaggcacctccccccagtctgccctctccaagcagac
tccctggcccttcagacactccaatcctcccacagtcctctagctctaaag
ctccacctcccagcctgcccagccctagtagactccccggaccactgatac
ccccatcttgccccagtgatgaggatccgcggccgc.
[0796] The amino acid sequence encoding OXM-CTPx5 is as
follows:
TABLE-US-00101 (SEQ ID NO: 64) M A T G S R T S L L L A F G L L C L
P W L Q E G S A H S Q G T F T S D Y S K Y L D S R R A Q D F V Q W L
M N T K R N R N N I A S S S S K A P P P S L P S P S R L P G P S D T
P I L P Q S S S S K A P P P S L P S P S R L P G P S D T P I L P Q S
S S S K A P P P S L P S P S R L P G P S D T P I L P Q S S S S K A P
P P S L P S P S R L P G P S D T P I L P Q S S S S K A P P P S L P S
P S R L P G P S D T P I L P Q.
[0797] Expression, Purification and Characterization of OXM-CTP
Variants
[0798] All seven variants of OXM-CTPs were transiently expressed
and produced in XLG's CHO-Express cell line (Excellgene Company,
Switzerland). Concentration levels of OXM-CTPs harvests were
determined using an OXM ELISA kit (Bachem Cat #S-1393.0001). The
harvest was purified using a DEAE column, followed by a Jacalin
column as a second purification step. The final fractions were
dialyzed against 10 mM buffer citrate, 147 mM NaCl, pH 6. The
concentration of purified variants was determined by absorbance at
280 nm using extinction coefficient of 1.9=1 mg/ml. 1.9 is the
extinction coefficient that theoretically was calculated for the
OXM peptide. Since CTP peptide does not absorb at 280 nm, the same
extinction coefficient was applied for OXM-CTP variants. OXM native
peptide was chemically synthesized (Almac Company, Ireland), and
its peptide content was determined by amino acid analysis.
[0799] Pharmacokinetic (PK) Profile of OXM-CTP Variants
[0800] The pharmacokinetic profiles of the OXM peptide and OXM-CTP
variants were assessed as follows. Male Sprague-Dawley (SD)-1 rats
were administered subcutaneously (SC) or intravenously (IV) with a
single dose of native OXM (n=6, 230 .mu.g/kg), of variants
CTP-OXM-CTP, CTP-OXM-CTP-CTP, OXM-CTP-CTP and CTP-CTP-OXM (n=6, 230
.mu.g/kg) or variants #OXM-CTP-CTP-CTP, OXM-CTP-CTP-CTP-CTP,
OXM-CTP-CTP-CTP-CTP-CTP (n=6, 153 .mu.g/kg). Cohorts of 3 animals
per group were bled at alternating time points. OXM serum
concentration was analyzed using commercial ELISA kit (Bachem, Cat
#S-1393.0001). The study design is summarized in Table 29. The
study was divided into 3 sequential experiments, Experiment #1
(Groups 1-4), Experiment #2 (Groups 5-9) and Experiment #3 (Groups
10-13).
TABLE-US-00102 TABLE 23 Summary of the PK study design. IPGTT study
in C57BL/6 mice Test No. of Dose Level Dose Level Group Article
animals/group/ Dose of OXM of OXM Time-Points # Variant # timepoint
Route Gender (.mu.g/rat) (.mu.g/kg) (hours post-dose) 1 OXM 6/3 IV
Male 34 230 0 (Pre-dose), 5 min, 15 min, 25 min, 35 min, 45 min, 60
min, 75 min, 1.5 h, 2 h 2 CTP-OXM- 6/3 IV Male 34 230 0 (Pre-dose),
10 min, 30 min, CTP 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 24 h, 36 h, 48 h
3 CTP-OXM- 6/3 IV Male 34 230 0 (Pre-dose), 10 min, 30 min, CTP-CTP
1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 24 h, 36 h, 48 h 4 CTP-CTP- 6/3 IV
Male 34 230 0 (Pre-dose), 10 min, 30 min, OXM 1 h, 2 h, 3 h, 4 h, 6
h, 8 h, 24 h, 36 h, 48 h 5 OXM 6/3 SC Male 34 230 0 (Pre-dose), 5
min, 15 min, 25 min, 35 min, 45 min, 60 min 75 min, 1.5 h, 2 h 6
CTP-OXM- 6/3 SC Male 34 230 0 (Pre-dose), 10 min, 30 min, CTP 1 h,
2 h, 4 h, 6 h, 8 h, 24 h, 36 h, 2 d, 3 d 7 CTP-OXM- 6/3 SC Male 34
230 0 (Pre-dose), 10 min, 30 min, CTP-CTP 1 h, 2 h, 4 h, 6 h, 8 h,
24 h, 36 h, 2 d, 3 d 8 CTP-CTP- 6/3 SC Male 34 230 0 (Pre-dose), 10
min, 30 min, OXM 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 36 h, 2 d, 3 d 9
OXM-CTP- 6/3 SC Male 34 230 0 (Pre-dose), 10 min, 30 min, CTP 1 h,
2 h, 4 h, 6 h, 8 h, 24 h, 36 h, 2 d, 3 d 10 OXM 6/3 SC Male 34 230
0 (Pre-dose), 5 min, 15 min, 25 min, 35 min, 45 min, 60 min 75 min,
1.5 h, 2 h 11 OXM- 6/3 SC Male 23 153 0 (Pre-dose), 10 min, 30 min,
CTPX3 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 36 h, 2 d, 3 d, 4 d 12 OXM-
6/3 SC Male 23 153 0 (Pre-dose), 10 min, 30 min, CTPX4 1 h, 2 h, 4
h, 6 h, 8 h, 24 h, 36 h, 2 d, 3 d, 4 d 13 OXM- 6/3 SC Male 23 153 0
(Pre-dose), 10 min, 30 min, CTPX5 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 36
h, 2 d, 3 d, 4 d
[0801] Overnight-fasted mice were measured for glucose (pre-dose
glucose). Immediately after, they were IP injected with one of the
test articles. Fifteen or 120 min post injection, glucose levels
were measured (zero time for glucose) followed by glucose
administration via IP injection (1.5 g/kg). Additional glucose
measurement at 10, 20, 30, 60, 90, 120 and 180 min were performed.
Blood glucose level were measured by tail vein sampling using a
handheld glucometer.
[0802] Food Intake Study in C57BL/6 Mice
[0803] At least one week before injection, mice were weighed and
were transferred to a mini cage for acclimatization, (one mouse per
cage). During the acclimatization period, they were handled daily
and received two injections of vehicle to minimize stress during
the study period. The day before the experiment, the mice were
fasted. Seventeen hours after fasting, at the early light phase
(900-1000 h), the mice were weighed again (prior to the IP
injection) followed by a single IP injection of 1700 nmol/kg (10
.mu.l/1 g mice) of OXM peptide or OXM-CTP variants OXM-CTPX3,
OXM-CTPX4 and OXM-CTPX5. After injection, the mice were returned to
their home cages (1 mouse per cage), and provided with a
pre-weighed amount of chow. Food intake were measured 0, 1, 2, 4,
6, 8, 21, 32 and 44, 55, 68, 80, 93 and 141 h post injection by
weighing the chow. At the end of experiment, rats were weighed
again.
Results
Example 8: Construction of CTP-Modified OXM
[0804] By genetic engineering, CTP peptide cDNA was fused to human
OXM cDNA, generating seven different OXM-CTP variant as detailed in
Table 28. The nucleotide sequences of the plasmids were verified,
and the plasmids were transiently transfected into XLG's
CHO-Express cell line (Excellgene Company, Switzerland). The
OXM-CTP variants were secreted into the growth medium, harvests
were collected and OXM-CTPs levels were determined.
Example 9: Expression, Purification and Characterization of OXM-CTP
Variants
[0805] The production media (harvests) were measured for secretion
level of OXM-CTP variants. The secretion levels are summarized in
Table 31. The secretion levels obtained were high considering the
standard transient transfection expression levels of recombinant
peptides.
TABLE-US-00103 TABLE 31 Summary of OXM-CTPs secretion level.
Variant CTP-OXM- CTP-OXM- OXM-CTP- CTP-CTP- OXM- OXM- OXM- CTP
CTP-CTP CTP OXM CTPX4 CTPX4 CTPX5 Harvest 31.1 15.3 3.3 51.9 13.9
15.3 14.4 concentration (.mu.g/ml)
[0806] The harvests were purified according to the method described
in Materials and Methods. The final samples of variants OXM-CTPX3,
OXM-CTPX4 and OXM-CTPX5 were analyzed by SDS-PAGE (Coomassie
staining (FIG. 11A) and anti-OXM Western Blot Analysis (FIG. 11B)).
For variants CTP-OXM-CTP, CTP-OXM-CTP-CTP, OXM-CTP-CTP and
CTP-CTP-OXM, samples from the purification process were analyzed by
both Coomassie staining and Western Blot analysis (FIG. 12). As
expected, OXM-CTP variants showed differences in size correlating
to the number of CTP cassettes fused to the peptide. The relatively
high molecular weights are an indication that there may be high
occupancy of the O-glycan chains on the potential serine sites on
the CTP peptide. The OXM peptide did not react with the anti-OXM
antibody, probably due to the technical difficulty in transferring
a small sized peptide. The Coomassie staining shows that the
OXM-CTP variants were highly purified and contained mainly the high
form.
Example 10: Pharmacokinetic (PK) Profile of OXM-CTP Variants
[0807] The pharmacokinetic profile of OXM peptide compared to
OXM-CTP variants was analyzed in male SD-1 rats. Animals were
administered with a single IV (Experiment 1, FIG. 13A) or single SC
(Experiments 2&3, FIGS. 13B-13C) injection of native OXM or
OXM-CTP variants CTP-OXM-CTP, CTP-OXM-CTP-CTP, OXM-CTP-CTP,
CTP-CTP-OXM (230 .mu.g/kg peptide) or variants OXM-CTPX3, OXM-CTPX4
and OXM-CTPX5 (153 .mu.g/kg peptide). Serum concentration of OXM or
OXM-CTP variants at the indicated time points was analyzed using
commercial ELISA. The PK profile is shown in FIG. 13, and the
conventional noncompartmental PK parameters are summarized in Table
32. Addition of CTP peptides to OXM resulted in prolonging the
half-life of native OXM, from 0.22 hr to 2.7-10 h for various
OXM-CTP variants (Table 32 SC administration; Experiments 2
&3).
[0808] Two copies of CTP were added to OXM peptide in three
different ways to produce variants CTP-OXM-CTP, OXM-CTP-CTP and
CTP-CTP-OXM. The most significant extension of serum half-life was
received when the CTPs were added in tandem to the C-terminus of
OXM; variant OXM-CTP-CTP with T1/2 of 4.76 h. Fusion of three CTPs
to the OXM C-terminus did not results in an elongated half-life as
compared to fusion of two CTPs. Surprisingly, addition of four and
five copies of CTPs to OXM c-terminus elongated the T 1/2 up to 10
h (Table 32).
[0809] Exposure as reflected by the AUC parameter, was most
increased by .about.17-fold for variant OXM-CTP-CTP (Experiment 2)
and .about.30-fold for variants OXM-CTPX4 and OXM-CTPX5 (Experiment
3). These results indicate that there is a superior prolonging
effect after the addition of four or five copies of CTP to the OXM
peptide.
[0810] The bioavailability as calculated from Experiments 1 & 2
was increased for CTP-OXM-CTP-CTP, but no significant improvement
was found for CTP-OXM-CTP and CTP-CTP-OXM (Table 32).
TABLE-US-00104 TABLE 32 Conventional noncompartmental PK parameters
of OXM peptide and OXM- CTP variants as were determined in three
different experiments. T1/2 T1/2.beta. Cmax Tmax AUC(0-t)
AUC.infin. CL Vd MRT Bioavail- hr hr ng/ml hr ng-hr/ml ng-hr/ml
ml/hr/kg ml/kg hr ability % Exp. OXM 0.29 NA NA NA 211.7 213.9
993.0 417.0 0.34 1 CTP-OXM- 0.25 1.57 NA NA 592.7 598.0 355.3 806.9
0.81 IV. CTP CTP-OXM- 0.28 1.61 NA NA 968.2 977.4 217.4 506.4 0.80
CTP-CTP CTP-CTP- 0.27 1.20 NA NA 488.6 490.4 433.3 748.1 0.86 OXM
Exp. OXM 0.22 118.40 0.08 42.52 42.84 4960.39 1564.40 0.37 20.09 2
CTP-OXM- 3.28 31.11 1.00 144.87 170.82 1243.97 5887.75 4.73 24.44
SC. CTP CTP-OXM- 4.39 30.18 6.00 313.98 320.86 662.28 4139.25 6.62
32.43 CTP-CTP OXM-CTP- 4.76 50.90 6.00 697.00 702.10 302.70 2078.90
8.20 NA CTP CTP-CTP- 2.74 27.77 1.00 114.15 129.85 1636.55 6459.76
4.10 23.36 OXM Exp. OXM 0.18 36.4 0.08 13.1 13.3 16999 4375 3
OXM-CTPX3 3.78 25.1 2.0 308.9 312 512.8 2797 SC. OXM-CTPX4 10.15
21.1 2.0 400.4 431.9 355.4 5379.6 OXM-CTPX5 9.63 21.2 8 431 442
346.2 4812
[0811] A comparison between the various groups showing the fold
increase of T1/2 and AUC.infin. demonstrated that OXM-CTPx4 and
OX-CTPx5 were superior in these parameters compared to other
variants (Table 33).
TABLE-US-00105 TABLE 33 Fold of increase of OXM-CTP variants's PK
parameters in compared to OXM peptide as calculated from
Experiments 1-3. T1/2 AUC.infin. Sample/OXM Sample/OXM Variant
peptide peptide Exp. 1 CTP-OXM-CTP NA 2.8 IV. CTP-OXM-CTP-CTP NA
5.6 CTP-CTP-OXM NA 2.29 Exp. 2 CTP-OXM-CTP 14.9 3.98 SC.
CTP-OXM-CTP-CTP 19.9 7.48 OXM-CTP-CTP 21.6 16.3 CTP-CTP-OXM 12.4
3.03 Exp. 3 OXM-CTP-CTP-CTP 21 23.45 SC. OXM-CTP-CTP- 56.4 32.478
CTP-CTP OXM-CTP-CTP- 53.5 33.23 CTP-CTP-CTP
Example 11: CTP-Modified OXM Induces Glucose Tolerance
[0812] An IPGTT study was carried out in C57BL/6 mice demonstrated
that OXM enhanced glucose clearance via stimulation of insulin
secretion. The IP glucose tolerance test (IPGTT) evaluates the
glucose lowering effect of OXM. In order to evaluate the in vivo
activity of OXM or OXM-CTP variants, the IPGTT model was applied.
Overnight fasted C57BL/6 mice were injected IP with OXM peptide or
OXM-CTP variants followed by IP injection of glucose (1.5 g/kg) and
measurement of blood glucose levels from the tail vein by
glucometer (FIG. 14). OXM-CTP variants were assessed in two
sequential experiments; Experiment 1 for variants CTP-OXM-CTP,
CTP-OXM-CTP-CTP, OXM-CTP-CTP, CTP-CTP-OXM (FIGS. 14A and 14C) and
Experiment 2 for variants OXM-CTP-CTP-CTP, OXM-CTPX4 and OXM-CTPX5
(FIGS. 14B and 14D). OXM (100 nmol/kg), or OXM-CTP variants (100
nmol/kg) were administered IP 15 min or 2 hrs (OXM peptide and
variant OXM-CTPX4) prior to glucose IP injection, and the induction
of glucose tolerance was compared to vehicle (buffer) group. A
marked effect was measured for OXM-CTP variants CTP-OXM-CTP-CTP,
OXM-CTP-CTP, OXM-CTPX3, OXM-CTPX4 and OXM-CTPX5 as reflected by
reduction of 20-30% of blood glucose AUC compared to vehicle group
as compared to 100 nmol/kg of OXM peptide which had low impact on
the calculated AUC (FIG. 14, reduction of 1%, Experiment 1 and
6.7%, Experiment 2). Surprisingly, CTP-OXM-CTP resulted in
increased glucose levels while CTP-CTP-OXM had a minor impact on
glucose tolerance. OXM-CTPX4 induced glucose tolerance activity
even when administered 120 min prior to glucose while OXM peptide
activity was no longer apparent. This result is aligned with the
improved pharmacokinetics profile of this variant.
Example 12: Production of CTP-Modified Polypeptide of Interest
Constructs
Expression of CTP-Modified Polypeptide of Interest
[0813] Cell transfection (nucleofection): Stable expression is
achieved by integration of a gene for a CTP-modified polypeptide of
interest into the target cell's chromosome: Initially the gene is
introduced into the cell, subsequently into the nucleus and finally
it is integrated into chromosomal DNA. After gene transfer, cells
are cultivated in medium containing a selection marker such as
dihydrofolate reductase (DHFR).
[0814] Clone Selection--Limiting Dilution of single cell by plating
in 96-well plates. The selection is applied to the single cell
culture and may be repeated several times to obtain 100% clonal
purity. Highest producing clone, based on growth curve, clone
characterization, specific productivity and protein profile, are
propagated in 1 L Shake flask prior to inoculation in bioreactor.
Briefly:
[0815] A polypeptide of interest protein-producing cell line is
manufactured by recombinant DNA technology Animal component-free
media is used throughout the derivation of the Master and Working
Cell Banks (MCB; WCB). Transfection of mammalian cells, which may
include selectable elements, for example dhfr-negative CD DG44
cells (CHO cells), which are adapted to protein-free medium and
suspension growth, is carried out using a transfection reagent, for
example FuGENE-6 (Roche Applied Science). Stable clones are
isolated by limiting dilution steps in cell culture. The highest
producing clones are amplified with increasing concentrations of a
selectable agent, for example methotrexate (MTX). Based on clone
population doubling level (PDL), productivity of CTP-modified
polypeptide of interest (picogram per cell per day, PCD), and
maximum attained cell density in the selected medium, the highest
producing clones are isolated and will be used to prepare the
R&D banks followed by manufacturing of a qualified Master Cell
Bank (MCB) and Working Cell Bank (WCB).
Upstream Process
[0816] The upstream process for a CTP-modified polypeptide of
interest consists of 2 types of medium formulations; growth medium
(Medium 1) and production medium (Medium 2). Medium 1 includes an
agent for selection, for example methotrexate (MTX), and Medium 2
is identical to Medium 1 excluding the agent for selection, for
example MTX. Medium 1 is used for the cell culture propagation
(Step 1-4 of FIG. 15) prior to seeding in a 200 Liter (L)
bioreactor, and Medium 2 is used in N-2 steps (two propagations
prior to the final product. "N" refers to the final product), a 200
L bioreactor (Step 5 of FIG. 15) and production bioreactor of 1000
L for up scaling (Step 6 of FIG. 15).
Manufacturing Process: Polypeptide of Interest
[0817] A selected clone is manufactured at a 1000-L scale in
serum-free culture medium. The culture of the given mammalian cell
line, for example a CHO cell line, is expanded in several steps
from a single vial of the working cell bank (WCB) to the final
production culture volume. The production cell culture supernatant
is tested for bioburden, bacterial endotoxin, productivity and
adventitious virus. The process is performed using 50 L and 200 L
bioreactors for seeding bioreactors and a 1000 Liter bioreactor for
scaling-up. All product contact surfaces are disposable, while
non-disposable product contact equipment are product dedicated.
These pieces of equipment are cleaned and sanitized between
batches. The culture is expanded to 50 L and 200 L bioreactors
prior to inoculation in a 1000 L bioreactor. Final scale-up and
fed-batch bioreactor production is performed in disposable
bioreactors of 1000 L. Removal of the cells is accomplished using a
disposable filter system (Millipore depth filter).
[0818] One or two vials of the WCB are thawed at 37.degree. C. The
cells are centrifuged at and the cell pellet is resuspended to a
target concentration with 10 mL fresh Media low volume shake flask
and incubated at 37.+-.0.5.degree. C., 5.+-.1% CO.sub.2. After the
2nd subcultivation the cell culture with the higher viability is
further expanded; the other cell culture is discarded. Four
sub-cultivation steps in shake flask with increasing volumes are
conducted with pre-defined step parameters such as seeding
concentration and working volume. Volumes followed by two seeding
bioreactor steps for a typical 1000 L bioreactor run.
[0819] The 50 L and 200 L seed bioreactors are used as seeding
bioreactor. Prior to inoculation the 200 L bioreactor is filled
with approx. 50 L of an appropriate cell medium, for example
PowerCHO 2CD Medium 2 (w/o MTX). The process control is set to the
parameters as follows: pH 7, Temperature 37.degree. C., DO 50%.
These parameters apply to the medium pre-conditioning and to the
seed train cultivation process.
[0820] When the process parameters are controlled within their
pre-defined ranges the inoculum transfer is started. During cell
mass expansion in the 50 L and 200 L seed bioreactors no feed
addition is applied to the process. The expected cultivation time
in the seed bioreactors is 3 to 4 days before cells are transferred
into the 1000 L production bioreactor. Samples for in process
control (IPC) are taken on a daily basis.
Cell Propagation in Production Bioreactor, 1000 Liter (Step 6, FIG.
15)
[0821] The culture is incubated in the bioreactor for about 11 days
(dependent on the viability of the cells) at 37.degree. C., 20%
dissolved oxygen (DO) and pH 7.2. On Day 3 the pH is shifted to 6.9
until the harvest. On day 4, Feed (Power Feed A without Lipids) is
added. Feed volume is equal to 33% of the final bioreactor volume.
On day 6, DMSO is added to the bioreactor. Glucose feed solution is
added to the culture in order to maintain a desired concentration
and a bolus of 1M of Sodium Bicarbonate is added in order to
maintain a desired culture concentration (HCO.sub.3). The harvest
is performed using predefined criteria. During the first four days,
the cell culture is sampled daily for cell count, viability and
metabolic analysis. From day 5, the culture is sampled twice-daily
for cell count, viability and metabolic analysis and from Day 9
also for specific productivity by Reverse-Phase HPLC. Productivity
of the CTP-modified polypeptide of interest is at least 500 gr/L
with the high glycosylated form consist of at least 70% of the
total polypeptide of interest-CTP protein in the harvest.
[0822] The example presented herein is manufacturing the
CTP-modified polypeptide of interest using a fedbatch mode but one
skilled in the art could develop a perfusion mode using in general,
similar purification scheme. Alternatively, one skilled in the art
could develop a perfusion method wherein duration of incubation
could be even up to 7-120 days.
Cell Harvest and Storage (Step 7 of FIG. 15)
[0823] The harvest is performed using a disposable filtration
process train. To clarify the harvest a depth filtration and 0.2
.mu.m filtration is performed. The clarification is followed by a
0.45/0.2 .mu.m filtration. The depth filters are flushed and the
residual liquid is blown out of the system with air. Filtration
process is run with a pump speed of .ltoreq.15 L/min and a maximal
defined pressure. Afterwards the filters are washed with Tris-HCl
buffer and blown out with pressurized air to increase the product
recovery.
[0824] The clarified harvest is tested for bioburden, bacterial
endotoxin, specific protein content by RP-HPLC, SDS-PAGE, Western
Blot, HPC Elisa assay, residual DNA, in vitro virus assay,
Virus-like particles, S+L- and Mycoplasma.
Purification Process
[0825] Purification scheme is described in FIG. 16.
Ultrafiltration and Diafiltration 1--UFDF1 (Step 8)
[0826] The Clarified Harvest is concentrated and diafiltered using
a hollow fiber cartridge, or equivalent TFN based UFDF step. The
cartridge nominal molecular weight cutoff size is 10,000 kDa. The
concentrated and diafiltered harvest is tested for specific protein
content by RP-HPLC and HCP Elisa.
Viral Inactivation by Incubation (Step 9)
[0827] The material is filtered through a Millipore 1.55 m2 filter
followed by a sterile Sartopore 2 XLG 10'' filter into a sterile
mixing bag (depth filtration step). Next, a solution is added to
inactivate viral content, for example Tris/10% Triton solution is
added to the final filtrate volume bringing the Triton
concentration to 1% (w/w). After incubation, before loading on an
anion exchange column, the product solution is filtered with a 0.2
.mu.m filter unit.
Anion Exchange-Sepharose Fast Flow Chromatography (Step 10)
[0828] An anion exchange column packed with resin is used for this
step. The column is packed in a pre-defined bed height. The
specific protein in the load is determined prior to the addition of
Triton due to the interference caused by the Triton in the assay.
The anion exchange column is equilibrated and loaded with the viral
inactivated pool and then washed. A second wash is conducted with
and the material is eluted and then stored at ambient temperature
(18-26.degree. C.) for processing the next day or at 2-8.degree. C.
for longer time. All chromatography steps are conducted in down
flow mode. Alternatively, steps could be run in an upflow mode. The
eluate is tested for specific protein by RP-HPLC (Target: main peak
elutes as single peak), HCP Elisa, SDS-PAGE, Western Blot and
residual DNA.
Hydrophobic Interaction Chromatography (HIC) Column Chromatography
(Step 11)
[0829] An HIC Resin is used for this step. The column is packed
column in a pre-defined bed height. The HIC chromatography is
performed in 1-5 cycles depending on product quantity.
Alternatively, up to 10 cycles could be run. The HIC load is
prepared by adjusting the anion exchange column eluate with
Ammonium Sulfate. The column is equilibrated and loaded with the
adjusted and 0.2 .mu.m filtered anion exchange eluate and then
washed and the material is eluted, and then stored at 2-8.degree.
C. until further processing.
HIC Eluate Ultrafiltration and Diafiltration 2 (Step 12)
[0830] The HIC eluate is concentrated and diafiltered reduce the
volume and prepare the material for the CHT Column Step. The HIC
eluate is concentrated and then diafiltered. Once the pH and
conductivity are determined to be in range, the system is drained
and 0.45/0.2 .mu.m filtered to a sterile bag. The final volume of
concentrated, diafiltered HIC eluate is stored at ambient room
temperature (18-26.degree. C.) overnight. The retentate is tested
for bioburden, bacterial endotoxin and specific protein by
A.sub.280.
Multimodel or Mixed-Mode Protein Chromatography (Step 13)
[0831] A multimodel or mixed-mode protein chromatography column
packed with resin is used for this step. The column is packed
column in a pre-defined bed height. The column is equilibrated and
loaded with the concentrated and diafiltrated, HIC eluate and
washed with 4 column volumes (CV) buffer. The flow-through and wash
material are collected and held overnight at ambient temperature
(18-26.degree. C.) until further processing.
Cation Exchange-Sepharose Chromatography (Step 14)
[0832] A column packed with Cation-Exchange Sepharose Resin is used
for this step. The column is packed column in a pre-defined bed
height. The load is prepared by adjusting the Multimodal or
Mixed-Mode Protein Chromatography flow-through fraction. Following
the pH adjustment the solution is loaded onto the column followed
by a wash step. The flow-through is collected for further
processing. The pH is adjusted and the material is filtered through
a 0.45/0.2 .mu.m filter. The material is stored at ambient
temperature (18-26.degree. C.) overnight or at 2-8.degree. C. for
up to 24 hours. All chromatography steps are done in downflow
mode.
Viral Inactivation by Nanofiltration (Step 15)
[0833] The Viral Filtration is performed using an Asahi Planova 20N
Virus filter. A Sartopore 2 filter with a 0.45/0.2 .mu.m or 0.1
.mu.m membrane is used as prefilter of the nanofilter. The Virus
filter is pre-equilibrated and primed with the final formulation
made with WFI. The adjusted SP-Flow-Through is passed through the
filter train at a continuous pressure and collected in a sterile
bioprocess bag. The filter train is flushed with formulation buffer
to maximize product recovery. The filter is integrity tested pre
and post use per the manufacturer's recommended procedures. The
Post use test includes a gold-particle test, also per the
manufacturer's procedure.
UFDF3 and Filtration and Storage of the Drug Substance (Step
16)
[0834] The Viral Filtrate is concentrated to a target the final DS
concentration (which can vary from 5-100 mg/ml) in preparation for
the Bulk Filtration and Fill. A single used or reusable cassette is
used for this step with a cut-off of 3-30 KDa. The product is
concentrated in a first step to 5-25 mg/ml and diafiltrated to 10
mM Citrate, 147 mM NaCl pH 6.4 (7 DF volumes). A final product
concentration is adjusted and filtered with a Millipak 100 filter.
The filtrated product solutions are aliquoted and frozen at a
temperature of -70.+-.5.degree. C.
Drug Product Manufacturing
[0835] The formulation of the drug product (DP) process starts with
the thawing of Drug substance (DS). Drug Product is achieved by
dilution of the Drug Substance (DS) to the required concentration
using the formulation buffer, aseptic filtration and filling in
standard 2R vials or other primary packaging such as cartridges or
pre-filled syringes. A skilled artisan would appreciate that the
term "drug substance" (DS) may encompass or be equivalent to the
active pharmaceutical ingredient (API). In one embodiment, a
CTP-modified polypeptide of interest, as set forth herein, is a
drug substance (DS) comprising the bulk purified drug. The skilled
artisan would also appreciate that the term "drug product" (DP) may
encompass the finally formulated drug once dispensed into a final
container, for example a vial, under aseptic conditions. In one
embodiment, a CTP-modified polypeptide of interest, as set forth
herein, is a drug product (DP) comprising the finally formulated
CTP-modified polypeptide of interest.
Characterization of CTP-Modified Polypeptide of Interest
[0836] CTP-modified polypeptide content in the harvest and the
percentage of the high glycosylated forms are determined by a
specific RP-HPLC method. Total protein in the harvest is determined
by Bradford analysis. The specific protein percentage in the
harvest produced by a selected clone is above 70% relative to the
total protein in the harvest. In addition, the manufacturing
upstream process is developed to enable high percentage of the
highly glycosylated CTP-modified protein compared to the low
glycosylated form. The highly glycosylated form is the target form,
as it results longer extension of the CTP-modified polypeptide
half-life.
O-Glycan Content
[0837] Glycoprofiling is performed by releasing glycans followed by
glycan labeling with 2-aminobenzamide (2AB), cleaned up and
analyzed by NP-HPLC. Briefly, an O-glycan content assay is
conducted to calculate the number of O-glycans mol per mol of a
CTP-modified polypeptide of interest. The terminal galactose units
of the O-glycans are enzymatically cleaved from the protein by
.beta.-galactosidase. These free galactose units are separated on a
CarboPac PA20-column and detected with pulsed amperometry.
Galactose (Gal) is quantified using external calibration with a
galactose reference standard. The content of galactose can be
directly related to the content of O-glycan structure, Gal-GalNAc.
Analysis of drug substance and drug product batches demonstrate a
robust batch to batch consistency. This unexpected robust
glycosylation content is significant, showing that the number of
O-glycans per CTP is improved over that known in the art.
Intact Molecular Weight Analysis Samples
[0838] Molecular weight analysis of different DS batches is
performed with the aim to obtain information on the number of
O-linked glycosylation sites. Intact samples as well as
de-sialylated samples using Neuramnidase, and de-O-glycosylated
samples using O-glycosidase, are analyzed by on-line LC/ES-MS. The
result showing a high % of serine occupancy is unexpected in
comparison with levels known in the art (only 4 serines
glycosylated compared with up to 6 in the CTP-modified polypeptide
of interest manufactured herein).
O-Linked Glycosylation Site Occupancy of CTP-Modified Protein
Samples
[0839] O-glycosylation site occupancy of 4 different DS batches is
performed at M-scan with the aim to obtain information on the
number of O-linked glycosylation sites per molecule. Samples are
de-sialylated using Neuramnidase followed by tryptic digestion of
reduced/carboxymethylaed samples. Finally an on line LC/ES-MS is
carried out for the treated samples and interpretation of the MS
data is conducted using a designated software. Evaluation of the
data obtained from analysis of the tryptic digest mixtures leads to
signals allowing 100% of the protein sequence being mapped.
O-glycosylation may take place on both the N-terminal and
C-terminal CTP region. Sites of occupancy are identified as serine
residues following proline as well as two of the four serines in
the regions of serine repeats. A total of up to 18 serine residues
may serve as attachment sites for O-glycans. No significant
differences between the batches are detected.
Purity
[0840] RP-HPLC separates molecules according to their polarity. A
mobile phase gradient from a more polar to a less polar solvent is
used to elute molecules with a strong polarity earlier than less
polar molecules. The related forms are separated from the native
protein using UV detection at 220 nm. The relative peak areas (area
%) of the related forms and the main peak can be calculated by
integrating the corresponding peak areas. The main peak of Drug
Substance and Drug Product consists of more than 97% peak area,
indicating a highly purified product and an effective purification
process.
[0841] Size Exclusion HPLC is a chromatographic technique that
separates molecules according to size. Within the fractionation
range chosen, larger molecules elute earlier than smaller
molecules. The separation mechanism is non-adsorptive and molecules
are eluted under isocratic conditions. SEC enables monomers to be
separated from higher molecular weight forms (such as dimers and
polymers) of the target molecule. The SEC method is developed to
analyze the content of dimers and polymers in Drug Substance and
Drug Product.
RP-HPLC Content Method
[0842] This method is being used for the content determination of
intermediate samples and the determination of %-unglycosylated
CTP-modified polypeptide in intermediate samples by reversed phase
chromatography. The reversed phase-HPLC separates molecules due to
their polarity. Relatively non-polar molecules ligate to the column
material while charged and polar molecules are eluted without
accomplishing an interaction with the column.
[0843] The ligated molecules are eluted with the aid of a gradient
from a polar to a less polar solution. Molecules of the strongest
polarity eluted first followed by the less polar molecules. The
detection is carried out via absorption at 214 nm.
Viral Clearance
[0844] The ability of the manufacturing process to address and
mitigate contamination of final drug product with endogenous and
adventitious virus has been the subject of a preliminary
evaluation. A GLP-compliant study has been conducted according to
applicable guidance for investigational products using three model
viruses spiked into scaled down segments of the manufacturing
process to quantify the ability of these steps to inactivate or
clear the spiked virus population. With the amounts of virus
expressed as log 10 Adjusted Titre, the log 10 clearance factor is
determined simply by subtracting the value for output from the
value for input. As log 10 numbers the clearance factors are
additive to derive an overall clearance factor for all evaluated
steps. A-MuLV is considered to be a model virus representing
possible presence of CHO retroviruses, the measures taken to
inactivate and remove contaminating A-MuLV virus achieved clearance
factor of at least antilog10, e.g. viral log reduction factor (LRF)
of about 22, demonstrating that the overall process has an
exceptional capacity for viral removal. For the PPV which is a
resistant non-enveloped small virus, robust removal by the
nanofiltration step is obtained.
[0845] While certain features disclosed herein have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended claims are intended to cover all such modifications
and changes as fall within the true spirit disclosed herein.
Sequence CWU 1
1
1151220PRTArtificial SequenceCTP-modified EPO 1Met Gly Val His Glu
Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu1 5 10 15Leu Ser Leu Pro
Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30Ile Cys Asp
Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45Ala Glu
Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60Asn
Ile Thr Val Pro Asp Thr Val Asn Phe Tyr Ala Trp Lys Arg Met65 70 75
80Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu
85 90 95Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser
Gln 100 105 110Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val
Ser Gly Leu 115 120 125Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly
Ala Gln Lys Glu Ala 130 135 140Ile Ser Pro Pro Asp Ala Ala Ser Ala
Ala Pro Leu Arg Thr Ile Thr145 150 155 160Ala Asp Thr Phe Arg Lys
Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg 165 170 175Gly Lys Leu Lys
Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg 180 185 190Ser Ser
Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 195 200
205Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 210 215
2202248PRTArtificial SequenceCTP-modified EPO 2Met Gly Val His Glu
Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu1 5 10 15Leu Ser Leu Pro
Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30Ile Cys Asp
Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45Ala Glu
Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60Asn
Ile Thr Val Pro Asp Thr Val Asn Phe Tyr Ala Trp Lys Arg Met65 70 75
80Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu
85 90 95Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser
Gln 100 105 110Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val
Ser Gly Leu 115 120 125Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly
Ala Gln Lys Glu Ala 130 135 140Ile Ser Pro Pro Asp Ala Ala Ser Ala
Ala Pro Leu Arg Thr Ile Thr145 150 155 160Ala Asp Thr Phe Arg Lys
Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg 165 170 175Gly Lys Leu Lys
Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg 180 185 190Ser Ser
Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 195 200
205Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser
210 215 220Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro
Gly Pro225 230 235 240Ser Asp Thr Pro Ile Leu Pro Gln
2453277PRTArtificial SequenceCTP-modified EPO 3Met Gly Val His Glu
Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu1 5 10 15Leu Ser Leu Pro
Leu Gly Leu Pro Val Leu Gly Ser Ser Ser Ser Lys 20 25 30Ala Pro Pro
Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser 35 40 45Asp Thr
Pro Ile Leu Pro Gln Ala Pro Pro Arg Leu Ile Cys Asp Ser 50 55 60Arg
Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile65 70 75
80Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val
85 90 95Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val
Gly 100 105 110Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu
Ser Glu Ala 115 120 125Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser
Ser Gln Pro Trp Glu 130 135 140Pro Leu Gln Leu His Val Asp Lys Ala
Val Ser Gly Leu Arg Ser Leu145 150 155 160Thr Thr Leu Leu Arg Ala
Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro 165 170 175Pro Asp Ala Ala
Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr 180 185 190Phe Arg
Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu 195 200
205Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg Ser Ser Ser
210 215 220Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu
Pro Gly225 230 235 240Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser
Ser Ser Lys Ala Pro 245 250 255Pro Pro Ser Leu Pro Ser Pro Ser Arg
Leu Pro Gly Pro Ser Asp Thr 260 265 270Pro Ile Leu Pro Gln
2754387PRTArtificial SequenceCTP-modified EPO 4Met Gly Val His Glu
Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu1 5 10 15Leu Ser Leu Pro
Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30Ile Cys Asp
Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45Ala Glu
Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60Asn
Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg65 70 75
80Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu
85 90 95Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser
Ser 100 105 110Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala
Val Ser Gly 115 120 125Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu
Gly Ala Gln Lys Glu 130 135 140Ala Ile Ser Pro Pro Asp Ala Ala Ser
Ala Ala Pro Leu Arg Thr Ile145 150 155 160Thr Ala Asp Thr Phe Arg
Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175Arg Gly Lys Leu
Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190Arg Ser
Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser 195 200
205Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ala Pro Pro
210 215 220Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu
Glu Ala225 230 235 240Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala
Glu His Cys Ser Leu 245 250 255Asn Glu Asn Ile Thr Val Pro Asp Thr
Lys Val Asn Phe Tyr Ala Trp 260 265 270Lys Arg Met Glu Val Gly Gln
Gln Ala Val Glu Val Trp Gln Gly Leu 275 280 285Ala Leu Leu Ser Glu
Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn 290 295 300Ser Ser Gln
Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val305 310 315
320Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln
325 330 335Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro
Leu Arg 340 345 350Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg
Val Tyr Ser Asn 355 360 365Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr
Gly Glu Ala Cys Arg Thr 370 375 380Gly Asp Arg3855221PRTArtificial
SequenceCTP-modified EPO 5Met Gly Val His Glu Cys Pro Ala Trp Leu
Trp Leu Leu Leu Ser Leu1 5 10 15Leu Ser Leu Pro Leu Gly Leu Pro Val
Leu Gly Ser Ser Ser Ser Lys 20 25 30Ala Pro Pro Pro Ser Leu Pro Ser
Pro Ser Arg Leu Pro Gly Pro Ser 35 40 45Asp Thr Pro Ile Leu Pro Gln
Ala Pro Pro Arg Leu Ile Cys Asp Ser 50 55 60Arg Val Leu Glu Arg Tyr
Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile65 70 75 80Thr Thr Gly Cys
Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val 85 90 95Pro Asp Thr
Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly 100 105 110Gln
Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala 115 120
125Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu
130 135 140Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg
Ser Leu145 150 155 160Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys
Glu Ala Ile Ser Pro 165 170 175Pro Asp Ala Ala Ser Ala Ala Pro Leu
Arg Thr Ile Thr Ala Asp Thr 180 185 190Phe Arg Lys Leu Phe Arg Val
Tyr Ser Asn Phe Leu Arg Gly Lys Leu 195 200 205Lys Leu Tyr Thr Gly
Glu Ala Cys Arg Thr Gly Asp Arg 210 215 2206249PRTArtificial
SequenceCTP-modified EPO 6Met Gly Val His Glu Cys Pro Ala Trp Leu
Trp Leu Leu Leu Ser Leu1 5 10 15Leu Ser Leu Pro Leu Gly Leu Pro Val
Leu Gly Ser Ser Ser Ser Lys 20 25 30Ala Pro Pro Pro Ser Leu Pro Ser
Pro Ser Arg Leu Pro Gly Pro Ser 35 40 45Asp Thr Pro Ile Leu Pro Gln
Ala Pro Pro Arg Leu Ile Cys Asp Ser 50 55 60Arg Val Leu Glu Arg Tyr
Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile65 70 75 80Thr Thr Gly Cys
Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val 85 90 95Pro Asp Thr
Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly 100 105 110Gln
Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala 115 120
125Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu
130 135 140Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg
Ser Leu145 150 155 160Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys
Glu Ala Ile Ser Pro 165 170 175Pro Asp Ala Ala Ser Ala Ala Pro Leu
Arg Thr Ile Thr Ala Asp Thr 180 185 190Phe Arg Lys Leu Phe Arg Val
Tyr Ser Asn Phe Leu Arg Gly Lys Leu 195 200 205Lys Leu Tyr Thr Gly
Glu Ala Cys Arg Thr Gly Asp Arg Ser Ser Ser 210 215 220Ser Lys Ala
Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly225 230 235
240Pro Ser Asp Thr Pro Ile Leu Pro Gln 2457220PRTArtificial
SequenceCTP-modified EPO 7Met Gly Val His Glu Cys Pro Ala Trp Leu
Trp Leu Leu Leu Ser Leu1 5 10 15Leu Ser Leu Pro Leu Gly Leu Pro Val
Leu Gly Ala Pro Pro Arg Leu 20 25 30Ile Cys Asp Ser Arg Val Leu Glu
Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45Ala Glu Asn Ile Thr Thr Gly
Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60Asn Ile Thr Val Pro Asp
Thr Val Asn Phe Tyr Ala Trp Lys Arg Met65 70 75 80Glu Val Gly Gln
Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu 85 90 95Ser Glu Ala
Val Leu Arg Ser Gln Ala Leu Leu Val Asn Ser Ser Gln 100 105 110Pro
Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu 115 120
125Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala
130 135 140Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr
Ile Thr145 150 155 160Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr
Ser Asn Phe Leu Arg 165 170 175Gly Lys Leu Lys Leu Tyr Thr Gly Glu
Ala Cys Arg Thr Gly Asp Arg 180 185 190Ser Ser Ser Ser Lys Ala Pro
Pro Pro Ser Leu Pro Ser Pro Ser Arg 195 200 205Leu Pro Gly Pro Ser
Asp Thr Pro Ile Leu Pro Gln 210 215 2208193PRTArtificial
SequenceCTP-modified EPO 8Met Gly Val His Glu Cys Pro Ala Trp Leu
Trp Leu Leu Leu Ser Leu1 5 10 15Leu Ser Leu Pro Leu Gly Leu Pro Val
Leu Gly Ala Pro Pro Arg Leu 20 25 30Ile Cys Asp Ser Arg Val Leu Glu
Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45Ala Glu Asn Ile Thr Thr Gly
Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60Asn Ile Thr Val Pro Asp
Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg65 70 75 80Met Glu Val Gly
Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95Leu Ser Glu
Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110Gln
Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120
125Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu
130 135 140Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg
Thr Ile145 150 155 160Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val
Tyr Ser Asn Phe Leu 165 170 175Arg Gly Lys Leu Lys Leu Tyr Thr Gly
Glu Ala Cys Arg Thr Gly Asp 180 185 190Arg9271PRTArtificial
SequenceCTP-mofied IFN 9Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu
Leu Leu Cys Phe Ser1 5 10 15Thr Thr Ala Leu Ser Ser Ser Ser Ser Lys
Ala Pro Pro Pro Ser Leu 20 25 30Pro Ser Pro Ser Arg Leu Pro Gly Pro
Ser Asp Thr Pro Ile Leu Pro 35 40 45Gln Met Ser Tyr Asn Leu Leu Gly
Phe Leu Gln Arg Ser Ser Asn Phe 50 55 60Gln Cys Gln Lys Leu Leu Trp
Gln Leu Asn Gly Arg Leu Glu Tyr Cys65 70 75 80Leu Lys Asp Arg Met
Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 85 90 95Gln Gln Phe Gln
Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 100 105 110Gln Asn
Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 115 120
125Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile
130 135 140Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu
Asp Phe145 150 155 160Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu
Lys Arg Tyr Tyr Gly 165 170 175Arg Ile Leu His Tyr Leu Lys Ala Lys
Glu Tyr Ser His Cys Ala Trp 180 185 190Thr Ile Val Arg Val Glu Ile
Leu Arg Asn Phe Tyr Phe Ile Asn Arg 195 200 205Leu Thr Gly Tyr Leu
Arg Asn Ser Ser Ser Ser Lys Ala Pro Pro Pro 210 215 220Ser Leu Pro
Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile225 230 235
240Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser
245 250 255Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro
Gln 260 265 27010243PRTArtificial SequenceCTP-modified IFN 10Met
Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser1 5 10
15Thr Thr Ala Leu Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu
20 25 30Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu
Pro 35 40 45Gln Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser
Asn Phe 50 55 60Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu
Glu Tyr Cys65 70 75 80Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu
Glu Ile Lys Gln Leu 85 90 95Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu
Thr Ile Tyr Glu Met Leu 100 105 110Gln Asn Ile Phe Ala Ile Phe Arg
Gln Asp Ser Ser Ser Thr Gly Trp 115 120 125Asn Glu
Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile 130 135
140Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp
Phe145 150 155 160Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys
Arg Tyr Tyr Gly 165 170 175Arg Ile Leu His Tyr Leu Lys Ala Lys Glu
Tyr Ser His Cys Ala Trp 180 185 190Thr Ile Val Arg Val Glu Ile Leu
Arg Asn Phe Tyr Phe Ile Asn Arg 195 200 205Leu Thr Gly Tyr Leu Arg
Asn Ser Ser Ser Ser Lys Ala Pro Pro Pro 210 215 220Ser Leu Pro Ser
Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile225 230 235 240Leu
Pro Gln11215PRTArtificial SequenceCTP-modified IFN 11Met Thr Asn
Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser1 5 10 15Thr Thr
Ala Leu Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 20 25 30Pro
Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 35 40
45Gln Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe
50 55 60Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr
Cys65 70 75 80Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile
Lys Gln Leu 85 90 95Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile
Tyr Glu Met Leu 100 105 110Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp
Ser Ser Ser Thr Gly Trp 115 120 125Asn Glu Thr Ile Val Glu Asn Leu
Leu Ala Asn Val Tyr His Gln Ile 130 135 140Asn His Leu Lys Thr Val
Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe145 150 155 160Thr Arg Gly
Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly 165 170 175Arg
Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp 180 185
190Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg
195 200 205Leu Thr Gly Tyr Leu Arg Asn 210 21512243PRTArtificial
SequenceCTP-modified IFN 12Met Thr Asn Lys Cys Leu Leu Gln Ile Ala
Leu Leu Leu Cys Phe Ser1 5 10 15Thr Thr Ala Leu Ser Met Ser Tyr Asn
Leu Leu Gly Phe Leu Gln Arg 20 25 30Ser Ser Asn Phe Gln Cys Gln Lys
Leu Leu Trp Gln Leu Asn Gly Arg 35 40 45Leu Glu Tyr Cys Leu Lys Asp
Arg Met Asn Phe Asp Ile Pro Glu Glu 50 55 60Ile Lys Gln Leu Gln Gln
Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile65 70 75 80Tyr Glu Met Leu
Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser 85 90 95Ser Thr Gly
Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val 100 105 110Tyr
His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu 115 120
125Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys
130 135 140Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu
Tyr Ser145 150 155 160His Cys Ala Trp Thr Ile Val Arg Val Glu Ile
Leu Arg Asn Phe Tyr 165 170 175Phe Ile Asn Arg Leu Thr Gly Tyr Leu
Arg Asn Ser Ser Ser Ser Lys 180 185 190Ala Pro Pro Pro Ser Leu Pro
Ser Pro Ser Arg Leu Pro Gly Pro Ser 195 200 205Asp Thr Pro Ile Leu
Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro 210 215 220Ser Leu Pro
Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile225 230 235
240Leu Pro Gln13215PRTArtificial SequenceCTP-modified IFN 13Met Thr
Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser1 5 10 15Thr
Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg 20 25
30Ser Ser Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg
35 40 45Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu
Glu 50 55 60Ile Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu
Thr Ile65 70 75 80Tyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg
Gln Asp Ser Ser 85 90 95Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn
Leu Leu Ala Asn Val 100 105 110Tyr His Gln Ile Asn His Leu Lys Thr
Val Leu Glu Glu Lys Leu Glu 115 120 125Lys Glu Asp Phe Thr Arg Gly
Lys Leu Met Ser Ser Leu His Leu Lys 130 135 140Arg Tyr Tyr Gly Arg
Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser145 150 155 160His Cys
Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr 165 170
175Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn Ser Ser Ser Ser Lys
180 185 190Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly
Pro Ser 195 200 205Asp Thr Pro Ile Leu Pro Gln 210
2151432PRTArtificial SequenceCTP-peptide 14Asp Pro Arg Phe Gln Asp
Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser1 5 10 15Leu Pro Ser Pro Ser
Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu 20 25
301528PRTArtificial SequenceCTP-peptide 15Ser Ser Ser Ser Lys Ala
Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg1 5 10 15Leu Pro Gly Pro Ser
Asp Thr Pro Ile Leu Pro Gln 20 251612PRTArtificial
SequenceCTP-peptide 16Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu
Pro1 5 1017786DNAArtificial SequenceCTP-modified EPO 17tctagaggtc
atcatggggg tgcacgaatg tcctgcctgg ctgtggcttc tcctgtccct 60tctgtcgctc
cctctgggcc tcccagtcct gggctcctct tcctcaaagg cccctccccc
120gagccttcca agtccatccc gactcccggg gccctcggac accccaatat
taccacaagc 180cccaccacgc ctcatctgtg acagccgagt cctggagagg
tacctcttgg aggccaagga 240ggccgagaat atcacgacgg gctgtgctga
acactgcagc ttgaatgaga atatcactgt 300cccagacacc aaagttaatt
tctatgcctg gaagaggatg gaggtcgggc agcaggccgt 360agaagtctgg
cagggcctgg ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt
420ggtcaactct tcccagccgt gggagcccct gcagctgcat gtggataaag
ccgtcagtgg 480ccttcgcagc ctcaccactc tgcttcgggc tctgggagcc
cagaaggaag ccatctcccc 540tccagatgcg gcctcagctg ctccactccg
aacaatcact gctgacactt tccgcaaact 600cttccgagtc tactccaatt
tcctccgggg aaagctgaag ctgtacacag gggaggcctg 660caggacaggg
gacagatcct cttcctcaaa ggcccctccc ccgagccttc caagtccatc
720ccgactcccg gggccctcgg acaccccgat cctcccacaa taaaggtctt
ctggatccgc 780ggccgc 78618873DNAArtificial SequenceCTP-modified EPO
18tctagaggtc atcatggggg tgcacgaatg tcctgcctgg ctgtggcttc tcctgtccct
60tctgtcgctc cctctgggcc tcccagtcct gggctcctct tcctcaaagg cccctccccc
120gagccttcca agtccatccc gactcccggg gccctcggac accccaatat
taccacaagc 180cccaccacgc ctcatctgtg acagccgagt cctggagagg
tacctcttgg aggccaagga 240ggccgagaat atcacgacgg gctgtgctga
acactgcagc ttgaatgaga atatcactgt 300cccagacacc aaagttaatt
tctatgcctg gaagaggatg gaggtcgggc agcaggccgt 360agaagtctgg
cagggcctgg ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt
420ggtcaactct tcccagccgt gggagcccct gcagctgcat gtggataaag
ccgtcagtgg 480ccttcgcagc ctcaccactc tgcttcgggc tctgggagcc
cagaaggaag ccatctcccc 540tccagatgcg gcctcagctg ctccactccg
aacaatcact gctgacactt tccgcaaact 600cttccgagtc tactccaatt
tcctccgggg aaagctgaag ctgtacacag gggaggcctg 660caggacaggg
gacagatcct cttcctcaaa ggcccctccc ccgagccttc caagtccatc
720ccgactcccg gggccctccg acacaccaat cctgccacag agcagctcct
ctaaggcccc 780tcctccatcc ctgccatccc cctcccggct gcctggcccc
tctgacaccc ctatcctgcc 840tcagtgatga aggtcttctg gatccgcggc cgc
8731927PRTArtificial SequenceEPO signal peptide 19Met Gly Val His
Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu1 5 10 15Leu Ser Leu
Pro Leu Gly Leu Pro Val Leu Gly 20 2520166PRTArtificial
SequenceIFN-beta 20Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser
Ser Asn Phe Gln1 5 10 15Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg
Leu Glu Tyr Cys Leu 20 25 30Lys Asp Arg Met Asn Phe Asp Ile Pro Glu
Glu Ile Lys Gln Leu Gln 35 40 45Gln Phe Gln Lys Glu Asp Ala Ala Leu
Thr Ile Tyr Glu Met Leu Gln 50 55 60Asn Ile Phe Ala Ile Phe Arg Gln
Asp Ser Ser Ser Thr Gly Trp Asn65 70 75 80Glu Thr Ile Val Glu Asn
Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95His Leu Lys Thr Val
Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110Arg Gly Lys
Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125Ile
Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135
140Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg
Leu145 150 155 160Thr Gly Tyr Leu Arg Asn 1652130PRTArtificial
SequenceGLP-1 21His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3022661DNAArtificial SequenceCTP-modified IFN
22tctagaggac atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac
60caccgccctg agcatgagct acaacctgct gggcttcctg cagaggtcca gcaacttcca
120gtgccagaag ctgctgtggc agctgaacgg caggctggaa tactgcctga
aggacaggat 180gaacttcgac atcccagagg aaatcaagca gctgcagcag
ttccagaagg aggacgccgc 240cctgaccatc tacgagatgc tgcagaacat
cttcgccatc ttcaggcagg acagcagcag 300caccggctgg aacgagacca
tcgtggagaa cctgctggcc aacgtgtacc accagatcaa 360ccacctgaaa
accgtgctgg aagagaagct ggaaaaggag gacttcacca ggggcaagct
420gatgagcagc ctgcacctga agaggtacta cggcagaatc ctgcactacc
tgaaggccaa 480ggagtacagc cactgcgcct ggaccatcgt gagggtggag
atcctgagga acttctactt 540catcaacagg ctgaccggct acctgaggaa
cagctccagc agcaaggccc ctccaccttc 600cctgcccagt ccaagccgac
tccctgggcc ctccgataca ccaattctgc cacagtgatg 660a
66123765DNAArtificial SequenceCTP-modified IFN 23tctagaggac
atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg
agcatgagct acaacctgct gggcttcctg cagaggtcca gcaacttcca
120gtgccagaag ctgctgtggc agctgaacgg caggctggaa tactgcctga
aggacaggat 180gaacttcgac atcccagagg aaatcaagca gctgcagcag
ttccagaagg aggacgccgc 240cctgaccatc tacgagatgc tgcagaacat
cttcgccatc ttcaggcagg acagcagcag 300caccggctgg aacgagacca
tcgtggagaa cctgctggcc aacgtgtacc accagatcaa 360ccacctgaaa
accgtgctgg aagagaagct ggaaaaggag gacttcacca ggggcaagct
420gatgagcagc ctgcacctga agaggtacta cggcagaatc ctgcactacc
tgaaggccaa 480ggagtacagc cactgcgcct ggaccatcgt gagggtggag
atcctgagga acttctactt 540catcaacagg ctgaccggct acctgaggaa
cagctccagc agcaaggccc ctccaccttc 600cctgcccagt ccaagccgac
tccctgggcc ctccgacaca ccaatcctgc cacagagcag 660ctcctctaag
gcccctcctc catccctgcc atccccctcc cggctgcctg gcccctctga
720cacccctatc ctgcctcagt gatgaaggtc tggatccgcg gccgc
76524849DNAArtificial SequenceCTP-modified IFN 24tctagaggac
atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg
agcagcagca gctccaaggc cccacccccc agcctgccca gccccagcag
120actgccaggc cccagcgaca cccccatcct gccccagatg agctacaacc
tgctgggctt 180cctgcagagg tccagcaact tccagtgcca gaagctgctg
tggcagctga acggcaggct 240ggaatactgc ctgaaggaca ggatgaactt
cgacatccca gaggaaatca agcagctgca 300gcagttccag aaggaggacg
ccgccctgac catctacgag atgctgcaga acatcttcgc 360catcttcagg
caggacagca gcagcaccgg ctggaacgag accatcgtgg agaacctgct
420ggccaacgtg taccaccaga tcaaccacct gaaaaccgtg ctggaagaga
agctggaaaa 480ggaggacttc accaggggca agctgatgag cagcctgcac
ctgaagaggt actacggcag 540aatcctgcac tacctgaagg ccaaggagta
cagccactgc gcctggacca tcgtgagggt 600ggagatcctg aggaacttct
acttcatcaa caggctgacc ggctacctga ggaacagctc 660cagcagcaag
gcccctccac cttccctgcc cagtccaagc cgactccctg ggccctccga
720cacaccaatc ctgccacaga gcagctcctc taaggcccct cctccatccc
tgccatcccc 780ctcccggctg cctggcccct ctgacacccc tatcctgcct
cagtgatgaa ggtctggatc 840cgcggccgc 84925409PRTArtificial
SequenceCTP-modified IFN 25Met Thr Asn Lys Cys Leu Leu Gln Ile Ala
Leu Leu Leu Cys Phe Ser1 5 10 15Thr Thr Ala Leu Ser Ser Ser Ser Ser
Lys Ala Pro Pro Pro Ser Leu 20 25 30Pro Ser Pro Ser Arg Leu Pro Gly
Pro Ser Asp Thr Pro Ile Leu Pro 35 40 45Gln Met Ser Tyr Asn Leu Leu
Gly Phe Leu Gln Arg Ser Ser Asn Phe 50 55 60Gln Cys Gln Lys Leu Leu
Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys65 70 75 80Leu Lys Asp Arg
Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 85 90 95Gln Gln Phe
Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 100 105 110Gln
Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 115 120
125Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile
130 135 140Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu
Asp Phe145 150 155 160Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu
Lys Arg Tyr Tyr Gly 165 170 175Arg Ile Leu His Tyr Leu Lys Ala Lys
Glu Tyr Ser His Cys Ala Trp 180 185 190Thr Ile Val Arg Val Glu Ile
Leu Arg Asn Phe Tyr Phe Ile Asn Arg 195 200 205Leu Thr Gly Tyr Leu
Arg Asn Ser Ser Ser Ser Lys Ala Pro Pro Pro 210 215 220Ser Leu Pro
Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile225 230 235
240Leu Pro Gln Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser
245 250 255Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg
Leu Glu 260 265 270Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro
Glu Glu Ile Lys 275 280 285Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala
Ala Leu Thr Ile Tyr Glu 290 295 300Met Leu Gln Asn Ile Phe Ala Ile
Phe Arg Gln Asp Ser Ser Ser Thr305 310 315 320Gly Trp Asn Glu Thr
Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His 325 330 335Gln Ile Asn
His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu 340 345 350Asp
Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr 355 360
365Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys
370 375 380Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr
Phe Ile385 390 395 400Asn Arg Leu Thr Gly Tyr Leu Arg Asn
405261261DNAArtificial SequenceCTP-modified IFN 26tctagaggac
atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg
agcagcagca gctccaaggc cccacccccc agcctgccca gccccagcag
120gctgccaggc cccagcgaca cccccatcct gccccagatg agctacaacc
tgctgggctt 180cctgcagagg tccagcaact tccagtgcca gaaactgctg
tggcagctga acggcaggct 240ggaatactgc ctgaaggacc ggatgaactt
cgacatcccc gaagagatca agcagctgca 300gcagttccag aaagaggacg
ccgccctgac catctacgag atgctgcaga acatcttcgc 360catcttcagg
caggacagca gcagcaccgg ctggaacgag accatcgtgg agaacctgct
420ggccaacgtg taccaccaga tcaaccacct gaaaaccgtg ctggaagaga
agctggaaaa 480agaggacttc accaggggca agctgatgag cagcctgcac
ctgaagaggt actacggcag 540aatcctgcac tacctgaagg ccaaagagta
cagccactgc gcctggacca tcgtgagggt 600ggagatcctg cggaacttct
acttcatcaa caggctgacc ggctacctga ggaacagctc 660cagcagcaag
gcccctccac cctccctgcc ctccccaagc agactgcccg gaccctccga
720cacaccaatt ctgccacaga tgtcctacaa tctgctcgga tttctgcagc
gctcctccaa 780ctttcagtgt cagaagctcc tctggcagct caatggccgc
ctggaatatt gtctgaaaga 840cagaatgaat tttgacatcc cagaggaaat
taaacagctc cagcagtttc agaaagaaga 900tgctgctctc acaatctatg
aaatgctcca gaatatcttt gcaatctttc gccaggacag 960ctcctccacc
gggtggaatg agacaattgt cgagaatctg ctcgccaatg tctatcatca
1020gatcaatcac ctcaagacag tcctcgaaga aaaactcgaa aaagaagatt
tcacacgcgg 1080caaactgatg tcctccctgc atctgaagcg ctactatggg
cgcatcctgc attatctgaa 1140agctaaagaa tactcccact gtgcttggac
aattgtgcgc gtcgagatcc tgagaaactt 1200ttatttcatt aaccgcctga
caggatacct gcgcaactga tgaaggtctg gatgcggccg 1260c
126127673DNAArtificial SequenceCTP-modified IFN 27tctagaggac
atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg
agcagcagca gctccaaggc cccacccccc agcctgccca gccccagcag
120gctgccaggc cccagcgaca cccccatcct gccccagatg agctacaacc
tgctgggctt 180cctgcagagg tccagcaact tccagtgcca gaaactgctg
tggcagctga acggcaggct 240ggaatactgc ctgaaggacc ggatgaactt
cgacatcccc gaagagatca agcagctgca 300gcagttccag aaagaggacg
ccgccctgac catctacgag atgctgcaga acatcttcgc 360catcttcagg
caggacagca gcagcaccgg ctggaacgag accatcgtgg agaacctgct
420ggccaacgtg taccaccaga tcaaccacct gaaaaccgtg ctggaagaga
agctggaaaa 480agaggacttc accaggggca agctgatgag cagcctgcac
ctgaagaggt actacggcag 540aatcctgcac tacctgaagg ccaaagagta
cagccactgc gcctggacca tcgtgagggt 600ggagatcctg cggaacttct
acttcatcaa caggctgacc ggctacctga ggaactgatg 660agtccgcggc cgc
67328763DNAArtificial SequenceCTP-modified IFN 28tctagaggac
atgaccaaca agtgcctgct gcagatcgcc ctgctgctgt gcttcagcac 60caccgccctg
agcagcagca gctccaaggc cccacccccc agcctgccca gccccagcag
120actgccaggc cccagcgaca cccccatcct gccccagatg agctacaacc
tgctgggctt 180cctgcagagg tccagcaact tccagtgcca gaagctgctg
tggcagctga acggcaggct 240ggaatactgc ctgaaggaca ggatgaactt
cgacatccca gaggaaatca agcagctgca 300gcagttccag aaggaggacg
ccgccctgac catctacgag atgctgcaga acatcttcgc 360catcttcagg
caggacagca gcagcaccgg ctggaacgag accatcgtgg agaacctgct
420ggccaacgtg taccaccaga tcaaccacct gaaaaccgtg ctggaagaga
agctggaaaa 480ggaggacttc accaggggca agctgatgag cagcctgcac
ctgaagaggt actacggcag 540aatcctgcac tacctgaagg ccaaggagta
cagccactgc gcctggacca tcgtgagggt 600ggagatcctg aggaacttct
acttcatcaa caggctgacc ggctacctga ggaacagctc 660cagcagcaag
gcccctccac cttccctgcc cagtccaagc cgactccctg ggccctccga
720tacaccaatt ctgccacagt gatgaaggtc tggatgcggc cgc
76329166PRTArtificial SequenceIFN-beta 29Met Ser Tyr Asn Leu Leu
Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln1 5 10 15Ser Gln Lys Leu Leu
Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30Lys Asp Arg Met
Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45Gln Phe Gln
Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60Asn Ile
Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn65 70 75
80Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn
85 90 95His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe
Thr 100 105 110Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr
Tyr Gly Arg 115 120 125Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser
His Cys Ala Trp Thr 130 135 140Ile Val Arg Val Glu Ile Leu Arg Asn
Phe Tyr Phe Ile Asn Arg Leu145 150 155 160Thr Gly Tyr Leu Arg Asn
165301356DNAArtificial SequenceFVII 30ctcgaggaca tggtctccca
ggccctcagg ctcctctgcc ttctgcttgg gcttcagggc 60tgcctggctg cagtcttcgt
aacccaggag gaagcccacg gcgtcctgca ccggcgccgg 120cgcgccaacg
cgttcctgga ggagctgcgg ccgggctccc tggagaggga gtgcaaggag
180gagcagtgct ccttcgagga ggcccgggag atcttcaagg acgcggagag
gacgaagctg 240ttctggattt cttacagtga tggggaccag tgtgcctcaa
gtccatgcca gaatgggggc 300tcctgcaagg accagctcca gtcctatatc
tgcttctgcc tccctgcctt cgagggccgg 360aactgtgaga cgcacaagga
tgaccagctg atctgtgtga acgagaacgg cggctgtgag 420cagtactgca
gtgaccacac gggcaccaag cgctcctgtc ggtgccacga ggggtactct
480ctgctggcag acggggtgtc ctgcacaccc acagttgaat atccatgtgg
aaaaatacct 540attctagaaa aaagaaatgc cagcaaaccc caaggccgaa
ttgtgggggg caaggtgtgc 600cccaaagggg agtgtccatg gcaggtcctg
ttgttggtga atggagctca gttgtgtggg 660gggaccctga tcaacaccat
ctgggtggtc tccgcggccc actgtttcga caaaatcaag 720aactggagga
acctgatcgc ggtgctgggc gagcacgacc tcagcgagca cgacggggat
780gagcagagcc ggcgggtggc gcaggtcatc atccccagca cgtacgtccc
gggcaccacc 840aaccacgaca tcgcgctgct ccgcctgcac cagcccgtgg
tcctcactga ccatgtggtg 900cccctctgcc tgcccgaacg gacgttctct
gagaggacgc tggccttcgt gcgcttctca 960ttggtcagcg gctggggcca
gctgctggac cgtggcgcca cggccctgga gctcatggtc 1020ctcaacgtgc
cccggctgat gacccaggac tgcctgcagc agtcacggaa ggtgggagac
1080tccccaaata tcacggagta catgttctgt gccggctact cggatggcag
caaggactcc 1140tgcaaggggg acagtggagg cccacatgcc acccactacc
ggggcacgtg gtacctgacg 1200ggcatcgtca gctggggcca gggctgcgca
accgtgggcc actttggggt gtacaccagg 1260gtctcccagt acatcgagtg
gctgcaaaag ctcatgcgct cagagccacg cccaggagtc 1320ctcctgcgag
ccccatttcc ctgaggatgc ggccgc 135631444PRTArtificial SequenceFVII
31Met Val Ser Gln Ala Leu Arg Leu Leu Cys Leu Leu Leu Gly Leu Gln1
5 10 15Gly Cys Leu Ala Ala Val Phe Val Thr Gln Glu Glu Ala His Gly
Val 20 25 30Leu His Arg Arg Arg Arg Ala Asn Ala Phe Leu Glu Glu Leu
Arg Pro 35 40 45Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu Gln Cys Ser
Phe Glu Glu 50 55 60Ala Arg Glu Ile Phe Lys Asp Ala Glu Arg Thr Lys
Leu Phe Trp Ile65 70 75 80Ser Tyr Ser Asp Gly Asp Gln Cys Ala Ser
Ser Pro Cys Gln Asn Gly 85 90 95Gly Ser Cys Lys Asp Gln Leu Gln Ser
Tyr Ile Cys Phe Cys Leu Pro 100 105 110Ala Phe Glu Gly Arg Asn Cys
Glu Thr His Lys Asp Asp Gln Leu Ile 115 120 125Cys Val Asn Glu Asn
Gly Gly Cys Glu Gln Tyr Cys Ser Asp His Thr 130 135 140Gly Thr Lys
Arg Ser Cys Arg Cys His Glu Gly Tyr Ser Leu Leu Ala145 150 155
160Asp Gly Val Ser Cys Thr Pro Thr Val Glu Tyr Pro Cys Gly Lys Ile
165 170 175Pro Ile Leu Glu Lys Arg Asn Ala Ser Lys Pro Gln Gly Arg
Ile Val 180 185 190Gly Gly Lys Val Cys Pro Lys Gly Glu Cys Pro Trp
Gln Val Leu Leu 195 200 205Leu Val Asn Gly Ala Gln Leu Cys Gly Gly
Thr Leu Ile Asn Thr Ile 210 215 220Trp Val Val Ser Ala Ala His Cys
Phe Asp Lys Ile Lys Asn Trp Arg225 230 235 240Asn Leu Ile Ala Val
Leu Gly Glu His Asp Leu Ser Glu His Asp Gly 245 250 255Asp Glu Gln
Ser Arg Arg Val Ala Gln Val Ile Ile Pro Ser Thr Tyr 260 265 270Val
Pro Gly Thr Thr Asn His Asp Ile Ala Leu Leu Arg Leu His Gln 275 280
285Pro Val Val Leu Thr Asp His Val Val Pro Leu Cys Leu Pro Glu Arg
290 295 300Thr Phe Ser Glu Arg Thr Leu Ala Phe Val Arg Phe Ser Leu
Val Ser305 310 315 320Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala Thr
Ala Leu Glu Leu Met 325 330 335Val Leu Asn Val Pro Arg Leu Met Thr
Gln Asp Cys Leu Gln Gln Ser 340 345 350Arg Lys Val Gly Asp Ser Pro
Asn Ile Thr Glu Tyr Met Phe Cys Ala 355 360 365Gly Tyr Ser Asp Gly
Ser Lys Asp Ser Cys Lys Gly Asp Ser Gly Gly 370 375 380Pro His Ala
Thr His Tyr Arg Gly Thr Trp Tyr Leu Thr Gly Ile Val385 390 395
400Ser Trp Gly Gln Gly Cys Ala Thr Val Gly His Phe Gly Val Tyr Thr
405 410 415Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln Lys Leu Met Arg
Ser Glu 420 425 430Pro Arg Pro Gly Val Leu Leu Arg Ala Pro Phe Pro
435 44032448PRTArtificial SequenceFVII 32Met Val Ser Gln Ala Leu
Arg Leu Leu Cys Leu Leu Leu Gly Leu Gln1 5 10 15Gly Cys Leu Ala Ala
Val Phe Val Thr Gln Glu Glu Ala His Gly Val 20 25 30Leu His Arg Arg
Arg Arg Ala Asn Ala Phe Leu Glu Glu Leu Arg Pro 35 40 45Gly Ser Leu
Glu Arg Glu Cys Lys Glu Glu Gln Cys Ser Phe Glu Glu 50 55 60Ala Arg
Glu Ile Phe Lys Asp Ala Glu Arg Thr Lys Leu Phe Trp Ile65 70 75
80Ser Tyr Ser Asp Gly Asp Gln Cys Ala Ser Ser Pro Cys Gln Asn Gly
85 90 95Gly Ser Cys Lys Asp Gln Leu Gln Ser Tyr Ile Cys Phe Cys Leu
Pro 100 105 110Ala Phe Glu Gly Arg Asn Cys Glu Thr His Lys Asp Asp
Gln Leu Ile 115 120 125Cys Val Asn Glu Asn Gly Gly Cys Glu Gln Tyr
Cys Ser Asp His Thr 130 135 140Gly Thr Lys Arg Ser Cys Arg Cys His
Glu Gly Tyr Ser Leu Leu Ala145 150 155 160Asp Gly Val Ser Cys Thr
Pro Thr Val Glu Tyr Pro Cys Gly Lys Ile 165 170 175Pro Ile Leu Glu
Lys Arg Asn Ala Ser Lys Pro Gln Gly Arg Ile Val 180 185 190Gly Gly
Lys Val Cys Pro Lys Gly Glu Cys Pro Trp Gln Val Leu Leu 195 200
205Leu Val Asn Gly Ala Gln Leu Cys Gly Gly Thr Leu Ile Asn Thr Ile
210 215 220Trp Val Val Ser Ala Ala His Cys Phe Asp Lys Ile Lys Asn
Trp Arg225 230 235 240Asn Leu Ile Ala Val Leu Gly Glu His Asp Leu
Ser Glu His Asp Gly 245 250 255Asp Glu Gln Ser Arg Arg Val Ala Gln
Val Ile Ile Pro Ser Thr Tyr 260 265 270Val Pro Gly Thr Thr Asn His
Asp Ile Ala Leu Leu Arg Leu His Gln 275 280 285Pro Val Val Leu Thr
Asp His Val Val Pro Leu Cys Leu Pro Glu Arg 290 295 300Thr Phe Ser
Glu Arg Thr Leu Ala Phe Val Arg Phe Ser Leu Val Ser305 310 315
320Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala Thr Ala Leu Glu Leu Met
325 330 335Val Leu Asn Val Pro Arg Leu Met Thr Gln Asp Cys Leu Gln
Gln Ser 340 345 350Arg Lys Val Gly Asp Ser Pro Asn Ile Thr Glu Tyr
Met Phe Cys Ala 355 360 365Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys
Lys Gly Asp Ser Gly Gly 370 375 380Pro His Ala Thr His Tyr Arg Gly
Thr Trp Tyr Leu Thr Gly Ile Val385 390 395 400Ser Trp Gly Gln Gly
Cys Ala Thr Val Gly His Phe Gly Val Tyr Thr 405 410 415Arg Val Ser
Gln Tyr Ile Glu Trp Leu Gln Lys Leu Met Arg Ser Glu 420 425 430Pro
Arg Pro Gly Val Leu Leu Arg Ala Pro Phe Pro Gly Cys Gly Arg 435 440
445331442DNAArtificial SequenceFVII-CTP 33ctcgaggaca tggtctccca
ggccctcagg ctcctctgcc ttctgcttgg gcttcagggc 60tgcctggctg cagtcttcgt
aacccaggag gaagcccacg gcgtcctgca ccggcgccgg 120cgcgccaacg
cgttcctgga ggagctgcgg ccgggctccc tggagaggga gtgcaaggag
180gagcagtgct ccttcgagga ggcccgggag atcttcaagg acgcggagag
gacgaagctg 240ttctggattt cttacagtga tggggaccag tgtgcctcaa
gtccatgcca gaatgggggc 300tcctgcaagg accagctcca gtcctatatc
tgcttctgcc tccctgcctt cgagggccgg 360aactgtgaga cgcacaagga
tgaccagctg atctgtgtga acgagaacgg cggctgtgag 420cagtactgca
gtgaccacac gggcaccaag cgctcctgtc ggtgccacga ggggtactct
480ctgctggcag acggggtgtc ctgcacaccc acagttgaat atccatgtgg
aaaaatacct 540attctagaaa aaagaaatgc cagcaaaccc caaggccgaa
ttgtgggggg caaggtgtgc 600cccaaagggg agtgtccatg gcaggtcctg
ttgttggtga atggagctca gttgtgtggg 660gggaccctga tcaacaccat
ctgggtggtc tccgcggccc actgtttcga caaaatcaag 720aactggagga
acctgatcgc ggtgctgggc gagcacgacc tcagcgagca cgacggggat
780gagcagagcc ggcgggtggc gcaggtcatc atccccagca cgtacgtccc
gggcaccacc 840aaccacgaca tcgcgctgct ccgcctgcac cagcccgtgg
tcctcactga ccatgtggtg 900cccctctgcc tgcccgaacg gacgttctct
gagaggacgc tggccttcgt gcgcttctca 960ttggtcagcg gctggggcca
gctgctggac cgtggcgcca cggccctgga gctcatggtc 1020ctcaacgtgc
cccggctgat gacccaggac tgcctgcagc agtcacggaa ggtgggagac
1080tccccaaata tcacggagta catgttctgt gccggctact cggatggcag
caaggactcc 1140tgcaaggggg acagtggagg cccacatgcc acccactacc
ggggcacgtg gtacctgacc 1200ggcatcgtga gctggggcca gggctgcgcc
accgtgggcc acttcggcgt gtacaccagg 1260gtgtcccagt acatcgagtg
gctgcagaaa ctgatgagaa gcgagcccag acccggcgtg 1320ctgctgagag
cccccttccc cagcagcagc tccaaggccc ctccccctag cctgcccagc
1380cctagcagac tgcctgggcc cagcgacacc cccatcctgc cccagtgagg
atccgcggcc 1440gc 144234472PRTArtificial SequenceFVII-CTP 34Met Val
Ser Gln Ala Leu Arg Leu Leu Cys Leu Leu Leu Gly Leu Gln1 5 10 15Gly
Cys Leu Ala Ala Val Phe Val Thr Gln Glu Glu Ala His Gly Val 20 25
30Leu His Arg Arg Arg Arg Ala Asn Ala Phe Leu Glu Glu Leu Arg Pro
35 40 45Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu Gln Cys Ser Phe Glu
Glu 50 55 60Ala Arg Glu Ile Phe Lys Asp Ala Glu Arg Thr Lys Leu Phe
Trp Ile65 70 75 80Ser Tyr Ser Asp Gly Asp Gln Cys Ala Ser Ser Pro
Cys Gln Asn Gly 85 90 95Gly Ser Cys Lys Asp Gln Leu Gln Ser Tyr Ile
Cys Phe Cys Leu Pro 100 105 110Ala Phe Glu Gly Arg Asn Cys Glu Thr
His Lys Asp Asp Gln Leu Ile 115 120 125Cys Val Asn Glu Asn Gly Gly
Cys Glu Gln Tyr Cys Ser Asp His Thr 130 135 140Gly Thr Lys Arg Ser
Cys Arg Cys His Glu Gly Tyr Ser Leu Leu Ala145 150 155 160Asp Gly
Val Ser Cys Thr Pro Thr Val Glu Tyr Pro Cys Gly Lys Ile 165 170
175Pro Ile Leu Glu Lys Arg Asn Ala Ser Lys Pro Gln Gly Arg Ile Val
180 185 190Gly Gly Lys Val Cys Pro Lys Gly Glu Cys Pro Trp Gln Val
Leu Leu 195 200 205Leu Val Asn Gly Ala Gln Leu Cys Gly Gly Thr Leu
Ile Asn Thr Ile 210 215 220Trp Val Val Ser Ala Ala His Cys Phe Asp
Lys Ile Lys Asn Trp Arg225 230 235 240Asn Leu Ile Ala Val Leu Gly
Glu His Asp Leu Ser Glu His Asp Gly 245 250 255Asp Glu Gln Ser Arg
Arg Val Ala Gln Val Ile Ile Pro Ser Thr Tyr 260 265 270Val Pro Gly
Thr Thr Asn His Asp Ile Ala Leu Leu Arg Leu His Gln 275 280 285Pro
Val Val Leu Thr Asp His Val Val Pro Leu Cys Leu Pro Glu Arg 290 295
300Thr Phe Ser Glu Arg Thr Leu Ala Phe Val Arg Phe Ser Leu Val
Ser305 310 315 320Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala Thr Ala
Leu Glu Leu Met 325 330 335Val Leu Asn Val Pro Arg Leu Met Thr Gln
Asp Cys Leu Gln Gln Ser 340 345 350Arg Lys Val Gly Asp Ser Pro Asn
Ile Thr Glu Tyr Met Phe Cys Ala 355 360 365Gly Tyr Ser Asp Gly Ser
Lys Asp Ser Cys Lys Gly Asp Ser Gly Gly 370 375 380Pro His Ala Thr
His Tyr Arg Gly Thr Trp Tyr Leu Thr Gly Ile Val385 390 395 400Ser
Trp Gly Gln Gly Cys Ala Thr Val Gly His Phe Gly Val Tyr Thr 405 410
415Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln Lys Leu Met Arg Ser Glu
420 425 430Pro Arg Pro Gly Val Leu Leu Arg Ala Pro Phe Pro Ser Ser
Ser Ser 435 440 445Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg
Leu Pro Gly Pro 450 455 460Ser Asp Thr Pro Ile Leu Pro Gln465
470351535DNAArtificial SequenceFVII-CTP-CTP 35ctcgaggaca tggtctccca
ggccctcagg ctcctctgcc ttctgcttgg gcttcagggc 60tgcctggctg cagtcttcgt
aacccaggag gaagcccacg gcgtcctgca ccggcgccgg 120cgcgccaacg
cgttcctgga ggagctgcgg ccgggctccc tggagaggga gtgcaaggag
180gagcagtgct ccttcgagga ggcccgggag atcttcaagg acgcggagag
gacgaagctg 240ttctggattt cttacagtga tggggaccag tgtgcctcaa
gtccatgcca gaatgggggc 300tcctgcaagg accagctcca gtcctatatc
tgcttctgcc tccctgcctt cgagggccgg 360aactgtgaga cgcacaagga
tgaccagctg atctgtgtga acgagaacgg cggctgtgag 420cagtactgca
gtgaccacac gggcaccaag cgctcctgtc ggtgccacga ggggtactct
480ctgctggcag acggggtgtc ctgcacaccc acagttgaat atccatgtgg
aaaaatacct 540attctagaaa aaagaaatgc cagcaaaccc caaggccgaa
ttgtgggggg caaggtgtgc 600cccaaagggg agtgtccatg gcaggtcctg
ttgttggtga atggagctca gttgtgtggg 660gggaccctga tcaacaccat
ctgggtggtc tccgcggccc actgtttcga caaaatcaag 720aactggagga
acctgatcgc ggtgctgggc gagcacgacc tcagcgagca cgacggggat
780gagcagagcc ggcgggtggc gcaggtcatc atccccagca cgtacgtccc
gggcaccacc 840aaccacgaca tcgcgctgct ccgcctgcac cagcccgtgg
tcctcactga ccatgtggtg 900cccctctgcc tgcccgaacg gacgttctct
gagaggacgc tggccttcgt gcgcttctca 960ttggtcagcg gctggggcca
gctgctggac cgtggcgcca cggccctgga gctcatggtc 1020ctcaacgtgc
cccggctgat
gacccaggac tgcctgcagc agtcacggaa ggtgggagac 1080tccccaaata
tcacggagta catgttctgt gccggctact cggatggcag caaggactcc
1140tgcaaggggg acagtggagg cccacatgcc acccactacc ggggcacgtg
gtacctgacc 1200ggcatcgtga gctggggcca gggctgcgcc accgtgggcc
acttcggcgt gtacaccagg 1260gtgtcccagt acatcgagtg gctgcagaaa
ctgatgagaa gcgagcccag acccggcgtg 1320ctgctgagag cccccttccc
cagcagcagc tccaaggccc ctccccctag cctgcccagc 1380cctagcagac
tgcctgggcc ctccgacaca ccaatcctgc cacagagcag ctcctctaag
1440gcccctcctc catccctgcc atccccctcc cggctgccag gcccctctga
cacccctatc 1500ctgcctcagt gatgaaggtc tggatccgcg gccgc
153536500PRTArtificial SequenceFVII-CTP-CTP 36Met Val Ser Gln Ala
Leu Arg Leu Leu Cys Leu Leu Leu Gly Leu Gln1 5 10 15Gly Cys Leu Ala
Ala Val Phe Val Thr Gln Glu Glu Ala His Gly Val 20 25 30Leu His Arg
Arg Arg Arg Ala Asn Ala Phe Leu Glu Glu Leu Arg Pro 35 40 45Gly Ser
Leu Glu Arg Glu Cys Lys Glu Glu Gln Cys Ser Phe Glu Glu 50 55 60Ala
Arg Glu Ile Phe Lys Asp Ala Glu Arg Thr Lys Leu Phe Trp Ile65 70 75
80Ser Tyr Ser Asp Gly Asp Gln Cys Ala Ser Ser Pro Cys Gln Asn Gly
85 90 95Gly Ser Cys Lys Asp Gln Leu Gln Ser Tyr Ile Cys Phe Cys Leu
Pro 100 105 110Ala Phe Glu Gly Arg Asn Cys Glu Thr His Lys Asp Asp
Gln Leu Ile 115 120 125Cys Val Asn Glu Asn Gly Gly Cys Glu Gln Tyr
Cys Ser Asp His Thr 130 135 140Gly Thr Lys Arg Ser Cys Arg Cys His
Glu Gly Tyr Ser Leu Leu Ala145 150 155 160Asp Gly Val Ser Cys Thr
Pro Thr Val Glu Tyr Pro Cys Gly Lys Ile 165 170 175Pro Ile Leu Glu
Lys Arg Asn Ala Ser Lys Pro Gln Gly Arg Ile Val 180 185 190Gly Gly
Lys Val Cys Pro Lys Gly Glu Cys Pro Trp Gln Val Leu Leu 195 200
205Leu Val Asn Gly Ala Gln Leu Cys Gly Gly Thr Leu Ile Asn Thr Ile
210 215 220Trp Val Val Ser Ala Ala His Cys Phe Asp Lys Ile Lys Asn
Trp Arg225 230 235 240Asn Leu Ile Ala Val Leu Gly Glu His Asp Leu
Ser Glu His Asp Gly 245 250 255Asp Glu Gln Ser Arg Arg Val Ala Gln
Val Ile Ile Pro Ser Thr Tyr 260 265 270Val Pro Gly Thr Thr Asn His
Asp Ile Ala Leu Leu Arg Leu His Gln 275 280 285Pro Val Val Leu Thr
Asp His Val Val Pro Leu Cys Leu Pro Glu Arg 290 295 300Thr Phe Ser
Glu Arg Thr Leu Ala Phe Val Arg Phe Ser Leu Val Ser305 310 315
320Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala Thr Ala Leu Glu Leu Met
325 330 335Val Leu Asn Val Pro Arg Leu Met Thr Gln Asp Cys Leu Gln
Gln Ser 340 345 350Arg Lys Val Gly Asp Ser Pro Asn Ile Thr Glu Tyr
Met Phe Cys Ala 355 360 365Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys
Lys Gly Asp Ser Gly Gly 370 375 380Pro His Ala Thr His Tyr Arg Gly
Thr Trp Tyr Leu Thr Gly Ile Val385 390 395 400Ser Trp Gly Gln Gly
Cys Ala Thr Val Gly His Phe Gly Val Tyr Thr 405 410 415Arg Val Ser
Gln Tyr Ile Glu Trp Leu Gln Lys Leu Met Arg Ser Glu 420 425 430Pro
Arg Pro Gly Val Leu Leu Arg Ala Pro Phe Pro Ser Ser Ser Ser 435 440
445Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro
450 455 460Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala
Pro Pro465 470 475 480Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly
Pro Ser Asp Thr Pro 485 490 495Ile Leu Pro Gln
500371621DNAArtificial SequenceFVII-CTP-CTP-CTP (MOD-5014) with SP
37ctcgaggaca tggtctccca ggccctcagg ctcctctgcc ttctgcttgg gcttcagggc
60tgcctggctg cagtcttcgt aacccaggag gaagcccacg gcgtcctgca ccggcgccgg
120cgcgccaacg cgttcctgga ggagctgcgg ccgggctccc tggagaggga
gtgcaaggag 180gagcagtgct ccttcgagga ggcccgggag atcttcaagg
acgcggagag gacgaagctg 240ttctggattt cttacagtga tggggaccag
tgtgcctcaa gtccatgcca gaatgggggc 300tcctgcaagg accagctcca
gtcctatatc tgcttctgcc tccctgcctt cgagggccgg 360aactgtgaga
cgcacaagga tgaccagctg atctgtgtga acgagaacgg cggctgtgag
420cagtactgca gtgaccacac gggcaccaag cgctcctgtc ggtgccacga
ggggtactct 480ctgctggcag acggggtgtc ctgcacaccc acagttgaat
atccatgtgg aaaaatacct 540attctagaaa aaagaaatgc cagcaaaccc
caaggccgaa ttgtgggggg caaggtgtgc 600cccaaagggg agtgtccatg
gcaggtcctg ttgttggtga atggagctca gttgtgtggg 660gggaccctga
tcaacaccat ctgggtggtc tccgcggccc actgtttcga caaaatcaag
720aactggagga acctgatcgc ggtgctgggc gagcacgacc tcagcgagca
cgacggggat 780gagcagagcc ggcgggtggc gcaggtcatc atccccagca
cgtacgtccc gggcaccacc 840aaccacgaca tcgcgctgct ccgcctgcac
cagcccgtgg tcctcactga ccatgtggtg 900cccctctgcc tgcccgaacg
gacgttctct gagaggacgc tggccttcgt gcgcttctca 960ttggtcagcg
gctggggcca gctgctggac cgtggcgcca cggccctgga gctcatggtc
1020ctcaacgtgc cccggctgat gacccaggac tgcctgcagc agtcacggaa
ggtgggagac 1080tccccaaata tcacggagta catgttctgt gccggctact
cggatggcag caaggactcc 1140tgcaaggggg acagtggagg cccacatgcc
acccactacc ggggcacgtg gtacctgacc 1200ggcatcgtga gctggggcca
gggctgcgcc accgtgggcc acttcggcgt gtacaccagg 1260gtgtcccagt
acatcgagtg gctgcagaaa ctgatgagaa gcgagcccag acccggcgtg
1320ctgctgagag cccccttccc cagcagcagc tccaaggccc ctccccctag
cctgcccagc 1380cctagcagac tgcctgggcc cagtgacacc cctatcctgc
ctcagtccag ctccagcaag 1440gccccacccc ctagcctgcc ttctccttct
cggctgcctg gccccagcga tactccaatt 1500ctgccccagt cctccagcag
taaggctccc cctccatctc tgccatcccc cagcagactg 1560ccaggccctt
ctgatacacc catcctccca cagtgatgag gatccgcggc cgcttaatta 1620a
162138528PRTArtificial SequenceFVII-CTP-CTP-CTP (MOD-5014) with
Signal Peptide 38Met Val Ser Gln Ala Leu Arg Leu Leu Cys Leu Leu
Leu Gly Leu Gln1 5 10 15Gly Cys Leu Ala Ala Val Phe Val Thr Gln Glu
Glu Ala His Gly Val 20 25 30Leu His Arg Arg Arg Arg Ala Asn Ala Phe
Leu Glu Glu Leu Arg Pro 35 40 45Gly Ser Leu Glu Arg Glu Cys Lys Glu
Glu Gln Cys Ser Phe Glu Glu 50 55 60Ala Arg Glu Ile Phe Lys Asp Ala
Glu Arg Thr Lys Leu Phe Trp Ile65 70 75 80Ser Tyr Ser Asp Gly Asp
Gln Cys Ala Ser Ser Pro Cys Gln Asn Gly 85 90 95Gly Ser Cys Lys Asp
Gln Leu Gln Ser Tyr Ile Cys Phe Cys Leu Pro 100 105 110Ala Phe Glu
Gly Arg Asn Cys Glu Thr His Lys Asp Asp Gln Leu Ile 115 120 125Cys
Val Asn Glu Asn Gly Gly Cys Glu Gln Tyr Cys Ser Asp His Thr 130 135
140Gly Thr Lys Arg Ser Cys Arg Cys His Glu Gly Tyr Ser Leu Leu
Ala145 150 155 160Asp Gly Val Ser Cys Thr Pro Thr Val Glu Tyr Pro
Cys Gly Lys Ile 165 170 175Pro Ile Leu Glu Lys Arg Asn Ala Ser Lys
Pro Gln Gly Arg Ile Val 180 185 190Gly Gly Lys Val Cys Pro Lys Gly
Glu Cys Pro Trp Gln Val Leu Leu 195 200 205Leu Val Asn Gly Ala Gln
Leu Cys Gly Gly Thr Leu Ile Asn Thr Ile 210 215 220Trp Val Val Ser
Ala Ala His Cys Phe Asp Lys Ile Lys Asn Trp Arg225 230 235 240Asn
Leu Ile Ala Val Leu Gly Glu His Asp Leu Ser Glu His Asp Gly 245 250
255Asp Glu Gln Ser Arg Arg Val Ala Gln Val Ile Ile Pro Ser Thr Tyr
260 265 270Val Pro Gly Thr Thr Asn His Asp Ile Ala Leu Leu Arg Leu
His Gln 275 280 285Pro Val Val Leu Thr Asp His Val Val Pro Leu Cys
Leu Pro Glu Arg 290 295 300Thr Phe Ser Glu Arg Thr Leu Ala Phe Val
Arg Phe Ser Leu Val Ser305 310 315 320Gly Trp Gly Gln Leu Leu Asp
Arg Gly Ala Thr Ala Leu Glu Leu Met 325 330 335Val Leu Asn Val Pro
Arg Leu Met Thr Gln Asp Cys Leu Gln Gln Ser 340 345 350Arg Lys Val
Gly Asp Ser Pro Asn Ile Thr Glu Tyr Met Phe Cys Ala 355 360 365Gly
Tyr Ser Asp Gly Ser Lys Asp Ser Cys Lys Gly Asp Ser Gly Gly 370 375
380Pro His Ala Thr His Tyr Arg Gly Thr Trp Tyr Leu Thr Gly Ile
Val385 390 395 400Ser Trp Gly Gln Gly Cys Ala Thr Val Gly His Phe
Gly Val Tyr Thr 405 410 415Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln
Lys Leu Met Arg Ser Glu 420 425 430Pro Arg Pro Gly Val Leu Leu Arg
Ala Pro Phe Pro Ser Ser Ser Ser 435 440 445Lys Ala Pro Pro Pro Ser
Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro 450 455 460Ser Asp Thr Pro
Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro465 470 475 480Pro
Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro 485 490
495Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro
500 505 510Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu
Pro Gln 515 520 525391607DNAArtificial SequenceFVII-CTP4X
39ctcgaggaca tggtctccca ggccctcagg ctcctctgcc ttctgcttgg gcttcagggc
60tgcctggctg cagtcttcgt aacccaggag gaagcccacg gcgtcctgca ccggcgccgg
120cgcgccaacg cgttcctgga ggagctgcgg ccgggctccc tggagaggga
gtgcaaggag 180gagcagtgct ccttcgagga ggcccgggag atcttcaagg
acgcggagag gacgaagctg 240ttctggattt cttacagtga tggggaccag
tgtgcctcaa gtccatgcca gaatgggggc 300tcctgcaagg accagctcca
gtcctatatc tgcttctgcc tccctgcctt cgagggccgg 360aactgtgaga
cgcacaagga tgaccagctg atctgtgtga acgagaacgg cggctgtgag
420cagtactgca gtgaccacac gggcaccaag cgctcctgtc ggtgccacga
ggggtactct 480ctgctggcag acggggtgtc ctgcacaccc acagttgaat
atccatgtgg aaaaatacct 540attctagaaa aaagaaatgc cagcaaaccc
caaggccgaa ttgtgggggg caaggtgtgc 600cccaaagggg agtgtccatg
gcaggtcctg ttgttggtga atggagctca gttgtgtggg 660gggaccctga
tcaacaccat ctgggtggtc tccgcggccc actgtttcga caaaatcaag
720aactggagga acctgatcgc ggtgctgggc gagcacgacc tcagcgagca
cgacggggat 780gagcagagcc ggcgggtggc gcaggtcatc atccccagca
cgtacgtccc gggcaccacc 840aaccacgaca tcgcgctgct ccgcctgcac
cagcccgtgg tcctcactga ccatgtggtg 900cccctctgcc tgcccgaacg
gacgttctct gagaggacgc tggccttcgt gcgcttctca 960ttggtcagcg
gctggggcca gctgctggac cgtggcgcca cggccctgga gctcatggtc
1020ctcaacgtgc cccggctgat gacccaggac tgcctgcagc agtcacggaa
ggtgggagac 1080tccccaaata tcacggagta catgttctgt gccggctact
cggatggcag caaggactcc 1140tgcaaggggg acagtggagg cccacatgcc
acccactacc ggggcacgtg gtacctgacc 1200ggcatcgtga gctggggcca
gggctgcgcc accgtgggcc acttcggcgt gtacaccagg 1260gtgtcccagt
acatcgagtg gctgcagaaa ctgatgagaa gcgagcccag acccggcgtg
1320ctgctgagag cccccttccc cagcagcagc tccaaggccc ctccccctag
cctgcccagc 1380cctagcagac tgcctgggcc cagtgacacc cctatcctgc
ctcagtccag ctccagcaag 1440gccccacccc ctagcctgcc ttctccttct
cggctgcctg gccccagcga tactccaatt 1500ctgccccagt cctccagcag
taaggctccc cctccatctc tgccatcccc cagcagactg 1560ccaggccctt
ctgatacacc catcctccca cagtgatgag gatccgc 160740532PRTArtificial
SequenceFVII-CTP4x 40Leu Glu Asp Met Val Ser Gln Ala Leu Arg Leu
Leu Cys Leu Leu Leu1 5 10 15Gly Leu Gln Gly Cys Leu Ala Ala Val Phe
Val Thr Gln Glu Glu Ala 20 25 30His Gly Val Leu His Arg Arg Arg Arg
Ala Asn Ala Phe Leu Glu Glu 35 40 45Leu Arg Pro Gly Ser Leu Glu Arg
Glu Cys Lys Glu Glu Gln Cys Ser 50 55 60Phe Glu Glu Ala Arg Glu Ile
Phe Lys Asp Ala Glu Arg Thr Lys Leu65 70 75 80Phe Trp Ile Ser Tyr
Ser Asp Gly Asp Gln Cys Ala Ser Ser Pro Cys 85 90 95Gln Asn Gly Gly
Ser Cys Lys Asp Gln Leu Gln Ser Tyr Ile Cys Phe 100 105 110Cys Leu
Pro Ala Phe Glu Gly Arg Asn Cys Glu Thr His Lys Asp Asp 115 120
125Gln Leu Ile Cys Val Asn Glu Asn Gly Gly Cys Glu Gln Tyr Cys Ser
130 135 140Asp His Thr Gly Thr Lys Arg Ser Cys Arg Cys His Glu Gly
Tyr Ser145 150 155 160Leu Leu Ala Asp Gly Val Ser Cys Thr Pro Thr
Val Glu Tyr Pro Cys 165 170 175Gly Lys Ile Pro Ile Leu Glu Lys Arg
Asn Ala Ser Lys Pro Gln Gly 180 185 190Arg Ile Val Gly Gly Lys Val
Cys Pro Lys Gly Glu Cys Pro Trp Gln 195 200 205Val Leu Leu Leu Val
Asn Gly Ala Gln Leu Cys Gly Gly Thr Leu Ile 210 215 220Asn Thr Ile
Trp Val Val Ser Ala Ala His Cys Phe Asp Lys Ile Lys225 230 235
240Asn Trp Arg Asn Leu Ile Ala Val Leu Gly Glu His Asp Leu Ser Glu
245 250 255His Asp Gly Asp Glu Gln Ser Arg Arg Val Ala Gln Val Ile
Ile Pro 260 265 270Ser Thr Tyr Val Pro Gly Thr Thr Asn His Asp Ile
Ala Leu Leu Arg 275 280 285Leu His Gln Pro Val Val Leu Thr Asp His
Val Val Pro Leu Cys Leu 290 295 300Pro Glu Arg Thr Phe Ser Glu Arg
Thr Leu Ala Phe Val Arg Phe Ser305 310 315 320Leu Val Ser Gly Trp
Gly Gln Leu Leu Asp Arg Gly Ala Thr Ala Leu 325 330 335Glu Leu Met
Val Leu Asn Val Pro Arg Leu Met Thr Gln Asp Cys Leu 340 345 350Gln
Gln Ser Arg Lys Val Gly Asp Ser Pro Asn Ile Thr Glu Tyr Met 355 360
365Phe Cys Ala Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys Lys Gly Asp
370 375 380Ser Gly Gly Pro His Ala Thr His Tyr Arg Gly Thr Trp Tyr
Leu Thr385 390 395 400Gly Ile Val Ser Trp Gly Gln Gly Cys Ala Thr
Val Gly His Phe Gly 405 410 415Val Tyr Thr Arg Val Ser Gln Tyr Ile
Glu Trp Leu Gln Lys Leu Met 420 425 430Arg Ser Glu Pro Arg Pro Gly
Val Leu Leu Arg Ala Pro Phe Pro Ser 435 440 445Ser Ser Ser Lys Ala
Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu 450 455 460Pro Gly Pro
Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys465 470 475
480Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser
485 490 495Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro
Pro Pro 500 505 510Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser
Asp Thr Pro Ile 515 520 525Leu Pro Gln Gly 530411775DNAArtificial
SequenceFVII-CTP5x 41ctcgaggaca tggtctccca ggccctcagg ctcctctgcc
ttctgcttgg gcttcagggc 60tgcctggctg cagtcttcgt aacccaggag gaagcccacg
gcgtcctgca ccggcgccgg 120cgcgccaacg cgttcctgga ggagctgcgg
ccgggctccc tggagaggga gtgcaaggag 180gagcagtgct ccttcgagga
ggcccgggag atcttcaagg acgcggagag gacgaagctg 240ttctggattt
cttacagtga tggggaccag tgtgcctcaa gtccatgcca gaatgggggc
300tcctgcaagg accagctcca gtcctatatc tgcttctgcc tccctgcctt
cgagggccgg 360aactgtgaga cgcacaagga tgaccagctg atctgtgtga
acgagaacgg cggctgtgag 420cagtactgca gtgaccacac gggcaccaag
cgctcctgtc ggtgccacga ggggtactct 480ctgctggcag acggggtgtc
ctgcacaccc acagttgaat atccatgtgg aaaaatacct 540attctagaaa
aaagaaatgc cagcaaaccc caaggccgaa ttgtgggggg caaggtgtgc
600cccaaagggg agtgtccatg gcaggtcctg ttgttggtga atggagctca
gttgtgtggg 660gggaccctga tcaacaccat ctgggtggtc tccgcggccc
actgtttcga caaaatcaag 720aactggagga acctgatcgc ggtgctgggc
gagcacgacc tcagcgagca cgacggggat 780gagcagagcc ggcgggtggc
gcaggtcatc atccccagca cgtacgtccc gggcaccacc 840aaccacgaca
tcgcgctgct ccgcctgcac cagcccgtgg tcctcactga ccatgtggtg
900cccctctgcc tgcccgaacg gacgttctct gagaggacgc tggccttcgt
gcgcttctca 960ttggtcagcg gctggggcca gctgctggac cgtggcgcca
cggccctgga gctcatggtc 1020ctcaacgtgc cccggctgat gacccaggac
tgcctgcagc agtcacggaa ggtgggagac 1080tccccaaata tcacggagta
catgttctgt gccggctact cggatggcag caaggactcc 1140tgcaaggggg
acagtggagg cccacatgcc acccactacc ggggcacgtg gtacctgacc
1200ggcatcgtga gctggggcca gggctgcgcc accgtgggcc acttcggcgt
gtacaccagg 1260gtgtcccagt acatcgagtg gctgcagaaa ctgatgagaa
gcgagcccag acccggcgtg 1320ctgctgagag cccccttccc cagcagcagc
tccaaggccc ctccccctag cctgcccagc 1380cctagcagac tgcctgggcc
ctctgacacc cctatcctgc ctcagtccag ctcctctaag 1440gctccaccac
cttccctgcc
tagcccttca agactgccag gccctagcga tacaccaatt 1500ctgccccagt
cctccagcag caaggctccc ccacctagcc tgccttctcc atcaaggctg
1560cctggcccat ccgatacccc aattttgcct cagagcagct ctagcaaggc
acctcccccc 1620agtctgccct ctccaagcag actccctggc ccttcagaca
ctccaatcct cccacagtcc 1680tctagctcta aagctccacc tcccagcctg
cccagcccta gtagactccc cggaccttct 1740gataccccca tcttgcccca
gtgatgagga tccgc 177542589PRTArtificial SequenceFVII-CTP5x 42Leu
Glu Asp Met Val Ser Gln Ala Leu Arg Leu Leu Cys Leu Leu Leu1 5 10
15Gly Leu Gln Gly Cys Leu Ala Ala Val Phe Val Thr Gln Glu Glu Ala
20 25 30His Gly Val Leu His Arg Arg Arg Arg Ala Asn Ala Phe Leu Glu
Glu 35 40 45Leu Arg Pro Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu Gln
Cys Ser 50 55 60Phe Glu Glu Ala Arg Glu Ile Phe Lys Asp Ala Glu Arg
Thr Lys Leu65 70 75 80Phe Trp Ile Ser Tyr Ser Asp Gly Asp Gln Cys
Ala Ser Ser Pro Cys 85 90 95Gln Asn Gly Gly Ser Cys Lys Asp Gln Leu
Gln Ser Tyr Ile Cys Phe 100 105 110Cys Leu Pro Ala Phe Glu Gly Arg
Asn Cys Glu Thr His Lys Asp Asp 115 120 125Gln Leu Ile Cys Val Asn
Glu Asn Gly Gly Cys Glu Gln Tyr Cys Ser 130 135 140Asp His Thr Gly
Thr Lys Arg Ser Cys Arg Cys His Glu Gly Tyr Ser145 150 155 160Leu
Leu Ala Asp Gly Val Ser Cys Thr Pro Thr Val Glu Tyr Pro Cys 165 170
175Gly Lys Ile Pro Ile Leu Glu Lys Arg Asn Ala Ser Lys Pro Gln Gly
180 185 190Arg Ile Val Gly Gly Lys Val Cys Pro Lys Gly Glu Cys Pro
Trp Gln 195 200 205Val Leu Leu Leu Val Asn Gly Ala Gln Leu Cys Gly
Gly Thr Leu Ile 210 215 220Asn Thr Ile Trp Val Val Ser Ala Ala His
Cys Phe Asp Lys Ile Lys225 230 235 240Asn Trp Arg Asn Leu Ile Ala
Val Leu Gly Glu His Asp Leu Ser Glu 245 250 255His Asp Gly Asp Glu
Gln Ser Arg Arg Val Ala Gln Val Ile Ile Pro 260 265 270Ser Thr Tyr
Val Pro Gly Thr Thr Asn His Asp Ile Ala Leu Leu Arg 275 280 285Leu
His Gln Pro Val Val Leu Thr Asp His Val Val Pro Leu Cys Leu 290 295
300Pro Glu Arg Thr Phe Ser Glu Arg Thr Leu Ala Phe Val Arg Phe
Ser305 310 315 320Leu Val Ser Gly Trp Gly Gln Leu Leu Asp Arg Gly
Ala Thr Ala Leu 325 330 335Glu Leu Met Val Leu Asn Val Pro Arg Leu
Met Thr Gln Asp Cys Leu 340 345 350Gln Gln Ser Arg Lys Val Gly Asp
Ser Pro Asn Ile Thr Glu Tyr Met 355 360 365Phe Cys Ala Gly Tyr Ser
Asp Gly Ser Lys Asp Ser Cys Lys Gly Asp 370 375 380Ser Gly Gly Pro
His Ala Thr His Tyr Arg Gly Thr Trp Tyr Leu Thr385 390 395 400Gly
Ile Val Ser Trp Gly Gln Gly Cys Ala Thr Val Gly His Phe Gly 405 410
415Val Tyr Thr Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln Lys Leu Met
420 425 430Arg Ser Glu Pro Arg Pro Gly Val Leu Leu Arg Ala Pro Phe
Pro Ser 435 440 445Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser
Pro Ser Arg Leu 450 455 460Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro
Gln Ser Ser Ser Ser Lys465 470 475 480Ala Pro Pro Pro Ser Leu Pro
Ser Pro Ser Arg Leu Pro Gly Pro Ser 485 490 495Asp Thr Pro Ile Leu
Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro 500 505 510Ser Leu Pro
Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile 515 520 525Leu
Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser 530 535
540Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln
Ser545 550 555 560Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser
Pro Ser Arg Leu 565 570 575Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro
Gln Gly Ser 580 585431404DNAArtificial SequenceFIX 43gcgatcgcca
tgcagcgcgt gaacatgatc atggcagaat caccaggcct catcaccatt 60gccttttagg
atatctactc agtgctgaat gtacagtttt tcttgatcat gaaaacgcca
120acaaaattct gaatcggcca aagaggtata attcaggtaa attggaagag
tttgttcaag 180ggaaccttga gagagaatgt atggaagaaa agtgtagttt
tgaagaagca cgagaagttt 240ttgaaaacac tgaaagaaca actgaatttt
ggaagcagta tgttgatgga gatcagtgtg 300agtccaatcc atgtttaaat
ggcggcagtt gcaaggatga cattaattcc tatgaatgtt 360ggtgtccctt
tggatttgaa ggaaagaact gtgaattaga tgtaacatgt aacattaaga
420atggcagatg cgagcagttt tgtaaaaata gtgctgataa caaggtggtt
tgctcctgta 480ctgagggata tcgacttgca gaaaaccaga agtcctgtga
accagcagtg ccatttccat 540gtggaagagt ttctgtttca caaacttcta
agctcacccg tgctgagact gtttttcctg 600atgtggacta tgtaaattct
actgaagctg aaaccatttt ggataacatc actcaaagca 660cccaatcatt
taatgacttc actcgagttg ttggtggaga agatgccaaa ccaggtcaat
720tcccttggca ggttgttttg aatggtaaag ttgatgcatt ctgtggaggc
tctatcgtta 780atgaaaaatg gattgtaact gctgcccact gtgttgaaac
tggtgttaaa attacagttg 840tcgcaggtga acataatatt gaggagacag
aacatacaga gcaaaagcga aatgtgattc 900gaattattcc tcaccacaac
tacaatgcag ctattaataa gtacaaccat gacattgccc 960ttctggaact
ggacgaaccc ttagtgctaa acagctacgt tacacctatt tgcattgctg
1020acaaggaata cacgaacatc ttcctcaaat ttggatctgg ctatgtaagt
ggctggggaa 1080gagtcttcca caaagggaga tcagctttag ttctccagta
ccttagagtt ccacttgttg 1140accgagccac atgtcttcga tctacaaagt
tcaccatcta taacaacatg ttctgtgctg 1200gcttccatga aggaggtaga
gattcatgtc aaggagatag tgggggaccc catgttactg 1260aagtggaagg
gaccagtttc ttaactggaa ttattagctg gggtgaagag tgtgcaatga
1320aaggcaaata tggaatatat accaaggtat cccggtatgt caactggatt
aaggaaaaaa 1380caaagctcac ttgaacgcgg ccgc 140444461PRTArtificial
SequenceFIX 44Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly
Leu Ile Thr1 5 10 15Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys
Thr Val Phe Leu 20 25 30Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg
Pro Lys Arg Tyr Asn 35 40 45Ser Gly Lys Leu Glu Glu Phe Val Gln Gly
Asn Leu Glu Arg Glu Cys 50 55 60Met Glu Glu Lys Cys Ser Phe Glu Glu
Ala Arg Glu Val Phe Glu Asn65 70 75 80Thr Glu Arg Thr Thr Glu Phe
Trp Lys Gln Tyr Val Asp Gly Asp Gln 85 90 95Cys Glu Ser Asn Pro Cys
Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile 100 105 110Asn Ser Tyr Glu
Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys 115 120 125Glu Leu
Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe 130 135
140Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu
Gly145 150 155 160Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro
Ala Val Pro Phe 165 170 175Pro Cys Gly Arg Val Ser Val Ser Gln Thr
Ser Lys Leu Thr Arg Ala 180 185 190Glu Thr Val Phe Pro Asp Val Asp
Tyr Val Asn Ser Thr Glu Ala Glu 195 200 205Thr Ile Leu Asp Asn Ile
Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe 210 215 220Thr Arg Val Val
Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp225 230 235 240Gln
Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile 245 250
255Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly
260 265 270Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu
Thr Glu 275 280 285His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile
Pro His His Asn 290 295 300Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His
Asp Ile Ala Leu Leu Glu305 310 315 320Leu Asp Glu Pro Leu Val Leu
Asn Ser Tyr Val Thr Pro Ile Cys Ile 325 330 335Ala Asp Lys Glu Tyr
Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr 340 345 350Val Ser Gly
Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val 355 360 365Leu
Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg 370 375
380Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe
His385 390 395 400Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly
Gly Pro His Val 405 410 415Thr Glu Val Glu Gly Thr Ser Phe Leu Thr
Gly Ile Ile Ser Trp Gly 420 425 430Glu Glu Cys Ala Met Lys Gly Lys
Tyr Gly Ile Tyr Thr Lys Val Ser 435 440 445Arg Tyr Val Asn Trp Ile
Lys Glu Lys Thr Lys Leu Thr 450 455 460451502DNAArtificial
SequenceFIX-CTP 45gcgatcgcca tgcagcgcgt gaacatgatc atggcagaat
caccaggcct catcaccatc 60tgccttttag gatatctact cagtgctgaa tgtacagttt
ttcttgatca tgaaaacgcc 120aacaaaattc tgaatcggcc aaagaggtat
aattcaggta aattggaaga gtttgttcaa 180gggaaccttg agagagaatg
tatggaagaa aagtgtagtt ttgaagaagc acgagaagtt 240tttgaaaaca
ctgaaagaac aactgaattt tggaagcagt atgttgatgg agatcagtgt
300gagtccaatc catgtttaaa tggcggcagt tgcaaggatg acattaattc
ctatgaatgt 360tggtgtccct ttggatttga aggaaagaac tgtgaattag
atgtaacatg taacattaag 420aatggcagat gcgagcagtt ttgtaaaaat
agtgctgata acaaggtggt ttgctcctgt 480actgagggat atcgacttgc
agaaaaccag aagtcctgtg aaccagcagt gccatttcca 540tgtggaagag
tttctgtttc acaaacttct aagctcaccc gtgctgagac tgtttttcct
600gatgtggact atgtaaattc tactgaagct gaaaccattt tggataacat
cactcaaagc 660acccaatcat ttaatgactt cactcgagtt gttggtggag
aagatgccaa accaggtcaa 720ttcccttggc aggttgtttt gaatggtaaa
gttgatgcat tctgtggagg ctctatcgtt 780aatgaaaaat ggattgtaac
tgctgcccac tgtgttgaaa ctggtgttaa aattacagtt 840gtcgcaggtg
aacataatat tgaggagaca gaacatacag agcaaaagcg aaatgtgatt
900cgaattattc ctcaccacaa ctacaatgca gctattaata agtacaacca
tgacattgcc 960cttctggaac tggacgaacc cttagtgcta aacagctacg
ttacacctat ttgcattgct 1020gacaaggaat acacgaacat cttcctcaaa
tttggatctg gctatgtaag tggctgggga 1080agagtcttcc acaaagggag
atcagcttta gttcttcagt accttagagt tccacttgtt 1140gaccgagcca
catgtcttcg atctacaaag ttcaccatct ataacaacat gttctgtgct
1200ggcttccatg aaggaggtag agattcatgt caaggagata gtgggggacc
ccatgttact 1260gaagtggaag ggaccagttt cttaactgga attattagct
ggggtgaaga gtgtgcaatg 1320aaaggcaaat atggaatata taccaaggta
tcccggtatg tcaactggat taaggaaaaa 1380acaaagctca ctagctccag
cagcaaggcc cctcccccga gcctgccctc cccaagcagg 1440ctgcctgggc
cctccgacac accaatcctg ccacagtgat gaaggtctgg atccgcggcc 1500gc
150246489PRTArtificial SequenceFIX-CTP 46Met Gln Arg Val Asn Met
Ile Met Ala Glu Ser Pro Gly Leu Ile Thr1 5 10 15Ile Cys Leu Leu Gly
Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu 20 25 30Asp His Glu Asn
Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn 35 40 45Ser Gly Lys
Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys 50 55 60Met Glu
Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn65 70 75
80Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln
85 90 95Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp
Ile 100 105 110Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly
Lys Asn Cys 115 120 125Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly
Arg Cys Glu Gln Phe 130 135 140Cys Lys Asn Ser Ala Asp Asn Lys Val
Val Cys Ser Cys Thr Glu Gly145 150 155 160Tyr Arg Leu Ala Glu Asn
Gln Lys Ser Cys Glu Pro Ala Val Pro Phe 165 170 175Pro Cys Gly Arg
Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala 180 185 190Glu Thr
Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu 195 200
205Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe
210 215 220Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe
Pro Trp225 230 235 240Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe
Cys Gly Gly Ser Ile 245 250 255Val Asn Glu Lys Trp Ile Val Thr Ala
Ala His Cys Val Glu Thr Gly 260 265 270Val Lys Ile Thr Val Val Ala
Gly Glu His Asn Ile Glu Glu Thr Glu 275 280 285His Thr Glu Gln Lys
Arg Asn Val Ile Arg Ile Ile Pro His His Asn 290 295 300Tyr Asn Ala
Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu305 310 315
320Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile
325 330 335Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser
Gly Tyr 340 345 350Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg
Ser Ala Leu Val 355 360 365Leu Gln Tyr Leu Arg Val Pro Leu Val Asp
Arg Ala Thr Cys Leu Arg 370 375 380Ser Thr Lys Phe Thr Ile Tyr Asn
Asn Met Phe Cys Ala Gly Phe His385 390 395 400Glu Gly Gly Arg Asp
Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val 405 410 415Thr Glu Val
Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly 420 425 430Glu
Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser 435 440
445Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Ser Ser Ser
450 455 460Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu
Pro Gly465 470 475 480Pro Ser Asp Thr Pro Ile Leu Pro Gln
485471585DNAArtificial SequenceFIX-CTP-CTP 47gcgatcgcca tgcagcgcgt
gaacatgatc atggcagaat caccaggcct catcaccatc 60tgccttttag gatatctact
cagtgctgaa tgtacagttt ttcttgatca tgaaaacgcc 120aacaaaattc
tgaatcggcc aaagaggtat aattcaggta aattggaaga gtttgttcaa
180gggaaccttg agagagaatg tatggaagaa aagtgtagtt ttgaagaagc
acgagaagtt 240tttgaaaaca ctgaaagaac aactgaattt tggaagcagt
atgttgatgg agatcagtgt 300gagtccaatc catgtttaaa tggcggcagt
tgcaaggatg acattaattc ctatgaatgt 360tggtgtccct ttggatttga
aggaaagaac tgtgaattag atgtaacatg taacattaag 420aatggcagat
gcgagcagtt ttgtaaaaat agtgctgata acaaggtggt ttgctcctgt
480actgagggat atcgacttgc agaaaaccag aagtcctgtg aaccagcagt
gccatttcca 540tgtggaagag tttctgtttc acaaacttct aagctcaccc
gtgctgagac tgtttttcct 600gatgtggact atgtaaattc tactgaagct
gaaaccattt tggataacat cactcaaagc 660acccaatcat ttaatgactt
cactcgagtt gttggtggag aagatgccaa accaggtcaa 720ttcccttggc
aggttgtttt gaatggtaaa gttgatgcat tctgtggagg ctctatcgtt
780aatgaaaaat ggattgtaac tgctgcccac tgtgttgaaa ctggtgttaa
aattacagtt 840gtcgcaggtg aacataatat tgaggagaca gaacatacag
agcaaaagcg aaatgtgatt 900cgaattattc ctcaccacaa ctacaatgca
gctattaata agtacaacca tgacattgcc 960cttctggaac tggacgaacc
cttagtgcta aacagctacg ttacacctat ttgcattgct 1020acaaggaata
cacgaacatc ttcctcaaat ttggatctgg ctatgtaagt ggctggggaa
1080gagtcttcca caaagggaga tcagctttag ttcttcagta ccttagagtt
ccacttgttg 1140accgagccac atgtcttcga tctacaaagt tcaccatcta
taacaacatg ttctgtgctg 1200gcttccatga aggaggtaga gattcatgtc
aaggagatag tgggggaccc catgttactg 1260aagtggaagg gaccagtttc
ttaactggaa ttattagctg gggtgaagag tgtgcaatga 1320aaggcaaata
tggaatatat accaaggtat cccggtatgt caactggatt aaggaaaaaa
1380caaagctcac tagctccagc agcaaggccc ctcccccgag cctgccctcc
ccaagcaggc 1440tgcctgggcc ctccgacaca ccaatcctgc cacagagcag
ctcctctaag gcccctcctc 1500catccctgcc atccccctcc cggctgcctg
gcccctctga cacccctatc ctgcctcagt 1560gatgaaggtc tggatccgcg gccgc
158548517PRTArtificial SequenceFIX-CTP-CTP 48Met Gln Arg Val Asn
Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr1 5 10 15Ile Cys Leu Leu
Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu 20 25 30Asp His Glu
Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn 35 40 45Ser Gly
Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys 50 55 60Met
Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn65 70 75
80Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp
Gly Asp Gln 85 90 95Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys
Lys Asp Asp Ile 100 105 110Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly
Phe Glu Gly Lys Asn Cys 115 120 125Glu Leu Asp Val Thr Cys Asn Ile
Lys Asn Gly Arg Cys Glu Gln Phe 130 135 140Cys Lys Asn Ser Ala Asp
Asn Lys Val Val Cys Ser Cys Thr Glu Gly145 150 155 160Tyr Arg Leu
Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe 165 170 175Pro
Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala 180 185
190Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu
195 200 205Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn
Asp Phe 210 215 220Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly
Gln Phe Pro Trp225 230 235 240Gln Val Val Leu Asn Gly Lys Val Asp
Ala Phe Cys Gly Gly Ser Ile 245 250 255Val Asn Glu Lys Trp Ile Val
Thr Ala Ala His Cys Val Glu Thr Gly 260 265 270Val Lys Ile Thr Val
Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu 275 280 285His Thr Glu
Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn 290 295 300Tyr
Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu305 310
315 320Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys
Ile 325 330 335Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly
Ser Gly Tyr 340 345 350Val Ser Gly Trp Gly Arg Val Phe His Lys Gly
Arg Ser Ala Leu Val 355 360 365Leu Gln Tyr Leu Arg Val Pro Leu Val
Asp Arg Ala Thr Cys Leu Arg 370 375 380Ser Thr Lys Phe Thr Ile Tyr
Asn Asn Met Phe Cys Ala Gly Phe His385 390 395 400Glu Gly Gly Arg
Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val 405 410 415Thr Glu
Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly 420 425
430Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser
435 440 445Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Ser
Ser Ser 450 455 460Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser
Arg Leu Pro Gly465 470 475 480Pro Ser Asp Thr Pro Ile Leu Pro Gln
Ser Ser Ser Ser Lys Ala Pro 485 490 495Pro Pro Ser Leu Pro Ser Pro
Ser Arg Leu Pro Gly Pro Ser Asp Thr 500 505 510Pro Ile Leu Pro Gln
515491673DNAArtificial SequenceFIX-CTP-CTP-CTP (MOD-3013) with SP
49tctagagtcg accccgccat gcagcgcgtg aacatgatca tggcagaatc accaggcctc
60atcaccatct gccttttagg atatctactc agtgctgaat gtacagtttt tcttgatcat
120gaaaacgcca acaaaattct gaatcggcca aagaggtata attcaggtaa
attggaagag 180tttgttcaag ggaaccttga gagagaatgt atggaagaaa
agtgtagttt tgaagaagca 240cgagaagttt ttgaaaacac tgaaagaaca
actgaatttt ggaagcagta tgttgatgga 300gatcagtgtg agtccaatcc
atgtttaaat ggcggcagtt gcaaggatga cattaattcc 360tatgaatgtt
ggtgtccctt tggatttgaa ggaaagaact gtgaattaga tgtaacatgt
420aacattaaga atggcagatg cgagcagttt tgtaaaaata gtgctgataa
caaggtggtt 480tgctcctgta ctgagggata tcgacttgca gaaaaccaga
agtcctgtga accagcagtg 540ccatttccat gtggaagagt ttctgtttca
caaacttcta agctcacccg tgctgaggca 600gtttttcctg atgtggacta
tgtaaattct actgaagctg aaaccatttt ggataacatc 660actcaaagca
cccaatcatt taatgacttc actcgagttg ttggtggaga agatgccaaa
720ccaggtcaat tcccttggca ggttgttttg aatggtaaag ttgatgcatt
ctgtggaggc 780tctatcgtta atgaaaaatg gattgtaact gctgcccact
gtgttgaaac tggtgttaaa 840attacagttg tcgcaggtga acataatatt
gaggagacag aacatacaga gcaaaagcga 900aatgtgattc gaattattcc
tcaccacaac tacaatgcag ctattaataa gtacaaccat 960gacattgccc
ttctggaact ggacgaaccc ttagtgctaa acagctacgt tacacctatt
1020tgcattgctg acaaggaata cacgaacatc ttcctcaaat ttggatctgg
ctatgtaagt 1080ggctggggaa gagtcttcca caaagggaga tcagctttag
ttcttcagta ccttagagtt 1140ccacttgttg accgagccac atgtcttcga
tctacaaagt tcaccatcta taacaacatg 1200ttctgtgctg gcttccatga
aggaggtaga gattcatgtc aaggagatag tgggggaccc 1260catgttactg
aagtggaagg gaccagtttc ttaactggaa ttattagctg gggtgaagag
1320tgtgcaatga aaggcaaata tggaatatat accaaggtat cccggtatgt
caactggatt 1380aaggaaaaaa caaagctcac tagctccagc agcaaggccc
ctcccccgag cctgccctcc 1440ccaagcaggc tgcctgggcc cagtgacacc
cctatcctgc ctcagtccag ctccagcaag 1500gccccacccc ctagcctgcc
ttctccttct cggctgcctg gccccagcga tactccaatt 1560ctgccccagt
cctccagcag taaggctccc cctccatctc tgccatcccc cagcagactg
1620ccaggccctt ctgatacacc catcctccca cagtgatgag gatccgcggc cgc
167350545PRTArtificial SequenceFIX-CTP-CTP-CTP (MOD-3013) with SP
50Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr1
5 10 15Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe
Leu 20 25 30Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg
Tyr Asn 35 40 45Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu
Arg Glu Cys 50 55 60Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu
Val Phe Glu Asn65 70 75 80Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln
Tyr Val Asp Gly Asp Gln 85 90 95Cys Glu Ser Asn Pro Cys Leu Asn Gly
Gly Ser Cys Lys Asp Asp Ile 100 105 110Asn Ser Tyr Glu Cys Trp Cys
Pro Phe Gly Phe Glu Gly Lys Asn Cys 115 120 125Glu Leu Asp Val Thr
Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe 130 135 140Cys Lys Asn
Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly145 150 155
160Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe
165 170 175Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr
Arg Ala 180 185 190Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser
Thr Glu Ala Glu 195 200 205Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr
Gln Ser Phe Asn Asp Phe 210 215 220Thr Arg Val Val Gly Gly Glu Asp
Ala Lys Pro Gly Gln Phe Pro Trp225 230 235 240Gln Val Val Leu Asn
Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile 245 250 255Val Asn Glu
Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly 260 265 270Val
Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu 275 280
285His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn
290 295 300Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu
Leu Glu305 310 315 320Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val
Thr Pro Ile Cys Ile 325 330 335Ala Asp Lys Glu Tyr Thr Asn Ile Phe
Leu Lys Phe Gly Ser Gly Tyr 340 345 350Val Ser Gly Trp Gly Arg Val
Phe His Lys Gly Arg Ser Ala Leu Val 355 360 365Leu Gln Tyr Leu Arg
Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg 370 375 380Ser Thr Lys
Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His385 390 395
400Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val
405 410 415Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser
Trp Gly 420 425 430Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr
Thr Lys Val Ser 435 440 445Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr
Lys Leu Thr Ser Ser Ser 450 455 460Ser Lys Ala Pro Pro Pro Ser Leu
Pro Ser Pro Ser Arg Leu Pro Gly465 470 475 480Pro Ser Asp Thr Pro
Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro 485 490 495Pro Pro Ser
Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr 500 505 510Pro
Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu 515 520
525Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro
530 535 540Gln545511757DNAArtificial SequenceFIX-CTP4x 51tctagagtcg
accccgccat gcagcgcgtg aacatgatca tggcagaatc accaggcctc 60atcaccatct
gccttttagg atatctactc agtgctgaat gtacagtttt tcttgatcat
120gaaaacgcca acaaaattct gaatcggcca aagaggtata attcaggtaa
attggaagag 180tttgttcaag ggaaccttga gagagaatgt atggaagaaa
agtgtagttt tgaagaagca 240cgagaagttt ttgaaaacac tgaaagaaca
actgaatttt ggaagcagta tgttgatgga 300gatcagtgtg agtccaatcc
atgtttaaat ggcggcagtt gcaaggatga cattaattcc 360tatgaatgtt
ggtgtccctt tggatttgaa ggaaagaact gtgaattaga tgtaacatgt
420aacattaaga atggcagatg cgagcagttt tgtaaaaata gtgctgataa
caaggtggtt 480tgctcctgta ctgagggata tcgacttgca gaaaaccaga
agtcctgtga accagcagtg 540ccatttccat gtggaagagt ttctgtttca
caaacttcta agctcacccg tgctgaggca 600gtttttcctg atgtggacta
tgtaaattct actgaagctg aaaccatttt ggataacatc 660actcaaagca
cccaatcatt taatgacttc actcgagttg ttggtggaga agatgccaaa
720ccaggtcaat tcccttggca ggttgttttg aatggtaaag ttgatgcatt
ctgtggaggc 780tctatcgtta atgaaaaatg gattgtaact gctgcccact
gtgttgaaac tggtgttaaa 840attacagttg tcgcaggtga acataatatt
gaggagacag aacatacaga gcaaaagcga 900aatgtgattc gaattattcc
tcaccacaac tacaatgcag ctattaataa gtacaaccat 960gacattgccc
ttctggaact ggacgaaccc ttagtgctaa acagctacgt tacacctatt
1020tgcattgctg acaaggaata cacgaacatc ttcctcaaat ttggatctgg
ctatgtaagt 1080ggctggggaa gagtcttcca caaagggaga tcagctttag
ttcttcagta ccttagagtt 1140ccacttgttg accgagccac atgtcttcga
tctacaaagt tcaccatcta taacaacatg 1200ttctgtgctg gcttccatga
aggaggtaga gattcatgtc aaggagatag tgggggaccc 1260catgttactg
aagtggaagg gaccagtttc ttaactggaa ttattagctg gggtgaagag
1320tgtgcaatga aaggcaaata tggaatatat accaaggtat cccggtatgt
caactggatt 1380aaggaaaaaa caaagctcac tagctccagc agcaaggccc
ctcccccgag cctgccctcc 1440ccaagcaggc tgcctgggcc ctctgacacc
cctatcctgc ctcagtccag ctcctctaag 1500gccccaccac cttccctgcc
tagcccttca agactgccag gccctagcga tacaccaatt 1560ctgccccagt
cctccagcag caaggctccc ccacctagcc tgccttctcc atcaaggctg
1620cctggcccat ccgatacccc aattttgcct cagagcagct ctagcaaggc
acctcccccc 1680agtctgccct ctccaagcag actccctggc ccttcagaca
ctcccattct gccacagtga 1740tgaggatccg cggccgc 175752583PRTArtificial
SequenceFIX-CTP4x 52Ser Arg Val Asp Pro Ala Met Gln Arg Val Asn Met
Ile Met Ala Glu1 5 10 15Ser Pro Gly Leu Ile Thr Ile Cys Leu Leu Gly
Tyr Leu Leu Ser Ala 20 25 30Glu Cys Thr Val Phe Leu Asp His Glu Asn
Ala Asn Lys Ile Leu Asn 35 40 45Arg Pro Lys Arg Tyr Asn Ser Gly Lys
Leu Glu Glu Phe Val Gln Gly 50 55 60Asn Leu Glu Arg Glu Cys Met Glu
Glu Lys Cys Ser Phe Glu Glu Ala65 70 75 80Arg Glu Val Phe Glu Asn
Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln 85 90 95Tyr Val Asp Gly Asp
Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly 100 105 110Ser Cys Lys
Asp Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly 115 120 125Phe
Glu Gly Lys Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn 130 135
140Gly Arg Cys Glu Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val
Val145 150 155 160Cys Ser Cys Thr Glu Gly Tyr Arg Leu Ala Glu Asn
Gln Lys Ser Cys 165 170 175Glu Pro Ala Val Pro Phe Pro Cys Gly Arg
Val Ser Val Ser Gln Thr 180 185 190Ser Lys Leu Thr Arg Ala Glu Ala
Val Phe Pro Asp Val Asp Tyr Val 195 200 205Asn Ser Thr Glu Ala Glu
Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr 210 215 220Gln Ser Phe Asn
Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys225 230 235 240Pro
Gly Gln Phe Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala 245 250
255Phe Cys Gly Gly Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala
260 265 270His Cys Val Glu Thr Gly Val Lys Ile Thr Val Val Ala Gly
Glu His 275 280 285Asn Ile Glu Glu Thr Glu His Thr Glu Gln Lys Arg
Asn Val Ile Arg 290 295 300Ile Ile Pro His His Asn Tyr Asn Ala Ala
Ile Asn Lys Tyr Asn His305 310 315 320Asp Ile Ala Leu Leu Glu Leu
Asp Glu Pro Leu Val Leu Asn Ser Tyr 325 330 335Val Thr Pro Ile Cys
Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu 340 345 350Lys Phe Gly
Ser Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys 355 360 365Gly
Arg Ser Ala Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp 370 375
380Arg Ala Thr Cys Leu Arg Ser Thr Lys Phe Thr Ile Tyr Asn Asn
Met385 390 395 400Phe Cys Ala Gly Phe His Glu Gly Gly Arg Asp Ser
Cys Gln Gly Asp 405 410 415Ser Gly Gly Pro His Val Thr Glu Val Glu
Gly Thr Ser Phe Leu Thr 420 425 430Gly Ile Ile Ser Trp Gly Glu Glu
Cys Ala Met Lys Gly Lys Tyr Gly 435 440 445Ile Tyr Thr Lys Val Ser
Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr 450 455 460Lys Leu Thr Ser
Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser465 470 475 480Pro
Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser 485 490
495Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu
500 505 510Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser
Ser Lys 515 520 525Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu
Pro Gly Pro Ser 530 535 540Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser
Ser Lys Ala Pro Pro Pro545 550 555 560Ser Leu Pro Ser Pro Ser Arg
Leu Pro Gly Pro Ser Asp Thr Pro Ile 565 570 575Leu Pro Gln Gly Ser
Ala Ala 580531840DNAArtificial SequenceFIX-CTP5x 53ctagagtcga
ccccgccatg cagcgcgtga acatgatcat ggcagaatca ccaggcctca 60tcaccatctg
ccttttagga tatctactca gtgctgaatg tacagttttt cttgatcatg
120aaaacgccaa caaaattctg aatcggccaa agaggtataa ttcaggtaaa
ttggaagagt 180ttgttcaagg gaaccttgag agagaatgta tggaagaaaa
gtgtagtttt gaagaagcac 240gagaagtttt tgaaaacact gaaagaacaa
ctgaattttg gaagcagtat gttgatggag 300atcagtgtga gtccaatcca
tgtttaaatg gcggcagttg caaggatgac attaattcct 360atgaatgttg
gtgtcccttt ggatttgaag gaaagaactg tgaattagat gtaacatgta
420acattaagaa tggcagatgc gagcagtttt gtaaaaatag tgctgataac
aaggtggttt 480gctcctgtac tgagggatat cgacttgcag aaaaccagaa
gtcctgtgaa ccagcagtgc 540catttccatg tggaagagtt tctgtttcac
aaacttctaa gctcacccgt gctgaggcag 600tttttcctga tgtggactat
gtaaattcta ctgaagctga aaccattttg gataacatca 660ctcaaagcac
ccaatcattt aatgacttca ctcgagttgt tggtggagaa gatgccaaac
720caggtcaatt cccttggcag gttgttttga atggtaaagt tgatgcattc
tgtggaggct 780ctatcgttaa tgaaaaatgg attgtaactg ctgcccactg
tgttgaaact ggtgttaaaa 840ttacagttgt cgcaggtgaa cataatattg
aggagacaga acatacagag caaaagcgaa 900atgtgattcg aattattcct
caccacaact acaatgcagc tattaataag tacaaccatg 960acattgccct
tctggaactg gacgaaccct tagtgctaaa cagctacgtt acacctattt
1020gcattgctga caaggaatac acgaacatct tcctcaaatt tggatctggc
tatgtaagtg 1080gctggggaag agtcttccac aaagggagat cagctttagt
tcttcagtac cttagagttc 1140cacttgttga ccgagccaca tgtcttcgat
ctacaaagtt caccatctat aacaacatgt 1200tctgtgctgg cttccatgaa
ggaggtagag attcatgtca aggagatagt gggggacccc 1260atgttactga
agtggaaggg accagtttct taactggaat tattagctgg ggtgaagagt
1320gtgcaatgaa aggcaaatat ggaatatata ccaaggtatc ccggtatgtc
aactggatta 1380aggaaaaaac aaagctcact agctccagca gcaaggcccc
tcccccgagc ctgccctccc 1440caagcaggct gcctgggccc tctgacaccc
ctatcctgcc tcagtccagc tcctctaagg 1500ctccaccacc ttccctgcct
agcccttcaa gactgccagg ccctagcgat acaccaattc 1560tgccccagtc
ctccagcagc aaggctcccc cacctagcct gccttctcca tcaaggctgc
1620ctggcccatc cgatacccca attttgcctc agagcagctc tagcaaggca
cctcccccca 1680gtctgccctc tccaagcaga ctccctggcc cttcagacac
tccaatcctc ccacagtcct 1740ctagctctaa agctccacct cccagcctgc
ccagccctag tagactcccc ggaccttctg 1800atacccccat cttgccccag
tgatgaggat ccgcggccgc 184054610PRTArtificial
SequenceFIX-CTP5x 54Arg Val Asp Pro Ala Met Gln Arg Val Asn Met Ile
Met Ala Glu Ser1 5 10 15Pro Gly Leu Ile Thr Ile Cys Leu Leu Gly Tyr
Leu Leu Ser Ala Glu 20 25 30Cys Thr Val Phe Leu Asp His Glu Asn Ala
Asn Lys Ile Leu Asn Arg 35 40 45Pro Lys Arg Tyr Asn Ser Gly Lys Leu
Glu Glu Phe Val Gln Gly Asn 50 55 60Leu Glu Arg Glu Cys Met Glu Glu
Lys Cys Ser Phe Glu Glu Ala Arg65 70 75 80Glu Val Phe Glu Asn Thr
Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr 85 90 95Val Asp Gly Asp Gln
Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser 100 105 110Cys Lys Asp
Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe 115 120 125Glu
Gly Lys Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly 130 135
140Arg Cys Glu Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val
Cys145 150 155 160Ser Cys Thr Glu Gly Tyr Arg Leu Ala Glu Asn Gln
Lys Ser Cys Glu 165 170 175Pro Ala Val Pro Phe Pro Cys Gly Arg Val
Ser Val Ser Gln Thr Ser 180 185 190Lys Leu Thr Arg Ala Glu Ala Val
Phe Pro Asp Val Asp Tyr Val Asn 195 200 205Ser Thr Glu Ala Glu Thr
Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln 210 215 220Ser Phe Asn Asp
Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro225 230 235 240Gly
Gln Phe Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe 245 250
255Cys Gly Gly Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala His
260 265 270Cys Val Glu Thr Gly Val Lys Ile Thr Val Val Ala Gly Glu
His Asn 275 280 285Ile Glu Glu Thr Glu His Thr Glu Gln Lys Arg Asn
Val Ile Arg Ile 290 295 300Ile Pro His His Asn Tyr Asn Ala Ala Ile
Asn Lys Tyr Asn His Asp305 310 315 320Ile Ala Leu Leu Glu Leu Asp
Glu Pro Leu Val Leu Asn Ser Tyr Val 325 330 335Thr Pro Ile Cys Ile
Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys 340 345 350Phe Gly Ser
Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys Gly 355 360 365Arg
Ser Ala Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg 370 375
380Ala Thr Cys Leu Arg Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met
Phe385 390 395 400Cys Ala Gly Phe His Glu Gly Gly Arg Asp Ser Cys
Gln Gly Asp Ser 405 410 415Gly Gly Pro His Val Thr Glu Val Glu Gly
Thr Ser Phe Leu Thr Gly 420 425 430Ile Ile Ser Trp Gly Glu Glu Cys
Ala Met Lys Gly Lys Tyr Gly Ile 435 440 445Tyr Thr Lys Val Ser Arg
Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys 450 455 460Leu Thr Ser Ser
Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro465 470 475 480Ser
Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser 485 490
495Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro
500 505 510Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser
Lys Ala 515 520 525Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro
Gly Pro Ser Asp 530 535 540Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser
Lys Ala Pro Pro Pro Ser545 550 555 560Leu Pro Ser Pro Ser Arg Leu
Pro Gly Pro Ser Asp Thr Pro Ile Leu 565 570 575Pro Gln Ser Ser Ser
Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro 580 585 590Ser Arg Leu
Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Gly Ser 595 600 605Ala
Ala 610552413DNAArtificial Sequencefurin 55tctagagtcg accccgccat
ggagctgagg ccctggttgc tatgggtggt agcagcaaca 60ggaaccttgg tcctgctagc
agctgatgct cagggccaga aggtcttcac caacacgtgg 120gctgtgcgca
tccctggagg cccagcggtg gccaacagtg tggcacggaa gcatgggttc
180ctcaacctgg gccagatctt cggggactat taccacttct ggcatcgagg
agtgacgaag 240cggtccctgt cgcctcaccg cccgcggcac agccggctgc
agagggagcc tcaagtacag 300tggctggaac agcaggtggc aaagcgacgg
actaaacggg acgtgtacca ggagcccaca 360gaccccaagt ttcctcagca
gtggtacctg tctggtgtca ctcagcggga cctgaatgtg 420aaggcggcct
gggcgcaggg ctacacaggg cacggcattg tggtctccat tctggacgat
480ggcatcgaga agaaccaccc ggacttggca ggcaattatg atcctggggc
cagttttgat 540gtcaatgacc aggaccctga cccccagcct cggtacacac
agatgaatga caacaggcac 600ggcacacggt gtgcggggga agtggctgcg
gtggccaaca acggtgtctg tggtgtaggt 660gtggcctaca acgcccgcat
tggaggggtg cgcatgctgg atggcgaggt gacagatgca 720gtggaggcac
gctcgctggg cctgaacccc aaccacatcc acatctacag tgccagctgg
780ggccccgagg atgacggcaa gacagtggat gggccagccc gcctcgccga
ggaggccttc 840ttccgtgggg ttagccaggg ccgagggggg ctgggctcca
tctttgtctg ggcctcgggg 900aacgggggcc gggaacatga cagctgcaac
tgcgacggct acaccaacag tatctacacg 960ctgtccatca gcagcgccac
gcagtttggc aacgtgccgt ggtacagcga ggcctgctcg 1020tccacactgg
ccacgaccta cagcagtggc aaccagaatg agaagcagat cgtgacgact
1080gacttgcggc agaagtgcac ggagtctcac acgggcacct cagcctctgc
ccccttagca 1140gccggcatca ttgctctcac cctggaggcc aataagaacc
tcacatggcg ggacatgcaa 1200cacctggtgg tacagacctc gaagccagcc
cacctcaatg ccaacgactg ggccaccaat 1260ggtgtgggcc ggaaagtgag
ccactcatat ggctacgggc ttttggacgc aggcgccatg 1320gtggccctgg
cccagaattg gaccacagtg gccccccagc ggaagtgcat catcgacatc
1380ctcaccgagc ccaaagacat cgggaaacgg ctcgaggtgc ggaagaccgt
gaccgcgtgc 1440ctgggcgagc ccaaccacat cactcggctg gagcacgctc
aggcgcggct caccctgtcc 1500tataatcgcc gtggcgacct ggccatccac
ctggtcagcc ccatgggcac ccgctccacc 1560ctgctggcag ccaggccaca
tgactactcc gcagatgggt ttaatgactg ggccttcatg 1620acaactcatt
cctgggatga ggatccctct ggcgagtggg tcctagagat tgaaaacacc
1680agcgaagcca acaactatgg gacgctgacc aagttcaccc tcgtactcta
tggcaccgcc 1740cctgaggggc tgcccgtacc tccagaaagc agtggctgca
agaccctcac gtccagtcag 1800gcctgtgtgg tgtgcgagga aggcttctcc
ctgcaccaga agagctgtgt ccagcactgc 1860cctccaggct tcgcccccca
agtcctcgat acgcactata gcaccgagaa tgacgtggag 1920accatccggg
ccagcgtctg cgccccctgc cacgcctcat gtgccacatg ccaggggccg
1980gccctgacag actgcctcag ctgccccagc cacgcctcct tggaccctgt
ggagcagact 2040tgctcccggc aaagccagag cagccgagag tccccgccac
agcagcagcc acctcggctg 2100cccccggagg tggaggcggg gcaacggctg
cgggcagggc tgctgccctc acacctgcct 2160gaggtggtgg ccggcctcag
ctgcgccttc atcgtgctgg tcttcgtcac tgtcttcctg 2220gtcctgcagc
tgcgctctgg ctttagtttt cggggggtga aggtgtacac catggaccgt
2280ggcctcatct cctacaaggg gctgccccct gaagcctggc aggaggagtg
cccgtctgac 2340tcagaagagg acgagggccg gggcgagagg accgccttta
tcaaagacca gagcgccctc 2400tgaacgcggc cgc 241356794PRTArtificial
Sequencefurin 56Met Glu Leu Arg Pro Trp Leu Leu Trp Val Val Ala Ala
Thr Gly Thr1 5 10 15Leu Val Leu Leu Ala Ala Asp Ala Gln Gly Gln Lys
Val Phe Thr Asn 20 25 30Thr Trp Ala Val Arg Ile Pro Gly Gly Pro Ala
Val Ala Asn Ser Val 35 40 45Ala Arg Lys His Gly Phe Leu Asn Leu Gly
Gln Ile Phe Gly Asp Tyr 50 55 60Tyr His Phe Trp His Arg Gly Val Thr
Lys Arg Ser Leu Ser Pro His65 70 75 80Arg Pro Arg His Ser Arg Leu
Gln Arg Glu Pro Gln Val Gln Trp Leu 85 90 95Glu Gln Gln Val Ala Lys
Arg Arg Thr Lys Arg Asp Val Tyr Gln Glu 100 105 110Pro Thr Asp Pro
Lys Phe Pro Gln Gln Trp Tyr Leu Ser Gly Val Thr 115 120 125Gln Arg
Asp Leu Asn Val Lys Ala Ala Trp Ala Gln Gly Tyr Thr Gly 130 135
140His Gly Ile Val Val Ser Ile Leu Asp Asp Gly Ile Glu Lys Asn
His145 150 155 160Pro Asp Leu Ala Gly Asn Tyr Asp Pro Gly Ala Ser
Phe Asp Val Asn 165 170 175Asp Gln Asp Pro Asp Pro Gln Pro Arg Tyr
Thr Gln Met Asn Asp Asn 180 185 190Arg His Gly Thr Arg Cys Ala Gly
Glu Val Ala Ala Val Ala Asn Asn 195 200 205Gly Val Cys Gly Val Gly
Val Ala Tyr Asn Ala Arg Ile Gly Gly Val 210 215 220Arg Met Leu Asp
Gly Glu Val Thr Asp Ala Val Glu Ala Arg Ser Leu225 230 235 240Gly
Leu Asn Pro Asn His Ile His Ile Tyr Ser Ala Ser Trp Gly Pro 245 250
255Glu Asp Asp Gly Lys Thr Val Asp Gly Pro Ala Arg Leu Ala Glu Glu
260 265 270Ala Phe Phe Arg Gly Val Ser Gln Gly Arg Gly Gly Leu Gly
Ser Ile 275 280 285Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Glu His
Asp Ser Cys Asn 290 295 300Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr
Leu Ser Ile Ser Ser Ala305 310 315 320Thr Gln Phe Gly Asn Val Pro
Trp Tyr Ser Glu Ala Cys Ser Ser Thr 325 330 335Leu Ala Thr Thr Tyr
Ser Ser Gly Asn Gln Asn Glu Lys Gln Ile Val 340 345 350Thr Thr Asp
Leu Arg Gln Lys Cys Thr Glu Ser His Thr Gly Thr Ser 355 360 365Ala
Ser Ala Pro Leu Ala Ala Gly Ile Ile Ala Leu Thr Leu Glu Ala 370 375
380Asn Lys Asn Leu Thr Trp Arg Asp Met Gln His Leu Val Val Gln
Thr385 390 395 400Ser Lys Pro Ala His Leu Asn Ala Asn Asp Trp Ala
Thr Asn Gly Val 405 410 415Gly Arg Lys Val Ser His Ser Tyr Gly Tyr
Gly Leu Leu Asp Ala Gly 420 425 430Ala Met Val Ala Leu Ala Gln Asn
Trp Thr Thr Val Ala Pro Gln Arg 435 440 445Lys Cys Ile Ile Asp Ile
Leu Thr Glu Pro Lys Asp Ile Gly Lys Arg 450 455 460Leu Glu Val Arg
Lys Thr Val Thr Ala Cys Leu Gly Glu Pro Asn His465 470 475 480Ile
Thr Arg Leu Glu His Ala Gln Ala Arg Leu Thr Leu Ser Tyr Asn 485 490
495Arg Arg Gly Asp Leu Ala Ile His Leu Val Ser Pro Met Gly Thr Arg
500 505 510Ser Thr Leu Leu Ala Ala Arg Pro His Asp Tyr Ser Ala Asp
Gly Phe 515 520 525Asn Asp Trp Ala Phe Met Thr Thr His Ser Trp Asp
Glu Asp Pro Ser 530 535 540Gly Glu Trp Val Leu Glu Ile Glu Asn Thr
Ser Glu Ala Asn Asn Tyr545 550 555 560Gly Thr Leu Thr Lys Phe Thr
Leu Val Leu Tyr Gly Thr Ala Pro Glu 565 570 575Gly Leu Pro Val Pro
Pro Glu Ser Ser Gly Cys Lys Thr Leu Thr Ser 580 585 590Ser Gln Ala
Cys Val Val Cys Glu Glu Gly Phe Ser Leu His Gln Lys 595 600 605Ser
Cys Val Gln His Cys Pro Pro Gly Phe Ala Pro Gln Val Leu Asp 610 615
620Thr His Tyr Ser Thr Glu Asn Asp Val Glu Thr Ile Arg Ala Ser
Val625 630 635 640Cys Ala Pro Cys His Ala Ser Cys Ala Thr Cys Gln
Gly Pro Ala Leu 645 650 655Thr Asp Cys Leu Ser Cys Pro Ser His Ala
Ser Leu Asp Pro Val Glu 660 665 670Gln Thr Cys Ser Arg Gln Ser Gln
Ser Ser Arg Glu Ser Pro Pro Gln 675 680 685Gln Gln Pro Pro Arg Leu
Pro Pro Glu Val Glu Ala Gly Gln Arg Leu 690 695 700Arg Ala Gly Leu
Leu Pro Ser His Leu Pro Glu Val Val Ala Gly Leu705 710 715 720Ser
Cys Ala Phe Ile Val Leu Val Phe Val Thr Val Phe Leu Val Leu 725 730
735Gln Leu Arg Ser Gly Phe Ser Phe Arg Gly Val Lys Val Tyr Thr Met
740 745 750Asp Arg Gly Leu Ile Ser Tyr Lys Gly Leu Pro Pro Glu Ala
Trp Gln 755 760 765Glu Glu Cys Pro Ser Asp Ser Glu Glu Asp Glu Gly
Arg Gly Glu Arg 770 775 780Thr Ala Phe Ile Lys Asp Gln Ser Ala
Leu785 7905737PRTArtificial Sequenceoxyntomodulin 57His Ser Gln Gly
Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser1 5 10 15Arg Arg Ala
Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn 20 25 30Arg Asn
Asn Ile Ala 3558119PRTArtificial SequenceCTP-OXM-CTP 58Met Ala Thr
Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu1 5 10 15Cys Leu
Pro Trp Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys Ala 20 25 30Pro
Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp 35 40
45Thr Pro Ile Leu Pro Gln His Ser Gln Gly Thr Phe Thr Ser Asp Tyr
50 55 60Ser Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val Gln Trp
Leu65 70 75 80Met Asn Thr Lys Arg Asn Arg Asn Asn Ile Ala Ser Ser
Ser Ser Lys 85 90 95Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu
Pro Gly Pro Ser 100 105 110Asp Thr Pro Ile Leu Pro Gln
11559147PRTArtificial SequenceCTP-OXM-CTP-CTP 59Met Ala Thr Gly Ser
Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu1 5 10 15Cys Leu Pro Trp
Leu Gln Glu Gly Ser Ala Ser Ser Ser Ser Lys Ala 20 25 30Pro Pro Pro
Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp 35 40 45Thr Pro
Ile Leu Pro Gln His Ser Gln Gly Thr Phe Thr Ser Asp Tyr 50 55 60Ser
Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val Gln Trp Leu65 70 75
80Met Asn Thr Lys Arg Asn Arg Asn Asn Ile Ala Ser Ser Ser Ser Lys
85 90 95Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro
Ser 100 105 110Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala
Pro Pro Pro 115 120 125Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro
Ser Asp Thr Pro Ile 130 135 140Leu Pro Gln14560122PRTArtificial
SequenceOXM-CTP-CTP 60Leu Glu Asp Met Ala Thr Gly Ser Arg Thr Ser
Leu Leu Leu Ala Phe1 5 10 15Gly Leu Leu Cys Leu Pro Trp Leu Gln Glu
Gly Ser Ala His Ser Gln 20 25 30Gly Thr Phe Thr Ser Asp Tyr Ser Lys
Tyr Leu Asp Ser Arg Arg Ala 35 40 45Gln Asp Phe Val Gln Trp Leu Met
Asn Thr Lys Arg Asn Arg Asn Asn 50 55 60Ile Ala Ser Ser Ser Ser Lys
Ala Pro Pro Pro Ser Leu Pro Ser Pro65 70 75 80Ser Arg Leu Pro Gly
Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser 85 90 95Ser Ser Lys Ala
Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro 100 105 110Gly Pro
Ser Asp Thr Pro Ile Leu Pro Gln 115 12061140PRTArtificial
SequenceCTP-CTP-OXM 61Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu
Ala Phe Gly Leu Leu1 5 10 15Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala
Ser Ser Ser Ser Lys Ala 20 25 30Pro Pro Pro Ser Leu Pro Ser Pro Ser
Arg Leu Pro Gly Pro Ser Asp 35 40 45Thr Pro Ile Leu Pro Gln Ser Ser
Ser Ser Lys Ala Pro Pro Pro Ser 50 55 60Leu Pro Ser Pro Ser Arg Leu
Pro Gly Pro Ser Asp Thr Pro Ile Leu65 70 75 80Pro Gln His Ser Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu 85 90 95Asp Ser Arg Arg
Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys 100 105 110Arg Asn
Arg Asn Asn Ile Ala Arg Ser Gly Cys Gly Arg Gly Thr Leu 115 120
125Gly Leu Met Gly Leu Pro Phe Thr Ala Arg Phe Pro 130 135
14062147PRTArtificial SequenceOXM-CTP-CTP-CTP 62Met Ala Thr Gly Ser
Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu1 5 10 15Cys Leu Pro Trp
Leu Gln Glu Gly Ser Ala His Ser Gln Gly Thr Phe 20 25 30Thr Ser Asp
Tyr Ser Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe 35 40 45Val Gln
Trp Leu Met Asn Thr Lys Arg Asn Arg Asn Asn Ile Ala Ser 50 55
60Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu65
70 75 80Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser
Lys 85 90 95Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly
Pro Ser 100 105 110Asp Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys
Ala Pro Pro Pro 115 120 125Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly
Pro Ser Asp Thr Pro Ile 130 135 140Leu Pro Gln14563175PRTArtificial
SequenceOXM-CTP-4x 63Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu
Ala Phe Gly Leu Leu1 5 10 15Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala
His Ser Gln Gly Thr Phe 20 25 30Thr Ser Asp Tyr Ser Lys Tyr Leu Asp
Ser Arg Arg Ala Gln Asp Phe 35 40 45Val Gln Trp Leu Met Asn Thr Lys
Arg Asn Arg Asn Asn Ile Ala Ser 50 55 60Ser Ser Ser Lys Ala Pro Pro
Pro Ser Leu Pro Ser Pro Ser Arg Leu65 70 75 80Pro Gly Pro Ser Asp
Thr Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys 85 90 95Ala Pro Pro Pro
Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser 100 105 110Asp Thr
Pro Ile Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro 115 120
125Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile
130 135 140Leu Pro Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu
Pro Ser145 150 155 160Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro
Ile Leu Pro Gln 165 170 17564203PRTArtificial SequenceOXM-CTP-5x
64Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu1
5 10 15Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala His Ser Gln Gly Thr
Phe 20 25 30Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser Arg Arg Ala Gln
Asp Phe 35 40 45Val Gln Trp Leu Met Asn Thr Lys Arg Asn Arg Asn Asn
Ile Ala Ser 50 55 60Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser
Pro Ser Arg Leu65 70 75 80Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro
Gln Ser Ser Ser Ser Lys 85 90 95Ala Pro Pro Pro Ser Leu Pro Ser Pro
Ser Arg Leu Pro Gly Pro Ser 100 105 110Asp Thr Pro Ile Leu Pro Gln
Ser Ser Ser Ser Lys Ala Pro Pro Pro 115 120 125Ser Leu Pro Ser Pro
Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile 130 135 140Leu Pro Gln
Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser145 150 155
160Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser
165 170 175Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser
Arg Leu 180 185 190Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 195
20065391DNAArtificial SequenceCTP-OXM-CTP 65tctagaggac atggccaccg
gcagcaggac cagcctgctg ctggccttcg gcctgctgtg 60cctgccatgg ctgcaggagg
gcagcgccag ctcttcttct aaggctccac ccccatctct 120gcccagcccc
agcagactgc cgggccccag cgacacaccc attctgcccc agcacagcca
180gggcaccttc accagcgact acagcaagta cctggacagc agaagggccc
aggacttcgt 240ccagtggctg atgaacacca agaggaacag gaacaacatc
gcttcctcta gctccaaggc 300ccctccaccc tctctgccta gcccctctcg
gctgcctggc ccatccgaca caccaatcct 360gccacagtga tgaaggtctg
gatgcggccg c 39166475DNAArtificial SequenceCTP-OXM-CTP-CTP
66tctagaggac atggccaccg gcagcaggac cagcctgctg ctggccttcg gcctgctgtg
60cctgccatgg ctgcaggagg gcagcgccag ctcttcttct aaggctccac ccccatctct
120gcccagcccc agcagactgc cgggccccag cgacacaccc attctgcccc
agcacagcca 180gggcaccttc accagcgact acagcaagta cctggacagc
agaagggccc aggacttcgt 240ccagtggctg atgaacacca agaggaacag
gaacaacatc gctagctcca gcagcaaggc 300ccctcccccg agcctgccct
ccccaagcag gctgcctggg ccctccgaca caccaatcct 360gccacagagc
agctcctcta aggcccctcc tccatccctg ccatccccct cccggctgcc
420tggcccctct gacaccccta tcctgcctca gtgatgaagg tctggatgcg gccgc
47567391DNAArtificial SequenceOXM-CTP-CTP 67tctagaggac atggccacag
ggagcaggac cagcctgctg ctggctttcg gcctgctgtg 60tctgccatgg ctgcaggagg
gcagcgctca cagccagggc accttcacca gcgactacag 120caagtacctg
gacagcagaa gggcccagga cttcgtccag tggctgatga acaccaagag
180gaacaggaac aacatcgcta gctccagcag caaggcccct cccccgagcc
tgccctcccc 240aagcaggctg cctgggccct ccgacacacc aatcctgcca
cagagcagct cctctaaggc 300ccctcctcca tccctgccat ccccctcccg
gctgcctggc ccctctgaca cccctatcct 360gcctcagtga tgaaggtctg
gatgcggccg c 39168481DNAArtificial SequenceCTP-CTP-OXM 68ctatagggcg
aattgaagga aggccgtcaa ggccgcatga gctctctaga ggacatggcc 60accggcagca
ggaccagcct gctgctggcc ttcggcctgc tgtgcctgcc atggctgcag
120gagggcagcg ccagctccag cagcaaggcc cctcccccga gcctgccctc
cccaagcagg 180ctgcctgggc cctccgacac accaatcctg ccacagagca
gctcctctaa ggcccctcct 240ccatccctgc catccccctc ccggctgcct
ggcccctctg acacccctat cctgcctcag 300cacagccagg gcaccttcac
cagcgactac agcaagtacc tggacagcag aagggcccag 360gacttcgtcc
agtggctgat gaacaccaag aggaacagga acaacatcgc ttgatgaagg
420tctggatgcg gccgcggtac cctgggcctc atgggccttc ctttcactgc
ccgctttcca 480g 48169481DNAArtificial SequenceOXM-CTP-CTP-CTP
69tctagactcg agcgatcgcc atggccaccg gctctaggac ctccctgctg ctggccttcg
60gcctgctgtg cctgccctgg ctgcaggaag gcagcgctca ctcccagggc accttcacct
120ccgactactc caagtacctg gactctcgga gagcccagga cttcgtgcag
tggctgatga 180acaccaagcg gaaccggaac aatatcgcct cctcaagctc
caaggcacct ccaccttccc 240tgcctagccc ttccagactc cctgggccca
gtgacacccc tatcctgcct cagtccagct 300ccagcaaggc cccaccccct
agcctgcctt ctccttctcg gctgcctggc cccagcgata 360ctccaattct
gccccagtcc tccagcagta aggctccccc tccatctctg ccatccccca
420gcagactgcc aggcccttct gatacaccca tcctcccaca gtgatgagga
tccgcggccg 480c 48170565DNAArtificial SequenceOXM-CTP-4x
70tctagactcg agcgatcgcc atggctaccg gctccagaac ctctctgctg ctggccttcg
60gcctgctgtg tctgccttgg ctgcaagagg gcagcgctca ttcccagggc accttcacct
120ccgactactc caagtacctg gactctcgca gagcccagga cttcgtgcag
tggctgatga 180acaccaagcg gaaccggaac aatatcgcct cctccagctc
caaggcccct cctccatctc 240tgccatcccc cagtagactg cctgggccct
ctgacacccc tatcctgcct cagtccagct 300cctctaaggc cccaccacct
tccctgccta gcccttcaag actgccaggc cctagcgata 360caccaattct
gccccagtcc tccagcagca aggctccccc acctagcctg ccttctccat
420caaggctgcc tggcccatcc gataccccaa ttttgcctca gagcagctct
agcaaggcac 480ctccccccag tctgccctct ccaagcagac tccctggccc
ttcagacact cccattctgc 540cacagtgatg aggatccgcg gccgc
56571650DNAArtificial SequenceOXM-CTP-5x 71ctctagactc gagcgatcgc
catggctacc ggctccagaa cctctctgct gctggccttc 60ggcctgctgt gtctgccttg
gctgcaagag ggcagcgctc attcccaggg caccttcacc 120tccgactact
ccaagtacct ggactctcgc agagcacagg acttcgtgca gtggctgatg
180aacaccaagc ggaaccggaa caatatcgcc tcctccagct ccaaggcccc
tcctccatct 240ctgccatccc ccagtagact gcctgggccc tctgacaccc
ctatcctgcc tcagtccagc 300tcctctaagg ctccaccacc ttccctgcct
agcccttcaa gactgccagg ccctagcgat 360acaccaattc tgccccagtc
ctccagcagc aaggctcccc cacctagcct gccttctcca 420tcaaggctgc
ctggcccatc cgatacccca attttgcctc agagcagctc tagcaaggca
480cctcccccca gtctgccctc tccaagcaga ctccctggcc cttcagacac
tccaatcctc 540ccacagtcct ctagctctaa agctccacct cccagcctgc
ccagccctag tagactcccc 600ggaccttctg atacccccat cttgccccag
tgatgaggat ccgcggccgc 6507225DNAArtificial SequencePrimer
72aatctagagg tcatcatggg ggtgc 257332DNAArtificial SequencePrimer
73attgcggccg cggatccaga agacctttat tg 327425DNAArtificial
SequencePrimer 74taaatattgg ggtgtccgag ggccc 257532DNAArtificial
SequencePrimer 75ccaatattac cacaagcccc accacgcctc at
327635DNAArtificial SequencePrimer 76tgcggccgcg gatccttatc
tgtcccctgt cctgc 357717DNAArtificial SequencePrimer 77gccctgctgt
cggaagc 177832DNAArtificial SequencePrimer 78attgcggccg cggatccaga
agacctttat tg 327932DNAArtificial SequencePrimer 79ctttgaggaa
gaggagccca ggactgggag gc 328024DNAArtificial SequencePrimer
80cctgggctcc tcttcctcaa aggc 248117DNAArtificial SequencePrimer
81gcttccgaca gcagggc 178220DNAArtificial SequencePrimer
82gtttagtgaa ccgtcagaat 208320DNAArtificial SequencePrimer
83ttgaggaaga tgttcgtgta 208420DNAArtificial SequencePrimer
84attacagttg tcgcaggtga 208530DNAArtificial SequencePrimer
85gctggagcta gtgagctttg ttttttcctt 308623DNAArtificial
SequencePrimer 86ttttcactgc attctagttg tgg 238720DNAArtificial
SequencePrimer 87ctcccagttc aattacagct 208827DNAArtificial
SequencePrimer 88ggaaaaactg cctcagcacg ggtgagc 278932DNAArtificial
SequencePrimer 89gtgctgaggc agtttttcct gatgtggact at
329018DNAArtificial SequencePrimer 90caacacagtg ggcagcag
189125DNAArtificial SequencePrimer 91ctcgaggaca tggtctccca ggccc
259225DNAArtificial SequencePrimer 92tctagaatag gtatttttcc acatg
259322DNAArtificial SequencePrimer 93tctagaaaaa agaaatgcca gc
229432DNAArtificial SequencePrimer 94gcggccgcat cctcagggaa
atggggctcg ca 329519DNAArtificial SequencePrimer 95ctctagagga
catggccac 199627DNAArtificial SequencePrimer 96ctggctgtgc
tggggcagaa tgggtgt 279717DNAArtificial SequencePrimer 97ccccagcaca
gccaggg 179818DNAArtificial SequencePrimer 98gcggccgcat ccagacct
189919DNAArtificial SequencePrimer 99ctctagagga catggccac
1910029DNAArtificial SequencePrimer 100gctggagcta gcgatgttgt
tcctgttcc 2910126DNAArtificial SequencePrimer 101acatcgctag
ctccagcagc aaggcc 2610218DNAArtificial SequencePrimer 102gcggccgcat
ccagacct 1810320DNAArtificial SequencePrimer 103ctcccagttc
aattacagct 2010424DNAArtificial SequencePrimer 104gctgtgagcg
ctgccctcct gcag 2410523DNAArtificial SequencePrimer 105gcgctcacag
ccagggcacc ttc 2310618DNAArtificial SequencePrimer 106gcggccgcat
ccagacct 1810725DNAArtificial SequencePrimer 107gctcactagc
tccagcagca aggcc 2510821PRTArtificial Sequencesignal peptide IFN
Beta1 108Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys
Phe Ser1 5 10 15Thr Thr Ala Leu Ser 20109250PRTArtificial
SequenceCTP-modified EPO without the SP 109Ser Ser Ser Ser Lys Ala
Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg1 5 10 15Leu Pro Gly Pro Ser
Asp Thr Pro Ile Leu Pro Gln Ala Pro Pro Arg 20 25 30Leu Ile Cys Asp
Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys 35 40 45Glu Ala Glu
Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn 50 55 60Glu Asn
Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys65 70 75
80Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala
85 90 95Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn
Ser 100 105 110Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys
Ala Val Ser 115 120 125Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala
Leu Gly Ala Gln Lys 130 135 140Glu Ala Ile Ser Pro Pro Asp Ala Ala
Ser Ala Ala Pro Leu Arg Thr145 150 155 160Ile Thr Ala Asp Thr Phe
Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe 165 170 175Leu Arg Gly Lys
Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly 180 185 190Asp Arg
Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro 195 200
205Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser
210 215 220Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg
Leu Pro225 230 235 240Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 245
25011038PRTArtificial SequenceSignal peptide of FVII 110Met Val Ser
Gln Ala Leu Arg Leu Leu Cys Leu Leu Leu Gly Leu Gln1 5 10 15Gly Cys
Leu Ala Ala Val Phe Val Thr Gln Glu Glu Ala His Gly Val 20 25 30Leu
His Arg Arg Arg Arg 35111490PRTArtificial SequenceCTP-modified
FVII/FVIIa without SP MOD-5014 111Ala Asn Ala Phe Leu Glu Glu Leu
Arg Pro Gly Ser Leu Glu Arg Glu1 5 10 15Cys Lys Glu Glu Gln Cys Ser
Phe Glu Glu Ala Arg Glu Ile Phe Lys 20 25 30Asp Ala Glu Arg Thr Lys
Leu Phe Trp Ile Ser Tyr Ser Asp Gly Asp 35 40 45Gln Cys Ala Ser Ser
Pro Cys Gln Asn Gly Gly Ser Cys Lys Asp Gln 50 55 60Leu Gln Ser Tyr
Ile Cys Phe Cys Leu Pro Ala Phe Glu Gly Arg Asn65 70 75 80Cys Glu
Thr His Lys Asp Asp Gln Leu Ile Cys Val Asn Glu Asn Gly 85 90 95Gly
Cys Glu Gln Tyr Cys Ser Asp His Thr Gly Thr Lys Arg Ser Cys 100 105
110Arg Cys His Glu Gly Tyr Ser Leu Leu Ala Asp Gly Val Ser Cys Thr
115 120 125Pro Thr Val Glu Tyr Pro Cys Gly Lys Ile Pro Ile Leu Glu
Lys Arg 130 135 140Asn Ala Ser Lys Pro Gln Gly Arg Ile Val Gly Gly
Lys Val Cys Pro145 150 155 160Lys Gly Glu Cys Pro Trp Gln Val Leu
Leu Leu Val Asn Gly Ala Gln 165 170 175Leu Cys Gly Gly Thr Leu Ile
Asn Thr Ile Trp Val Val Ser Ala Ala 180 185 190His Cys Phe Asp Lys
Ile Lys Asn Trp Arg Asn Leu Ile Ala Val Leu 195 200 205Gly Glu His
Asp Leu Ser Glu His Asp Gly Asp Glu Gln Ser Arg Arg 210 215 220Val
Ala Gln Val Ile Ile Pro Ser Thr Tyr Val Pro Gly Thr Thr Asn225 230
235 240His Asp Ile Ala Leu Leu Arg Leu His Gln Pro Val Val Leu Thr
Asp 245 250 255His Val Val Pro Leu Cys Leu Pro Glu Arg Thr Phe Ser
Glu Arg Thr 260 265 270Leu Ala Phe Val Arg Phe Ser Leu Val Ser Gly
Trp Gly Gln Leu Leu 275 280 285Asp Arg Gly Ala Thr Ala Leu Glu Leu
Met Val Leu Asn Val Pro Arg 290 295 300Leu Met Thr Gln Asp Cys Leu
Gln Gln Ser Arg Lys Val Gly Asp Ser305 310 315 320Pro Asn Ile Thr
Glu Tyr Met Phe Cys Ala Gly Tyr Ser Asp Gly Ser 325 330 335Lys Asp
Ser Cys Lys Gly Asp Ser Gly Gly Pro His Ala Thr His Tyr 340 345
350Arg Gly Thr Trp Tyr Leu Thr Gly Ile Val Ser Trp Gly Gln Gly Cys
355 360 365Ala Thr Val Gly His Phe Gly Val Tyr Thr Arg Val Ser Gln
Tyr Ile 370 375 380Glu Trp Leu Gln Lys Leu Met Arg Ser Glu Pro Arg
Pro Gly Val Leu385 390 395 400Leu Arg Ala Pro Phe Pro Ser Ser Ser
Ser Lys Ala Pro Pro Pro Ser 405 410 415Leu Pro Ser Pro Ser Arg Leu
Pro Gly Pro Ser Asp Thr Pro Ile Leu 420 425 430Pro Gln Ser Ser Ser
Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro 435 440 445Ser Arg Leu
Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser Ser 450 455 460Ser
Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro465 470
475 480Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln
485 49011246PRTArtificial SequenceSignal peptide of FIX 112Met Gln
Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr1 5 10 15Ile
Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu 20 25
30Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg 35 40
45113499PRTArtificial SequenceCTP-modified FIX without SP MOD-3013
113Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg1
5 10 15Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val
Phe 20 25 30Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val
Asp Gly 35 40 45Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser
Cys Lys Asp 50 55 60Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly
Phe Glu Gly Lys65 70 75 80Asn Cys Glu Leu Asp Val Thr Cys Asn Ile
Lys Asn Gly Arg Cys Glu 85 90 95Gln Phe Cys Lys Asn Ser Ala Asp Asn
Lys Val Val Cys Ser Cys Thr 100 105 110Glu Gly Tyr Arg Leu Ala Glu
Asn Gln Lys Ser Cys Glu Pro Ala Val 115 120 125Pro Phe Pro Cys Gly
Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr 130 135 140Arg Ala Glu
Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu145 150 155
160Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn
165 170 175Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly
Gln Phe 180 185 190Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala
Phe Cys Gly Gly 195 200 205Ser Ile Val Asn Glu Lys Trp Ile Val Thr
Ala Ala His Cys Val Glu 210 215 220Thr Gly Val Lys Ile Thr Val Val
Ala Gly Glu His Asn Ile Glu Glu225 230 235 240Thr Glu His Thr Glu
Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His 245 250 255His Asn Tyr
Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu 260 265 270Leu
Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile 275 280
285Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser
290 295 300Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg
Ser Ala305 310 315 320Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val
Asp Arg Ala Thr Cys 325 330 335Leu Arg Ser Thr Lys Phe Thr Ile Tyr
Asn Asn Met Phe Cys Ala Gly 340 345 350Phe His Glu Gly Gly Arg Asp
Ser Cys Gln Gly Asp Ser Gly Gly Pro 355 360 365His Val Thr Glu Val
Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser 370 375 380Trp Gly Glu
Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys385 390 395
400Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Ser
405 410 415Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser
Arg Leu 420 425 430Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Ser
Ser Ser Ser Lys 435 440 445Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser
Arg Leu Pro Gly Pro Ser 450 455 460Asp Thr Pro Ile Leu Pro Gln Ser
Ser Ser Ser Lys Ala Pro Pro Pro465 470 475 480Ser Leu Pro Ser Pro
Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile 485 490 495Leu Pro
Gln114121PRTArtificial SequenceCTP-modified OXM without SP 114His
Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser1 5 10
15Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn
20 25 30Arg Asn Asn Ile Ala Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser
Leu 35 40 45Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile
Leu Pro 50 55 60Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro
Ser Pro Ser65 70 75 80Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu
Pro Gln Ser Ser Ser 85 90 95Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser
Pro Ser Arg Leu Pro Gly 100 105 110Pro Ser Asp Thr Pro Ile Leu Pro
Gln 115 12011526PRTArtificial SequenceSP used for CTP-modified OXM
115Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu1
5 10 15Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala 20 25
* * * * *