U.S. patent application number 13/945265 was filed with the patent office on 2014-11-06 for albumin fusion proteins.
This patent application is currently assigned to Human Genome Sciences, Inc.. The applicant listed for this patent is Human Genome Sciences, Inc.. Invention is credited to William A. Haseltine, Craig A. Rosen, Steven M. Ruben.
Application Number | 20140328794 13/945265 |
Document ID | / |
Family ID | 27585554 |
Filed Date | 2014-11-06 |
United States Patent
Application |
20140328794 |
Kind Code |
A1 |
Rosen; Craig A. ; et
al. |
November 6, 2014 |
ALBUMIN FUSION PROTEINS
Abstract
The present invention encompasses albumin fusion proteins.
Nucleic acid molecules encoding the albumin fusion proteins of the
invention are also encompassed by the invention, as are vectors
containing these nucleic acids, host cells transformed with these
nucleic acids vectors, and methods of making the albumin fusion
proteins of the invention and using these nucleic acids, vectors,
and/or host cells. Additionally the present invention encompasses
pharmaceutical compositions comprising albumin fusion proteins and
methods of treating or preventing diseases, disorders or conditions
related to diabetes mellitus using albumin fusion proteins of the
invention.
Inventors: |
Rosen; Craig A.; (Pasadena,
MD) ; Haseltine; William A.; (Washington, DC)
; Ruben; Steven M.; (Brookeville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Human Genome Sciences, Inc. |
Rockville |
MD |
US |
|
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
27585554 |
Appl. No.: |
13/945265 |
Filed: |
July 18, 2013 |
Related U.S. Patent Documents
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Application
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13078804 |
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13945265 |
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11772591 |
Jul 2, 2007 |
7977306 |
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13078804 |
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11393893 |
Mar 31, 2006 |
7238660 |
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11772591 |
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10775180 |
Feb 11, 2004 |
7189690 |
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11393893 |
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PCT/US02/40892 |
Dec 23, 2002 |
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10775180 |
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60423623 |
Nov 5, 2002 |
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60420246 |
Oct 23, 2002 |
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60417611 |
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60414984 |
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60411355 |
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60402708 |
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60402131 |
Aug 9, 2002 |
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60398008 |
Jul 24, 2002 |
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60378950 |
May 10, 2002 |
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60370227 |
Apr 8, 2002 |
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60367500 |
Mar 27, 2002 |
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60360000 |
Feb 28, 2002 |
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60359370 |
Feb 26, 2002 |
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60350358 |
Jan 24, 2002 |
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60341811 |
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Current U.S.
Class: |
424/85.4 ;
424/134.1; 424/94.1; 435/183; 435/252.3; 435/254.21; 435/320.1;
435/325; 514/11.7; 514/15.2; 514/4.8; 514/5.9; 514/6.8; 514/6.9;
514/7.3; 514/8.5; 530/351; 530/362; 536/23.4 |
Current CPC
Class: |
C07K 14/76 20130101;
C12N 15/62 20130101; C07K 2319/00 20130101; A61P 3/10 20180101;
A61P 3/00 20180101; A61P 3/04 20180101; C07K 14/765 20130101; C07K
14/4713 20130101 |
Class at
Publication: |
424/85.4 ;
530/362; 514/15.2; 514/6.9; 514/7.3; 514/6.8; 514/4.8; 536/23.4;
435/320.1; 435/325; 530/351; 435/254.21; 435/252.3; 435/183;
424/94.1; 424/134.1; 514/8.5; 514/5.9; 514/11.7 |
International
Class: |
C07K 14/76 20060101
C07K014/76 |
Claims
33-38. (canceled)
39. An albumin fusion protein comprising a member selected from the
group consisting of: (a) a Therapeutic protein: X and albumin
comprising the amino acid sequence of SEQ ID NO:327; (b) a
Therapeutic protein: X and a fragment or a variant of the amino
acid sequence of SEQ ID NO:327, wherein said fragment or variant
has albumin activity; (c) a Therapeutic protein: X and a fragment
or a variant of the amino acid sequence of SEQ ID NO:327, wherein
said fragment or variant has albumin activity, and further wherein
said albumin activity is the ability to prolong the shelf life of
the Therapeutic protein: X compared to the shelf-life of the
Therapeutic protein: X in an unfused state; (d) a Therapeutic
protein: X and a fragment or a variant of the amino acid sequence
of SEQ ID NO:327, wherein said fragment or variant has albumin
activity, and further wherein the fragment or variant comprises the
amino acid sequence of amino acids 1-387 of SEQ ID NO:327; (e) a
fragment or variant of a Therapeutic protein: X and albumin
comprising the amino acid sequence of SEQ ID NO:327, wherein said
fragment or variant has a biological activity of the Therapeutic
protein: X; (f) a Therapeutic protein: X, or fragment or variant
thereof, and albumin, or fragment or variant thereof, of (a) to
(e), wherein the Therapeutic protein: X, or fragment or variant
thereof, is fused to the N-terminus of albumin, or the N-terminus
of the fragment or variant of albumin; (g) a Therapeutic protein:
X, or fragment or variant thereof, and albumin, or fragment or
variant thereof, of (a) to (e), wherein the Therapeutic protein: X,
or fragment or variant thereof, is fused to the C-terminus of
albumin, or the C-terminus of the fragment or variant of albumin;
(h) a Therapeutic protein: X, or fragment or variant thereof, and
albumin, or fragment or variant thereof, of (a) to (e), wherein the
Therapeutic protein: X, or fragment or variant thereof, is fused to
the N-terminus and C-terminus of albumin, or the N-terminus and the
C-terminus of the fragment or variant of albumin; (i) a Therapeutic
protein: X, or fragment or variant thereof, and albumin, or
fragment or variant thereof, of (a) to (e), which comprises a first
Therapeutic protein: X, or fragment or variant thereof, and a
second Therapeutic protein: X, or fragment or variant thereof,
wherein said first Therapeutic protein: X, or fragment or variant
thereof, is different from said second Therapeutic protein: X, or
fragment or variant thereof; (j) a Therapeutic protein: X, or
fragment or variant thereof, and albumin, or fragment or variant
thereof, of (a) to (i), wherein the Therapeutic protein: X, or
fragment or variant thereof, is separated from the albumin or the
fragment or variant of albumin by a linker; and (k) a Therapeutic
protein: X, or fragment or variant thereof, and albumin, or
fragment or variant thereof, of (a) to (j), wherein the albumin
fusion protein has the following formula: R1-L-R2; R2-L-R1; or
R1-L-R2-L-R1, and further wherein R1 is Therapeutic protein: X, or
fragment or variant thereof, L is a peptide linker, and R2 is
albumin comprising the amino acid sequence of SEQ ID NO:327 or a
fragment or variant of albumin.
40. The albumin fusion protein of claim 39, wherein the shelf-life
of the albumin fusion protein is greater than the shelf-life of the
Therapeutic protein: X, or fragment or variant thereof, in an
unfused state.
41. The albumin fusion protein of claim 39, wherein the in vitro
biological activity of the Therapeutic protein: X, or fragment or
variant thereof, fused to albumin, or fragment or variant thereof,
is greater than the in vitro biological activity of the Therapeutic
protein: X, or fragment or variant thereof, in an unfused
state.
42. The albumin fusion protein of claim 39, wherein the in vivo
biological activity of the Therapeutic protein: X, or fragment or
variant thereof, fused to albumin, or fragment or variant thereof,
is greater than the in vivo biological activity of the Therapeutic
protein: X, or fragment or variant thereof, in an unfused
state.
43. An albumin fusion protein comprising a Therapeutic protein: X,
or fragment or variant thereof, inserted into an albumin, or
fragment or variant thereof, comprising the amino acid sequence of
SEQ ID NO:327 or fragment or variant thereof.
44. The albumin fusion protein of claim 43, wherein said albumin
fusion protein comprises a portion of albumin sufficient to prolong
the shelf-life of the Therapeutic protein: X, or fragment or
variant thereof, as compared to the shelf-life of the Therapeutic
protein: X, or fragment or variant thereof, in an unfused
state.
45. The albumin fusion protein of claim 43, wherein said albumin
fusion protein comprises a portion of albumin sufficient to prolong
the in vitro biological activity of the Therapeutic protein: X, or
fragment or variant thereof, fused to albumin as compared to the in
vitro biological activity of the Therapeutic protein: X, or
fragment or variant thereof, in an unfused state.
46. The albumin fusion protein of claim 43 wherein said albumin
fusion protein comprises a portion of albumin sufficient to prolong
the in vivo biological activity of the Therapeutic protein: X, or
fragment or variant thereof, fused to albumin compared to the in
vivo biological activity of the Therapeutic protein: X, or fragment
or variant thereof, in an unfused state.
47. The albumin fusion protein of claim 1, which is expressed in
yeast.
48. The albumin fusion protein of claim 1, wherein the albumin
fusion protein further comprises a secretion leader sequence.
49. A composition comprising the albumin fusion protein of claim 1
and a pharmaceutically acceptable carrier.
50. A kit comprising the composition of claim 49.
51. A method of treating a disease or disorder in a patient,
comprising the step of administering the albumin fusion protein of
claim 1.
52. A method of treating a metabolic/endocrine disorder in a
patient, comprising administering the albumin fusion protein of
claim 1.
53. A method of treating diabetes or a condition associated with
diabetes in a patient, comprising administering the albumin fusion
protein of claim 1.
54. A method of treating obesity in a patient, comprising
administering the albumin fusion protein of claim 1.
55. A method of maintaining a basal glucose level in a patient,
comprising administering the albumin fusion protein of claim 1.
56. A nucleic acid molecule comprising a polynucleotide sequence
encoding the albumin fusion protein of claim 1.
57. A vector comprising the nucleic acid molecule of claim 56.
58. A host cell comprising the nucleic acid molecule of claim 56.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/775,180 filed Feb. 11, 2004, which is a
continuation of International Application No. PCT/US02/40892, flied
Dec. 23, 2002, which claims benefit under 35 USC 119(e) of U.S.
Provisional Application Nos. 60/341,811, filed Dec. 21, 2001;
60/360,000, filed Feb. 28, 2002; 60/378,950, filed May 10, 2002;
60/398,008, filed Jul. 24, 2002; 60/411,355, filed Sep. 18, 2002;
60/414,984, filed Oct. 2, 2002; 60/417,611, filed Oct. 11, 2002;
60/420,246, filed Oct. 23, 2002; 60/423,623, filed Nov. 5, 2002;
60/350,358, filed Jan. 24, 2002; 60/359,370, filed Feb. 26, 2002;
60/367,500, filed Mar. 27, 2002; 60/402,131, filed Aug. 9, 2002;
60/402,708, filed Aug. 13, 2002; and 60/370,227, filed Apr. 8,
2002. All of the above listed applications are incorporated by
reference herein in their entirety.
REFERENCE TO SEQUENCE LISTING ON COMPACT DISC
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as an electronic document on three
identical compact discs (CD-R), labeled "Copy 1," Copy 2," and
"Computer Readable Format" These compact discs each contain the
file "Sequence Listing.txt" (1,387,726 bytes, created on Mar. 28,
2006), which is incorporated by reference in its entirety. The
Sequence Listing may be viewed on an IBM-PC machine running the
MS-Windows operating system.
BACKGROUND OF THE INVENTION
[0003] The invention relates generally to Therapeutic proteins
(including, but not limited to, at least one polypeptide, antibody,
peptide, or fragment and variant thereof) fused to albumin or
fragments or variants of albumin. The invention encompasses
polynucleotides encoding therapeutic albumin fusion proteins,
therapeutic albumin fusion proteins, compositions, pharmaceutical
compositions, formulations and kits. Host cells transformed with
the polynucleotides encoding therapeutic albumin fusion proteins
are also encompassed by the invention, as are methods of making the
albumin fusion proteins of the invention using these
polynucleotides, and/or host cells.
[0004] Over the past few decades, an increasing percentage of the
population has become diabetic. Diabetes mellitus is categorized
into two types: Type I, known as Insulin-Dependent Diabetes
Mellitus (IDDM), or Type II, known as Non-Insulin-Dependent
Diabetes Mellitus (NIDDM). IDDM is an autoimmune disorder in which
the insulin-secreting pancreatic beta cells of the islets of
Langerhans are destroyed. In these individuals, recombinant insulin
therapy is employed to maintain glucose homeostasis and normal
energy metabolism. NIDDM, on the other hand, is a polygenic
disorder with no one gene responsible for the progression of the
disease.
[0005] In NIDDM, insulin resistance eventually leads to the
abolishment of insulin secretion resulting in insulin deficiency.
Insulin resistance, at least in part, ensues from a block at the
level of glucose uptake and phosphorylation in humans. Diabetics
demonstrate a decrease in expression in adipose tissue of
insulin-receptor substrate 1 ("IRS1") (Carvalho et al., FASEB J
13(15):2173-8 (1999)), glucose transporter 4 ("GLUT4") (Garvey et
al., Diabetes 41(4):465-75 (1992)) and the novel abundant protein M
gene transcript 1 ("apM1") (Statniek et al., Int. J. Exp. Diabetes
1(2):81-8 (2000)), as well as other as of yet unidentified factors.
Insulin deficiency in NIDDM leads to failure of normal pancreatic
beta-cell function and eventually to pancreatic-beta cell
death.
[0006] Insulin affects fat, muscle, and liver. Insulin is the major
regulator of energy metabolism. Malfunctioning of any step(s) in
insulin secretion and/or action can lead to many disorders,
including for example the dysregulation of oxygen utilization,
adipogenesis, glycogenesis, lipogenesis, glucose uptake, protein
synthesis, thermogenesis, and maintenance of the basal metabolic
rate. This malfunctioning results in diseases and/or disorders that
include, but are not limited to, hyperinsulinemia, insulin
resistance, insulin deficiency, hyperglycemia, hyperlipidemia,
hyperketonemia, and diabetes.
[0007] Numerous debilitating diabetes-related secondary effects
include, but are not limited to, obesity, farms of blindness
(cataracts and diabetic retinopathy), limb amputations, kidney
failure, fatty liver, coronary artery disease, and neuropathy.
[0008] Some of the current drugs used to treat insulin resistance
and/or diabetes (e.g., insulin secratogogues--sulfonylurea, insulin
sensitizers--thiazolidenediones and metformin, and
.alpha.-glucosidase and lipase inhibitors) are inadequate due to
the dosage amounts and frequency with which they have to be
administered as a result of poor pharmacokinetic properties, the
lack of effective control over blood sugar levels, and potential
side effects, among other reasons. Diabetes Therapeutic proteins in
their native state or when recombinantly produced exhibit a rapid
in vivo clearance. Typically, significant amounts of therapeutics
are required to be effective during therapy. In addition, small
molecules smaller than the 20 kDa range can be readily filtered
through the renal tubules (glomerulus) leading to dose-dependent
nephrotoxicity.
[0009] Diabetes Therapeutic proteins in their native state or when
recombinantly produced are typically labile molecules exhibiting
short shelf-lives, particularly when formulated in aqueous
solutions. The instability in these molecules when formulated for
administration dictates that many of the molecules must be
lyophilized and refrigerated at all times during storage, thereby
rendering the molecules difficult to transport and/or stoic.
Storage problems are particularly acute when pharmaceutical
formulations must be stored and dispensed outside of the hospital
environment. Many protein and peptide drug compositions also
require the addition of high concentrations of other protein such
as albumin to reduce or prevent loss of protein due to binding to
the container. For this reason, many therapeutic proteins are
formulated in combination with a large proportion of albumin
carrier molecule (100-1000 fold excess), though this is an
undesirable and expensive feature of the formulation.
[0010] Few practical solutions to the storage problems of labile
protein molecules have been proposed. Accordingly, there is a need
for stabilized, long lasting formulations of proteinaceous
therapeutic molecules that are easily dispensed, preferably with a
simple formulation requiring minimal post storage manipulation.
[0011] Human serum albumin possesses many desirable
characteristics. HSA is found throughout the body, but more
specifically in the interstitial space and in blood at serum
concentrations of 40 g/L which is equivalent to 0.7 mal (Yeh et
al., Proc. Natl. Acad. Sci. USA, 89:1904-1908 (1992)). HSA is
considered to be the meet abundant protein of the serum and is
responsible for maintaining osmolarity. HSA has favorable
pharmacokinetic properties and is cleared very slowly by the liver
displaying in vivo half-lives up to several weeks (Yeh et al.,
Proc. Natl. Acad. Sci. USA, 89:1904-1908 (1992); Waldmann, T. A.,
Albumin Structure, Function and Uses, pp. 255-273 (1977)). HSA
lacks enzymatic activity and antigenicity thereby eliminating
potentially undesirable side effects. HSA acts as a carrier for
endogenous as well as exogenous ligands. Combined, these features
can be extended, at least partially, onto albumin fusion proteins.
The poor pharmacokinetic properties displayed by Diabetes
Therapeutic proteins can then be circumvented.
[0012] Human serum albumin (HSA, or HA), a protein of 585 amino
acids in its mature form (SEQ ID NO:327) of approximately 66 kDa,
is responsible for a significant proportion of the osmotic pressure
of serum and also functions as a carrier of endogenous and
exogenous ligands. At present, HA for clinical use is produced by
extraction from human blood. The production of recombinant HA (rHA)
in microorganisms has been disclosed in EP 330 451 and EP 361
991.
[0013] The present invention is directed to albumin fusion proteins
comprising a Therapeutic protein (e.g., a polypeptide, antibody, or
fragment or variant thereof) fused to albumin or a fragment
(portion) or variant of albumin. The present invention also
encompasses polynucleotides comprising, or alternatively consisting
of, nucleic acid molecules encoding a Therapeutic protein (e.g., a
polypeptide, antibody, or fragment or variant thereof) fused to
albumin or a fragment (portion) or variant of albumin that is
sufficient to prolong the shelf life of the Therapeutic protein,
increase serum half-life, and/or stabilize the Therapeutic protein
and/or its activity in solution (or in a pharmaceutical
composition) in vitro and/or in vivo. In one embodiment, an albumin
fusion protein encoded by a polynucleotide described in Table 1 or
2 has extended shelf life. In a second embodiment, an albumin
fusion protein encoded by a polynucleotide described in Table 1 or
2 has a longer serum half-life and/or stabilized activity in
solution (or in a pharmaceutical composition) in vitro and/or in
vivo than the corresponding unfused Therapeutic molecule described
in Table 1 or 2. The present invention also encompasses
polynucleotides, comprising, or alternatively consisting of,
nucleic acid molecules encoding proteins comprising a Therapeutic
protein (e.g., a polypeptide, antibody, or fragment or variant
thereof) fused to albumin or a fragment (portion) or variant of
albumin. Albumin fusion proteins encoded by polynucleotides of the
invention are also encompassed by the invention, as are host cells
containing these polynucleotides, and methods of making the albumin
fusion proteins of the invention and using these polynucleotides,
and/or host cells. The invention also encompasses polynucleotides
encoding therapeutic albumin fusion proteins, therapeutic albumin
fusion proteins, compositions, pharmaceutical compositions,
formulations and kits.
SUMMARY OF THE INVENTION
[0014] In a preferred embodiment, the albumin fusion protein of the
invention comprises one or more of the Therapeutic proteins
disclosed in the "Therapeutic Protein: X" column of Table 1. Fusion
proteins comprising fragments or variants of one or more of the
Therapeutic proteins as disclosed in the "Construct Name" column of
Table 2 are also encompassed by the invention. Polynucleotides
comprising, or alternatively consisting of, nucleic acid molecules
encoding the above albumin fusion proteins are also encompassed by
the invention, as are host cells containing these polynucleotides.
In one embodiment, an albumin fusion protein encoded by a
polynucleotide described in Table 1 or 2 has extended shelf life.
In a second embodiment, an albumin fusion protein encoded by a
polynucleotide described in Table 1 or 2 has a longer serum
half-life and/or stabilized activity in solution (or in a
pharmaceutical composition) in vitro and/or in vivo than the
corresponding unfused Therapeutic molecule described in Table
1.
[0015] In a preferred aspect of the invention, albumin fusion
proteins include, but are not limited to, those encoded by the
polynucleotides described in Table 2.
[0016] The invention also encompasses pharmaceutical formulations
comprising an albumin fusion protein of the invention and a
pharmaceutically acceptable diluent or carrier. Such formulations
may be in a kit or container. Such kit or container may be packaged
with instructions pertaining to the extended shelf life of the
Therapeutic protein. Such formulations may be used in methods of
treating (e.g., ameliorating) preventing, or diagnosing a disease
or disease symptom in a patient, preferably a mammal, most
preferably a human, comprising the step of administering the
pharmaceutical formulation to the patient.
[0017] In other embodiments, the present invention encompasses
methods of preventing or treating (e.g., ameliorating) a disease or
disorder. In preferred embodiments, the present invention
encompasses a method of treating a disease or disorder listed in
the "Preferred Indication Y" column of Table 1 comprising
administering to a patient in which such treatment or prevention is
desired an albumin fusion protein of the invention that comprises a
Therapeutic protein portion corresponding so a Therapeutic protein
(or fragment or variant thereof) disclosed in the "Therapeutic
Protein: X" column of Table 3 in the same row as the disease or
disorder to be treated is listed in the "Preferred Indication Y"
column of Table 1) in an amount effective to treat (e.g.,
ameliorate) or prevent the disease or disorder. Moreover, diseases
or disorders that can be treated or prevented with an albumin
fusion protein of the invention include, but are not limited to,
diabetes (e.g., Non-Insulin-Dependent Diabetes Mellitus (NIDDM) or
Insulin-Dependent Diabetes Mellitus (IDDM)), insulin resistance,
insulin deficiency, hyperinsulinemia, hyperglycemia, dyslipidemia,
hyperlipidemia hyperketonemia, hypertension, coronary artery
disease, atherosclerosis, renal failure, neuropathy (e.g.,
autonomic neuropathy, parasympathetic neuropathy, and
polyneuropathy), retinopathy, cataracts, metabolic disorders (e.g.,
insulin and/or glucose metabolic disorders), endocrine disorders,
obesity, weight loss, liver disorders (e.g., liver disease,
cirrhosis of the liver, and disorders associated with liver
transplant), and conditions associated with these diseases or
disorders.
[0018] In one embodiment, an albumin fusion protein described in
Table 1 or 2 has extended shelf life.
[0019] In a second embodiment, an albumin fusion protein described
in Table 1 or 2 is more stable than the corresponding unfused
Therapeutic molecule described in Table 1.
[0020] In another preferred embodiment, the "Therapeutic protein"
is a protein that is useful to treat (e.g., ameliorate) or prevent
a metabolic/endocrine disorder. In a highly preferred embodiment,
the metabolic/endocrine disorder is diabetes and/or a condition
associated with diabetes. As a non-limiting example, a "Therapeutic
protein" may be one that regulates glucose uptake by cells, that
binds specifically to a particular cell type (e.g., normal
adipocytes, myotubes, hepatocytes, and pancreatic beta cells of the
Islet of Langerhans, and/or abnormal (e.g., cancer cell or
insulin-resistant adipocytes, myotubes, and hepatocytes)), that
enhances insulin sensitivity in insulin-responsive tissues, and/or
that regulates hepatin glucose output, and therefore may be used to
target a compound (drug, or cytotoxic agent) to that cell type
specifically.
[0021] In highly preferred embodiments, the present invention
encompasses methods of preventing or treating (e.g., ameliorating)
diabetes and/or a condition associated with diabetes comprising
administering to a patient (preferably a human) in which such
prevention or treatment is desired an albumin fusion protein of the
invention that comprises a Therapeutic protein portion
corresponding to a Therapeutic protein (or fragment or variant
thereof) disclosed in the "Therapeutic Protein: X" column of Table
1 in an amount effective to treat or prevent diabetes and/or a
condition associated with diabetes, Conditions associated with
diabetes that can be prevented or treated with an albumin fusion
protein of the invention include, but are not limited to,
hyperglycemia, obesity, diabetic retinopathy, mononeuropathy,
polyneuropathy, atherosclerosis, ulcers, heart disease, stroke,
anemia, gangrene (e.g., of the feet and hands), impotence,
infection, cataract, poor kidney function, malfunctioning of the
autonomic nervous system, impaired white blood cell function,
Carpal tunnel syndrome, Dupuytren's contracture, and diabetic
ketoacidosis.
[0022] In a most preferred embodiment, an albumin fusion protein of
the invention is administered to a patient to modulate or maintain
glucose homeostasis.
[0023] In other embodiments, the present invention encompasses a
method of maintaining a basal level of insulin in a patient
comprising administering an albumin fusion protein of the
invention.
[0024] The invention also relates to methods of regulating (e.g.,
suppressing or decreasing) appetite, and methods of altering
nutritional partitioning in a patient (e.g., methods of increasing
muscle mass and/or methods of decreasing fat mass) comprising
administering to a patient (preferably a human) an albumin fusion
protein of the invention.
[0025] The invention also relates to methods of treating or
preventing insulin-related disorders comprising administering to a
patient (preferably a human) an albumin fusion protein of the
invention.
[0026] The invention further relates to methods of regulating
insulin responsiveness in a patient, as well as methods of
increasing glucose uptake by a cell, and methods of regulating
insulin sensitivity of a cell, using the albumin fusion proteins of
the invention.
[0027] The albumin fusion proteins of the invention may be
administered alone on in combination with other Therapeutic
proteins or molecules (e.g., insulin and/or other proteins
(including antibodies), peptides, or small molecules that regulate
insulin sensitivity, weight, heart disease, hypertension,
neuropathy, cell metabolism and/or glucose, insulin, or other
hormone levels, in a patient). In specific embodiments, the albumin
fusion proteins of the invention are administered in combination
with insulin (or an insulin derivative, analog, fusion protein, or
secretagogue).
[0028] The present invention further includes transgenic organisms
modified to contain or express compositions of the invention
(including, but not limited to, fusion proteins and/or the
polynucleotides described in Tables 1 and 2), preferably modified
to express an albumin fusion protein of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1 depicts the ability of GLP-1 albumin fusion proteins
and exendin-4 albumin fusion proteins to enhance glucose
sensitivity and uptake into 3T3-L1 adipocytes as compared to GLP-1
and exendin-4 alone, respectively.
[0030] FIG. 2 shows the effectiveness of insulin albumin fusion
proteins (CID 2250 and 2276) to allow for glucose uptake into
3T3-L1 adipocytes.
[0031] FIG. 3 is a map of the vector pPPC0005 cloning vector (ATCC
Deposit Number PTA-3278).
[0032] FIG. 4 is a map of the pSAC35 yeast S. Cerevisiae expression
vector (Sleep at al., Biotechnology 8:42 (1990)).
[0033] FIG. 5A-D shows the amino acid sequence of the mature form
of human albumin (SEQ ID NO:327) and a polynucleotide encoding it
(SEQ ID NO:326).
[0034] FIG. 6 shows the effect of various dilutions of IFNb albumin
fusion proteins encoded by DNA comprised in CID 2011 and 2053 on
SEAP activity in the ISRE-SEAP/293f reporter cells (see Example
19). Proteins were serially diluted front 5e-7 to 1e-14 g/ml in
DMEM/10% FBS and used to treat ISRE-SEAP/293f reporter cells. After
24 hours supernatants were removed from reporter cells and assayed
for SEAP activity. IFNb albumin fusion protein was purified from
three stable clones: 293f/#2011, CHO/#2011 and NS0/#2053. Mammalian
derived IFNb, Avonex, came from Biogen and was reported to have a
specific activity of 2.0e5 IU/ug.
[0035] FIG. 7 illustrates the steady-state levels of insulin mRNA
in INS-1 (832/13) cells after treatment with GLP-1 or GLP-1 albumin
fusion protein encoded by construct ID 3070 (CID 3070 protein).
Both GLP-1 and the CID 3070 protein stimulate transcription of the
insulin gene in INS-1 cells. The first bar (black) represents the
untreated cells. Bars 2-4 (white) represent cells treated with the
indicated concentrations of GLP-1. Bars 5-7 (gray) represent cells
treated with the indicated concentrations of an 3070 protein.
[0036] FIG. 8 compares the anti-proliferative activity of IFN
albumin fusion protein encoded by CID 3165 (CID 3165 protein) and
recombinant IFNa (rIFNa) on Hs294T melanoma cells. The cells were
cultured with varying concentrations of either CID 3165 protein or
rIFNa and proliferation was measured by BrdU incorporation after 3
days of culture. CID 3165 protein caused measurable inhibition of
cell proliferation at concentrations above 10 ng/ml with 50%
inhibition achieved at approximately 200 ng/ml. (.box-solid.)=CID
3165 protein, (.diamond-solid.)=rIFNa.
[0037] FIG. 9 shows the effect of various dilutions of IFNa albumin
fusion proteins on SEAP activity in the ISRE-SEAP/293F reporter
cells. One preparation of IFNa fused upstream of albumin
(.diamond-solid.) was tested, as well as two different preparations
of IFNa fused downstream of albumin (.tangle-solidup.) and
(.box-solid.).
[0038] FIG. 10 shows the effect of time and dose of IFNa albumin
fusion protein encoded by DNA comprised in construct 2249 (CID 2249
protein) on the mRNA level of OAS (p41) in treated monkeys (see
Example 92). Per time point: first bar=Vehicle control, 2.sup.nd
bar=30 ug/kg CID 2249 protein day 1 iv, third bar=30 ug/kg CID 2249
protein day 1 sc, 4.sup.th bar=300 ug/kg CID 2249 protein day 1 sc,
5.sup.th bar=40 ug/kg recombinant IFNa day 1, 3 and 5 sc.
DETAILED DESCRIPTION
Definitions
[0039] The following definitions are provided to facilitate
understanding of certain terms used throughout this
specification,
[0040] As used herein, "polynucleotide" refers to a nucleic acid
molecule having a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO:Y (wherein Y is a number shown in column 6 of
Table 2) or a fragment or variant thereof; a nucleotide sequence
generated as described in Table 2 or in the Examples; a nucleotide
sequence contained in an albumin fusion construct described in
Table 2; a nucleotide sequence contained in an albumin fusion
construct deposited with the ATCC (as described in Table 3); and/or
more generally to a nucleotide sequence encoding a Therapeutic
albumin fusion protein of the invention. Also as used herein,
"polynucleotide" refers to a nucleic acid molecule having a
nucleotide sequence encoding a fusion protein comprising, or
alternatively consisting of, at least one molecule of albumin (or a
fragment or variant thereof) joined in frame to at least one
Therapeutic protein X (or fragment or variant thereof); a nucleic
acid molecule having a nucleotide sequence encoding a fusion
protein comprising, or alternatively consisting of, the amino acid
sequence of SEQ ID NO:Y (as described in column 6 of Table 2) or a
fragment or variant thereof; a nucleic acid molecule having a
nucleotide sequence comprising or alternatively consisting of the
sequence shown in SEQ ID NO:X; a nucleic acid molecule having a
nucleotide sequence encoding a fusion protein comprising, or
alternatively consisting of, the amino acid sequence of SEQ ID
NO:Z; a nucleic acid molecule having a nucleotide sequence encoding
an albumin fission protein of the invention generated as described
in Table 2 or in the Examples; a nucleic acid molecule having a
nucleotide sequence encoding a Therapeutic albumin fusion protein
of the invention, a nucleic acid molecule having a nucleotide
sequence contained in an albumin fusion construct described in
Table 2.
[0041] As used herein, "albumin fusion construct" refers to: a
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide encoding at least one molecule of albumin (or a
fragment or variant thereof) joined in frame to at least one
polynucleotide encoding at least one molecule of a Therapeutic
protein of the invention (including fragments and variants); a
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide encoding at least one molecule of albumin (or a
fragment or variant thereof) joined in frame to at least one
polynucleotide encoding at least one molecule of a Therapeutic
protein of the invention (including fragments and variants)
generated as described in Table 2 or in the Examples; and/or a
nucleic acid molecule comprising, or alternatively consisting of,
one or more of the above polynucleotides and further comprising, or
alternatively consisting of, one or more of the following: (1) a
functional self-replicating vector (including but not limited to, a
shuttle vector, an expression vector, an integration vector, and/or
a replication system), (2) a region for initiation of transcription
(e.g., a promoter region, such as for example, a regulatable or
inducible promoter, a constitutive promoter), (3) a region for
termination of transcription, (4) a leader sequence, and (5) a
selectable marker. The polynucleotide encoding the Therapeutic
protein and albumin protein, once part of the albumin fusion
construct, may each be referred to herein as a "portion," "region"
or "moiety" of the albumin fusion construct.
[0042] As used herein, "mature albumin fusion protein" refers to:
the processed form of an albumin fusion protein. An albumin fusion
protein of the invention is processed by a host cell and secreted
into the surrounding culture medium. Processing of the nascent
albumin fusion protein that occurs in the secretory pathways of the
host used for expression may include, but is not limited to signal
peptide cleavage; formation of disulfide bonds; proper folding;
addition and processing of carbohydrates (such as for example, N-
and O-linked glycosylation); specific proteolytic cleavages; and
assembly into multimeric proteins. An albumin fusion protein of the
invention is preferably in the processed form. In a most preferred
embodiment, the "processed form of an albumin fusion protein"
refers to an albumin fusion protein product which has undergone
N-terminal signal peptide cleavage, herein also referred to as a
"mature albumin fusion protein".
[0043] The present invention relates generally to polynucleotides
encoding albumin fusion proteins; albumin fusion proteins; and
methods of treating (e.g., ameliorating) or preventing a disease or
disorder using albumin fusion proteins or polynucleotides encoding
albumin fusion proteins of the invention. As used herein, "albumin
fusion protein" refers to a protein formed by the fusion of at
least one molecule of albumin (or a fragment or variant thereof) to
at least one molecule of a Therapeutic protein (or fragment or
variant thereof). In preferred embodiments, the present invention
relates to treating (e.g., ameliorating) or preventing a
metabolic/endocrine disorder using a polynucleotide and/or albumin
fusion protein of the invention. In highly preferred embodiments,
the present invention relates to a method of treating or preventing
diabetes and/or a condition associated with diabetes using a
polynucleotide and/or albumin fusion protein of the invention. In a
highly preferred embodiment, an albumin fusion protein of the
invention comprises at least one molecule of a Therapeutic protein
X or fragment or variant of thereof (including, but not limited to
a mature form of the Therapeutic protein X) and at least one
molecule of albumin or fragment or variant thereof (including but
not limited to a mature form of albumin).
[0044] As used herein, "albumin fusion protein" refers to a protein
formed by the fusion of at least one molecule of albumin (or a
fragment or variant thereof) to at least one molecule of a
Therapeutic protein (or fragment or variant thereof). An albumin
fusion protein of the invention comprises at least a fragment or
variant of a Therapeutic protein and at least a fragment or variant
of human serum albumin, which are associated with one another,
preferably by genetic fusion (i.e., the albumin fusion protein is
generated lay translation of a nucleic acid in which a
polynucleotide encoding all or a portion of a Therapeutic protein
is joined in-frame with a polynucleotide encoding all or a portion
of albumin). The Therapeutic protein and albumin protein, once part
of the albumin fusion protein, may each be referred to herein as a
"portion", "region" or "moiety" of the albumin fusion protein
(e.g., a "Therapeutic protein portion" or an "albumin protein
portion").
[0045] An additional embodiment includes a protein containing at
least one protein, fragment, or variant of a Therapeutic protein of
the invention and at least a protein fragment or valiant of human
serum albumin, which are associated with one another, via chemical
conjugation.
[0046] In several instances, a representative clone containing an
albumin fusion construct of the invention was deposited with the
American Type Culture Collection (herein referred to as
"ATCC.RTM."). Furthermore, it is possible to retrieve a given
albumin fission construct from the deposit by techniques known in
the an and described elsewhere herein. The ATCC.RTM. is located at
10801 University Boulevard, Manassas, Va. 20110-2209, USA. The
ATCC.RTM. deposits were made pursuant to the terms of the Budapest
Treaty on the international recognition of the deposit of
microorganisms for the purposes of patent procedure.
[0047] In one embodiment, the invention provides a polynucleotide
encoding an albumin fusion protein comprising, or alternatively
consisting of a Therapeutic protein and a serum albumin protein. In
a further embodiment, the invention provides an albumin fusion
protein comprising, or alternatively consisting of, a Therapeutic
protein and a serum albumin protein. In a preferred embodiment, the
invention provides an albumin fusion protein comprising, or
alternatively consisting of, a Therapeutic protein and a serum
albumin protein encoded by a polynucleotide described in Table 2.
In a further preferred embodiment, the invention provides a
polynucleotide encoding an albumin fusion protein whose sequence is
shown as SEQ ID NO:Y in Table 2. In other embodiments, the
invention provides an albumin fusion protein comprising, or
alternatively consisting of, a biologically active and/or
therapeutically active fragment of a Therapeutic protein and a
serum albumin protein. In other embodiments, the invention provides
an albumin fusion protein comprising, or alternatively consisting
of, a biologically active and/or therapeutically active variant of
a Therapeutic protein and a serum albumin protein. In preferred
embodiments, the serum albumin protein component of the albumin
fusion protein is the mature portion of serum albumin. The
invention further encompasses polynucleotides comprising, or
alternatively consisting of, nucleic acid molecules and host cells
containing these nucleic acids encoding these albumin fusion
proteins.
[0048] In a further embodiment, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
Therapeutic protein, and a biologically active and/or
therapeutically active fragment of serum albumin. In a further
embodiment, the invention provides an albumin fusion protein
comprising, or alternatively consisting of, a Therapeutic protein
and a biologically active and/or therapeutically active variant of
serum albumin. In preferred embodiments, the Therapeutic protein
portion of the albumin fusion protein is the mature portion of the
Therapeutic protein. In a further preferred embodiment, the
Therapeutic protein portion of the albumin fusion protein is the
extracellular soluble domain of the Therapeutic protein. In an
alternative embodiment, the Therapeutic protein portion of the
albumin fusion protein is the active form of the Therapeutic
protein. The invention further encompasses polynucleotides
comprising, or alternatively consisting of, nucleic acid molecules
encoding the albumin fusion proteins of the invention. Host cells
containing these polynucleotides are also encompassed by the
invention as are methods of making albumin fusion proteins using
these host cells.
[0049] In a further embodiment, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
biologically active and/or therapeutically active fragment or
variant of a Therapeutic protein and a biologically active and/or
therapeutically active fragment or variant of serum albumin. In a
preferred embodiment, the invention provides an albumin fusion
protein comprising, or alternatively consisting of, the mature
portion of a Therapeutic protein and the mature portion of serum
albumin. The invention further encompasses polynucleotides
comprising, or alternatively consisting of, nucleic acid molecules
encoding the albumin fusion proteins of the invention. Host cells
containing these polynucleotides are also encompassed by the
invention as are methods of making albumin fusion proteins using
these host cells.
[0050] Therapeutic Proteins
[0051] As stated above, a polynucleotide of the invention encodes a
protein comprising or alternatively consisting of, at least a
fragment or variant of a Therapeutic protein and at least a
fragment or variant of human serum albumin, which are associated
with one another, preferably by genetic fusion.
[0052] As used herein, "Therapeutic protein" refers to proteins,
polypeptides, antibodies, peptides or fragments or variants
thereof, having one or more therapeutic and/or biological
activities. Therapeutic proteins encompassed by the invention
include but are not limited to, proteins, polypeptides, peptides,
antibodies, and biologics. (The terms peptides, proteins, and
polypeptides are used interchangeably herein.) It is specifically
contemplated that the term "Therapeutic protein" encompasses
antibodies and fragments and variants thereof. Thus a protein of
the invention may contain at least a fragment or variant of a
Therapeutic protein, and/or at least a fragment or variant of an
antibody. Additionally, the term "Therapeutic protein" may refer to
the endogenous or naturally occurring correlate of a Therapeutic
protein.
[0053] By a polypeptide displaying a "therapeutic activity" or a
protein that is "therapeutically active" is meant a polypeptide
that possesses one or more known biological and/or therapeutic
activities associated with a therapeutic protein such as one or
more of the Therapeutic proteins described herein or otherwise
known in the art. In a preferred embodiment, the "Therapeutic
protein" is a protein that is useful to treat (ameliorate) or
prevent a metabolic/endocrine disorder. As a non-limiting example,
a "Therapeutic protein" may be one that binds specifically to a
particular cell type (normal (e.g., lymphocytes) or abnormal e.g.,
(cancer cells)) and therefore may be used to target a compound
(drug, or cytotoxic agent) to that cell type specifically.
[0054] In a highly preferred embodiment, the metabolic/endocrine
disorder is diabetes mellitus and/or one or more conditions
associated with diabetes. In preferred embodiments, the
"Therapeutic protein" is a protein that is useful to treat (e.g.,
ameliorate) or prevent Type II Non-Insulin-Dependent Diabetes
Mellitus (NIDDM) and/or a condition or conditions associated with
NIDDM. In other preferred embodiments, the "Therapeutic protein" is
a protein that is useful to treat (e.g., ameliorate) or prevent
Type I Insulin-Dependent Diabetes Mellitus (IDDM) and/or a
condition or conditions associated with IDDM. In other preferred
embodiments, the "Therapeutic protein" is a protein that is useful
to treat (e.g., ameliorate) or prevent a condition including, but
not limited to, hyperglycemia, obesity, diabetic retinopathy,
mononeuropathy, polyneuropathy, atherosclerosis, ulcers, heart
disease, stroke, anemia, gangrene (e.g. of the feet and hands),
impotence, infection, cataract, poor kidney function,
malfunctioning of the autonomic nervous system, impaired white
blood cell function, Carpal tunnel syndrome, Dupuytren's
contracture, and diabetic ketoacidosis.
[0055] As a non-limiting example, a "Therapeutic protein" may be
one that regulates glucose uptake by cells and/or that binds
specifically to a particular cell type (e.g., normal adipocytes,
myotubes, hepatocytes, and pancreatic beta cells of the Islet of
Langerhans, and/or abnormal (e.g., cancer cell or insulin-resistant
adipocytes, myotubes, and hepatocytes)), that enhances insulin
sensitivity in insulin-responsive tissues, and/or that regulates
hepatic glucose output, and therefore may be used to target a
compound (drug, or cytotoxic agent) to that cell type
specifically.
[0056] For example, a non-exhaustive list of "Therapeutic protein"
portions which may be comprised by an albumin fusion protein of the
invention includes, but is not limited to, proteins comprising, a
polypeptide having an amino acid sequence disclosed in Table 2,
column 8 as SEQ ID NO: Z and fragments or variants thereof; or in
Table 1, column 1 as "Therapeutic Protein X" and fragments and
variants thereof.
[0057] Interferon hybrids may also be fused to the amino or carboxy
terminus of albumin to form an interferon hybrid albumin fusion
protein. Interferon hybrid albumin fusion protein may have
enhanced, or alternatively, suppressed interferon activity, such as
antiviral responses, regulation of cell growth, and modulation of
immune response (Lebleu at al., PNAS USA, 73:3107-3111 (1976);
greater at al Nature, 251:543-545 (1974); and Johnson, Texas
Reports Biol Med, 35357-369 (1977)). Each interferon hybrid albumin
fusion protein can be used to treat, prevent, or ameliorate viral
infections (e.g., hepatitis (e.g., HCV); or HIV), multiple
sclerosis, or cancer.
[0058] In one embodiment, the interferon hybrid portion of the
interferon hybrid albumin fusion protein comprises an interferon
alpha-interferon alpha hybrid (herein referred to as an alpha-alpha
hybrid). For example, the alpha-alpha hybrid portion of the
interferon hybrid albumin fusion protein consists, or alternatively
comprises, of interferon alpha A fused to interferon alpha D. In a
further embodiment, the A/D hybrid is fused at the common BgIII
restriction site to interferon alpha D, wherein the N-terminal
portion of the A/D hybrid corresponds to amino acids 1-62 of
interferon alpha A and the C-terminal portion corresponds to amino
acids 64-166 of interferon alpha D. For example, this A/D hybrid
would comprise the amino acid sequence:
CDLPQTHSLGSRRTLMLLAQMRX.sub.1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLF-
TKDSSAAWDEDLLDKFC
TELYQQLNDLEACVMQEERVGETPLMNX.sub.2DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLS-
TNLQERLRRKE (SEQ ID NO:472), wherein the X.sub.1 is R or K and the
X.sub.2 is A or V (see, for example, Construct ID #2875). In an
additional embodiment, the A/D hybrid is fused at the common PvuIII
restriction site, wherein the N-terminal portion of the A/D hybrid
corresponds to amino acids 1-91 of interferon alpha A and the
C-terminal portion corresponds to amino acids 93-166 of interferon
alpha D. For example, this A/D hybrid would comprise the amino acid
sequence:
CDLPQTHSLGSRRTLMLLAQMRX.sub.1ISLFSCLKDRHDFGPPQEEFGNQFQKAETIPVLIIEMIQQIFNL-
FSTKDSSAAWDETLLDKFY
TELYQQLNDLEACVMQEERVGETPLMNX.sub.2DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSSL-
STNLQERLRRKE (SEQ ID NO:457), wherein the X.sub.1 is R or K and the
X.sub.2 is A or V (see, for example, Construct ID #2872). These
hybrids are further described in U.S. Pat. No. 4,414,510, which is
hereby incorporated by reference in its entirety.
[0059] In an additional embodiment, the alpha-alpha hybrid portion
of the interferon hybrid albumin fusion protein consists, or
alternatively comprises, of interferon alpha A fused to interferon
alpha F. In a further embodiment, the A/F hybrid is fused al the
common PvuIII restriction site, wherein the N-terminal portion of
the A/F hybrid corresponds to amino acids 1-91 of interferon alpha
A and the C-terminal portion corresponds to amino acids 93-166 of
interferon alpha F. For example, this A/B hybrid would comprise the
amino acid sequence:
CDLPQTHSLGSRRTLMLLAQMRXISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSS-
AAWDETLLDKFYT
ELYQQLNDMEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQE-
RLRRKE (SEQ ID NO:467), wherein X is either R or K (see, for
example, Construct ID #2874). These hybrids are further described
in U.S. Pat. No. 4,414,510, which is hereby incorporated by
reference in its entirety. In a further embodiment, the alpha-alpha
hybrid portion of the interferon hybrid albumin fusion protein
consists, or alternatively comprises, of interferon alpha A fused
to interferon alpha B. In an additional embodiment, the A/B hybrid
is fused at the common PvuIII restriction site, wherein the
N-terminal portion of the A/B hybrid corresponds to amino acids
1-91 of interferon alpha A and the C-terminal portion corresponds
to amino acids 93-166 of interferon alpha B. For example, this A/B
hybrid would comprise an amino acid sequence:
CDLPQTHSLGSRRTLMLLAQMRX.sub.1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLF-
STKDSS AAWDETLLDKFY
TELYQQLNDLEX.sub.1X.sub.3X.sub.4X.sub.5QEVGVIESPLMYEDSILAVRKYFQRITLYLTEKK-
YSSCAWEVVRAEIMRSFSLSINLQKRLKSKE (SEQ ID NO:462), wherein the
X.sub.1 is R or K and X.sub.2 through X.sub.5 is SCVM or VLCD (see,
for example, Construct ID #2873). These hybrids are further
described in U.S. Pat. No. 4,414,530, which is hereby incorporated
by reference in its entirety.
[0060] In another embodiment, the interferon hybrid portion of the
interferon hybrid albumin fusion protein comprises an interferon
beta-interferon alpha hybrid (herein referred to as a beta-alpha
hybrid). For example, the beta-alpha hybrid portion of the
interferon hybrid albumin fusion protein consists, or alternatively
comprises, of interferon beta-1 fused to interferon alpha D (also
referred to as interferon alpha-1).
[0061] In a further embodiment, the beta-1/alpha D hybrid is fused
wherein the N-terminal portion corresponds to amino acids 1-73 of
interferon beta-1 and the C-terminal portion corresponds to amino
acids 74-167 of interferon alpha D. For example, this beta-I/alpha
D hybrid would comprise an amino acid sequence:
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYENILQNIFAIFRQ-
DSSAAWDEDLLDK
FCTELYQQLNDLEACVMQEERVGETPLMNXDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTN-
LQERLRRKE (SEQ ID NO:823), wherein X is A or V. These hybrids are
further described in U.S. Pat. No. 4,758,428, which is hereby
incorporated by reference in its entirety.
[0062] In another embodiment, the interferon hybrid portion of the
interferon hybrid albumin fusion protein comprises an interferon
alpha-interferon beta hybrid (herein referred to as a alpha-beta
hybrid). For example, the alpha-beta hybrid portion of the
interferon hybrid albumin fusion protein consists, or alternatively
comprises, of interferon alpha D (also referred to as interferon
alpha-1) fused to interferon beta-1. In a further embodiment, the
alpha D/beta-1 hybrid is fused wherein the N-terminal portion
corresponds to amino acids 1-73 of interferon alpha D and the
C-terminal portion corresponds to amino acids 74-166 of interferon
beta-1. Pot example, this alpha D/beta-1 hybrid would have an amino
acid sequence:
MCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKD-
SSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSUILKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINR-
LTOYLRN (SEQ ID NO:824). These hybrids are further described in
U.S. Pat. No. 4,758,428, which is hereby incorporated by reference
in its entirety.
[0063] In further embodiments, the interferon hybrid portion of the
interferon hybrid albumin fusion proteins may comprise additional
combinations of alpha-alpha interferon hybrids, alpha-beta
interferon hybrids, and beta-alpha interferon hybrids. In
additional embodiments, the interferon hybrid portion of the
interferon hybrid albumin fusion protein may be modified to include
mutations, substitutions, deletions, or additions to the amino acid
sequence of the interferon hybrid. Such modifications to the
interferon hybrid albumin fusion proteins may be made, for example,
to improve levels of production, increase stability, increase or
decrease activity, or confer new biological properties.
[0064] The above-described interferon hybrid albumin fusion
proteins are encompassed by the invention, as are host cells and
vectors containing polynucleotides encoding the polypeptides. In
one embodiment, a interferon hybrid albumin fusion protein encoded
by a polynucleotide as described above has extended shelf life. In
an additional embodiment, a interferon hybrid albumin fusion
protein encoded by a polynucleotide described above has a longer
serum half-life and/or more stabilized activity in solution (or in
a pharmaceutical composition) in vitro and/or in vivo than the
corresponding unfused interferon hybrid molecule.
[0065] In another non-limiting example, a "Therapeutic protein" is
a protein that has a biological activity, and in particular, a
biological activity. A non-exhaustive list of biological activities
that may be possessed by a Therapeutic protein includes, inducing
pancreatic ductal epithelial cell differentiation into
insulin-secreting pancreatic beta cells, stimulating synthesis and
release of insulin, enhancing glucose sensitivity, enhancing
insulin sensitivity, maintaining metabolic homeostasis, regulating
the activation of the complement system, enhancing or suppressing
an immune response, promoting or inhibiting angiogenesis,
regulating hematopoietic functions, stimulating nerve growth, or
any one or more of the biological activities described herein
(e.g., in the "Biological Activities" section below and/or as
disclosed for a given Therapeutic protein in Table 1).
[0066] As used herein, "therapeutic activity" or "activity" may
refer to an activity whose effect is consistent with a desirable
therapeutic outcome in humans, or to desired effects in non-human
mammals or in other species or organisms. Therapeutic activity may
be measured in vivo or in vitro. For example, a desirable effect
may be assayed in cell culture. As an example, the ability of a
Therapeutic protein and/or albumin fusion protein of the invention
(including fragments and variants thereat) to regulate glucose
uptake may be routinely assayed using, or routinely modified,
techniques described herein (e.g., Example 28) or otherwise known
in the art. In another example, the ability of a Therapeutic
protein and/or albumin fusion protein of the invention (including
fragments and variants thereof) to promote expression of the
H4IleSEAP reporters may be routinely assayed using, or routinely
modified, techniques described herein (e.g., Example 35) or
otherwise known in the art. Such in vitro or cell culture assays
are known for many Therapeutic proteins. Additional example of
assays include, but are not limited to, those described herein in
the Examples section or in the "Exemplary Activity Assay" column of
Table 1.
[0067] Therapeutic proteins corresponding to a Therapeutic protein
portion of an albumin fusion protein of the invention, such as cell
surface and secretory proteins, are often modified by the
attachment of one or more oligosaccharide groups. The modification,
referred to as glycosylation, can dramatically affect the physical
properties of proteins and can be important in protein stability,
secretion, and localization. Glycosylation occurs at specific
locations along the polypeptide backbone. There are usually two
major types of glycosylation: glycosylation characterized by
O-linked of oligosaccharides, which are attached to serine or
threonine residues; and glycosylation characterized by N-linked
oligosaccharides, which are attached to asparagine residues in an
Asn-X-Ser/Thr sequence, where X can be any amino acid except
praline. N-acetylneuramic acid also known as sialic acid) is
usually the terminal residue of both N-linked and O-linked
oligosaccharides. Variables such as protein structure and cell type
influence the number and nature of the carbohydrate units within
the chains at different glycosylation sites. Glycosylation isomers
are also common at the same site within a given cell type.
[0068] For example, several types of human interferon are
glycosylated. Natural human interferon-.alpha.2 is O-glycosylated
at threonine 106, and N-glycosylation occurs as asparagine 72 in
interferon-.alpha.14 (Adolf et al., J. Biochem 276:511 (1991);
Nyman T A et al., J. Biochem 329:295 (1998)). The oligosaccharides
at asparagine 80 in natural interferon-.beta.1.alpha. may play an
important factor in the solubility and stability of the protein,
but may not be essential for its biological activity. This permits
the production of an unglycosylated analog (interferon-.beta.1b)
engineered with sequence modifications to enhance stability (Hosoi
et al., J. Interferon Res. 8:375 (1988; Karpusas et al., Cell Mol
Life Sci 54:1203 (1998); Knight, J. Interferon Res. 2:421 (1982);
Runkel et al., Pharm Res 15:641 (1998); Lin, Dev. Biol. Stand.
96:97 (1998)). Interferon-.gamma. contains two N-linked
oligosaccharide chains at positions 25 and 97, both important for
the efficient formation of the bioactive recombinant protein, and
having an influence on the pharmacokinetic properties of the
protein (Sareneva et al., Eur. J. Biochem 242:191 (1996); Sareneva
et al., Biochem J. 303:831 (1994); Sareneva at al., J. Interferon
Res. 13:267 (1993)). Mixed O-linked and N-linked glycosylation also
occurs, far example in human erythropoietin, N-linked glycosylation
occurs at asparagine residues located at positions 24, 38 and 83
while O-linked glycosylation occurs at a serine residue located at
position 126 (Lai at al., J, Biol. Chem. 261:3116 (1986); Broody et
al., Arch. Biochem. Biophys. 265:329 (1988)).
[0069] Therapeutic proteins corresponding to a Therapeutic protein
portion of an albumin fusion protein of the invention, as well as
analogs and variants thereof, may be modified so that glycosylation
at one or more sites is altered as a result of manipulation(s) of
their nucleic acid sequence, by the host cell in which they are
expressed, or due to other conditions of their expression. For
example, glycosylation isomers may be produced by abolishing or
introducing glycosylation sites, e.g., by substitution or deletion
of amino acid residues, such as substitution of glutamine for
asparagine, or unglycosylated recombinant proteins may be produced
by expressing the proteins in host cells that will not glycosylate
them, e.g. in E. coli or glycosylation-deficient yeast. These
approaches are described in more detail below and are known in the
art.
[0070] Therapeutic proteins that may be used, for example, in
treating (e.g., ameliorating) or preventing diabetes and a
condition associated with diabetes are known in the art (e.g.,
those disclosed in Table 1 and nucleic acid sequences encoding
these proteins). Many of the sequences corresponding to these
Therapeutic proteins are available in public databases such as
Chemical Abstracts Services Databases (e.g., the CAS Registry),
GenBank, and subscription provided databases such as GeneSeq (e.g.,
Derwent). Exemplary nucleotide sequences of Therapeutic proteins
which encode a polynucleotide of the invention are disclosed in
column 7, "SEQ ID NO:X," of Table 2. Sequences shown as SEQ ID NO:X
may be a wild type polynucleotide sequence encoding a given
Therapeutic protein, or in some instances the sequence may be a
variant of the wild type polynucleotide sequence (e.g., a
polynucleotide which encodes the wild type Therapeutic protein
optimized, for example, for expression in a particular host; or a
polynucleotide encoding a variant of the wild type Therapeutic
protein (e.g., a site directed mutant; an allelic variant)). It is
well within the ability of the skilled artisan to use the sequence
shown as SEQ ID NO:X to derive the construct described in the same
row. For example, if SEQ ID NO:X corresponds to a full length
protein, but only a portion of that protein is used to generate the
specific CID, it is within the skill of the art to rely on
molecular biology techniques, such as PCR, to amplify the specific
fragment and clone it into the appropriate vector.
[0071] Additional Therapeutic proteins corresponding to a
Therapeutic protein portion of an albumin fusion protein of the
invention include, but are not limited to, one or more of the
Therapeutic proteins or peptides disclosed in the "Therapeutic
Protein X" column of Table 1, or fragment or variable thereof.
[0072] Table 1 provides a non-exhaustive list of Therapeutic
proteins that correspond to a Therapeutic protein portion of an
albumin fusion protein of the invention, and thus, an albumin
fusion protein encoded by a polynucleotide of the invention. The
first column, "Therapeutic Protein X," discloses Therapeutic
protein molecules that may be followed by parentheses containing
scientific and brand names of proteins that comprise, or
alternatively consist of, that Therapeutic protein molecule or a
fragment or variant thereof. "Therapeutic protein X" as used herein
may refer either to an individual Therapeutic protein molecule, or
to the entire group of Therapeutic proteins associated with a given
Therapeutic protein molecule disclosed in this column. The
"Biological activity" column (column 2) describes Biological
activities associated with the Therapeutic protein molecule (and
fragments and variants of the Therapeutic protein). Column 3,
"Exemplary Activity Assay," provides references that describe
assays which may be used or routinely modified to test the
therapeutic and/or biological activity of a Therapeutic Protein X
or an albumin fusion protein comprising a Therapeutic protein X
portion. Each of the references cited in the "Exemplary Activity
Assay" column are herein incorporated by reference in their
entireties, particularly with respect to the description of the
respective activity assay described in the reference (see Methods
section therein, for example) for assaying the corresponding
biological activity set forth in the "Biological Activity" column
of Table 1. The fourth column, "Preferred Indication Y," describes
disease, disorders, and/or conditions that may be treated (e.g.,
ameliorated), prevented, or diagnosed, by Therapeutic protein X or
an albumin fusion protein comprising a Therapeutic protein X
portion. The fifth column, "Therapeutic Protein Z" provides an
amino acid sequence associated with the Therapeutic protein
molecule (and fragments and variants of the Therapeutic protein).
The "Construct ID" column (column 6) provides a link to an
exemplary albumin fusion construct disclosed in Table 2 which
encodes an albumin fusion protein comprising the referenced
Therapeutic Protein X portion.
TABLE-US-00001 TABLE 1 Therapeutic Protein: X Biological Activity
Exemplary Activity Assay Preferred Indication: Y Therapeutic
Protein: Z Construct ID Insulin (Human insulin; Stimulates glucose
Insulin activity may be assayed in Hyperglycemia; Diabetes;
Diabetes Insipidus; Diabetes *See TABLE 2 for the 2250, 2255, 2276,
Insulin aspart; Insulin uptake and promotes vitro using a
[3-H]-glucose uptake mellitus; Type 1 diabetes; Type 2 diabetes;
Insulin Therapeutic protein description 2278, 2656, 2668, Glargine;
Insulin lispro; Lys- glycogenesis and assay. (J Biol Chem 1999 Oct
22; resistance; Insulin deficiency; Hyperlipidemia; under the
corresponding 2669, 2671, 2821, B28 Pro-B29; lyspro; LY
lipogenesis. 274(43): 30864-30873). Hyperketonemia; Non-insulin
dependent Diabetes Construct ID number 2822, 2832, 2877, 275585;
diarginylinsulin; Mellitus (NIDDM); Insulin-dependent Diabetes
2878, 2882, 2885, Des-B26-B30-insulin-B25- Mellitus (IDDM); A
Condition Associated With 2891, 2897, 2930, amide; Insulin detemir;
Diabetes Including, But Not Limited To Obesity, Heart 2931, 2942,
2986, LABI; NOVOLIN; Disease, Hyperglycemia, Infections,
Retinopathy, 3025, 3133, 3134, NOVORAPID; HUMULIN; And/Or Ulcers;
Metabolic Disorders; Immune 3197, 3198, 2726, NOVOMIX 30;
Disorders; Obesity; Vascular Disorders; Suppression of 2727, 2784,
2789 VELOSULIN; NOVOLOG; Body Weight; Suppression of Appetite;
Syndrome X. LANTUS; ILETIN; HUMALOG; MACRULIN; EXUBRA; INSUMAN;
ORALIN; ORALGEN; HUMAHALE; HUMAHALIN) Glucagon-Like-Peptide 1
Stimulates the synthesis GLP1 activity may be assayed in
Hyperglycemia; Diabetes; Diabetes Insipidus; Diabetes *See TABLE 2
for the 2448, 2455, 2456, (GLP1; Insulinotropin) and release of
insulin; vitro using a [3-H]-glucose uptake mellitus; Type 1
diabetes; Type 2 diabetes; Insulin Therapeutic protein description
2457, 2803, 2804, enhances the sensitivity assay. (J Biol Chem 1999
Oct 22; resistance; Insulin deficiency; Hyperlipidemia; under the
corresponding 2900, 2904, 2945, of adipose, muscle, and 274(43):
30864-30873). Hyperketonemia; Non-insulin dependent Diabetes
Construct ID number 2964, 2982, 3070, liver tissues towards
Mellitus (NIDDM); Insulin-dependent Diabetes 2802, 3027, 3028,
insulin; stimulates Mellitus (IDDM); A Condition Associated With
3045, 3046, 3069, glucose uptake; slows Diabetes Including, But Not
Limited To Obesity, Heart 3071, 3072, 3085, the digestive process;
Disease, Hyperglycemia, Infections, Retinopathy, 3086, 3087, 3140,
suppresses appetite; And/Or Ulcers; Metabolic Disorders; Immune
3309 blocks the secretion of Disorders; Obesity; Vascular
Disorders; Suppression of glucagon. Body Weight; Suppression of
Appetite; Syndrome X. Exendin-4 (AC-2993) Stimulates the synthesis
Exendin-4 activity may be assayed Hyperglycemia; Diabetes; Diabetes
Insipidus; Diabetes *See TABLE 2 for the 2469 and 2470. and release
of insulin; in vitro using a [3-H]-glucose mellitus; Type 1
diabetes; Type 2 diabetes; Insulin Therapeutic protein description
enhances the sensitivity uptake assay. (J Biol Chem 1999
resistance; Insulin deficiency; Hyperlipidemia; under the
corresponding of adipose, muscle, and Oct 22; 274(43):
30864-30873). Hyperketonemia; Non-insulin dependent Diabetes
Construct ID number liver tissues towards Mellitus (NIDDM);
Insulin-dependent Diabetes insulin; stimulates Mellitus (IDDM); A
Condition Associated With glucose uptake; slows Diabetes Including,
But Not Limited To Obesity, Heart the digestive process; Disease,
Hyperglycemia, Infections, Retinopathy, suppresses appetite; And/Or
Ulcers; Metabolic Disorders; Immune blocks the secretion of
Disorders; Obesity; Vascular Disorders; Suppression of glucagon.
Body Weight; Suppression of Appetite; Syndrome X. Resistin Mediates
insulin Ability of resistin to influence type Hyperglycemia;
Diabetes; Diabetes Insipidus; Diabetes *See TABLE 2 for the 2295,
2296, 2297, resistance in Type II II diabetes can be determined
using mellitus; Type 1 diabetes; Type 2 diabetes; Insulin
Therapeutic protein description 2300, and 2309. diabetes; inhibits
assays known in the art: Pontoglio resistance; Insulin deficiency;
Hyperlipidemia; under the corresponding insulin-stimulated et al.,
J Clin Invest 1998 May Hyperketonemia; Non-insulin dependent
Diabetes Construct ID number glucose uptake 15; 101(10): 2215-22.
Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A
Condition Associated With Diabetes Including, But Not Limited To
Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy,
And/Or Ulcers; Metabolic Disorders; Immune Disorders; Obesity;
Vascular Disorders; Suppression of Body Weight; Suppression of
Appetite; Syndrome X. Leptin Controls obesity through In vivo
modulation of food intake, Hyperglycemia; Diabetes; Diabetes
Insipidus; Diabetes *See TABLE 2 for the 2146, 2184, 2186,
regulation of appetite, reduction in body weight, and mellitus;
Type 1 diabetes; Type 2 diabetes; Insulin Therapeutic protein
description and 2187. reduction of body lowering of insulin and
glucose resistance; Insulin deficiency; Hyperlipidemia; under the
corresponding weight, and lowering of levels in ob/ob mice,
Hyperketonemia; Non-insulin dependent Diabetes Construct ID number
insulin and glucose radioimmunoassay (RIA) and Mellitus (NIDDM);
Insulin-dependent Diabetes level. activation of the leptin receptor
in a Mellitus (IDDM); a Condition Associated With cell-based assay.
Protein Expr Purif Diabetes Including, But Not Limited To Obesity,
Heart 1998 Dec; 14(3): 335-42 Disease, Hyperglycemia, Infections,
Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;
Obesity; Vascular Disorders; Suppression of Body Weight;
Suppression of Appetite; Syndrome X; Immunological Disorders;
Immunosuppression. HLDOU18 (Bone Activates L6/GSK3 Assays for
activation of GSK3 Hyperglycemia; Diabetes; Diabetes Insipidus;
Diabetes *See TABLE 2 for the 22328, 2340, Morphogenic Protein 9
kinase assay. kinase activity are well known in the mellitus; Type
1 diabetes; Type 2 diabetes; Insulin Therapeutic protein
description 22350, 2351, (BMP9); Growth art. For example, Biol.
Chem. resistance; Insulin deficiency; Hyperlipidemia; under the
corresponding 22359, 2362, differentiation factor-2 379(8-9):
(1998) 1101-1110.; Hyperketonemia; Non-insulin dependent Diabetes
Construct ID number 22367, 2369, precursor (GDF-2 Biochem J. 1993
Nov 15; 296 (Pt Mellitus (NIDDM); Insulin-dependent Diabetes
Mellitus 22370, 2473, precursor)) 1): 15-9. (IDDM); A Condition
Associated With Diabetes 2623, 2624, 2625, Including, But Not
Limited To Obesity, Heart Disease, 2631, 2632, 2633. Hyperglycemia,
Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune
Disorders; Obesity; Vascular Disorders; Suppression of Body Weight;
Suppression of Appetite; Syndrome X. IL-22 (IL22, interleukin-22;
Stimulates glucose IL-22 activity may be assayed in Hyperglycemia;
Diabetes; Diabetes Insipidus; Diabetes *See TABLE 2 for the 2901,
2903 IL17D, IL27) uptake in skeletal muscle vitro using a
[3-H]-glucose uptake mellitus; Type 1 diabetes; Type 2 diabetes;
Insulin Therapeutic protein description cells; increases skeletal
assay. (J Biol Chem 1999 Oct 22; resistance; Insulin deficiency;
Hyperlipidemia; under the corresponding muscle insulin 274(43):
30864-30873). Hyperketonemia; Non-insulin dependent Diabetes
Construct ID number sensitivity. Mellitus (NIDDM);
Insulin-dependent Diabetes Mellitus (IDDM); A Condition Associated
With Diabetes Including, But Not Limited To Obesity, Heart Disease,
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic
Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
RegIV (Colon Specific Stimulates glucose RegIV activity may be
assayed in Hyperglycemia; Diabetes; Diabetes Insipidus; Diabetes
*See TABLE 2 for the 2910. Gene; Colon Specific uptake; increases
insulin vitro using a [3-H]-glucose uptake mellitus; Type 1
diabetes; Type 2 diabetes; Insulin Therapeutic protein description
Protein) sensitivity. assay. (J Biol Chem 1999 Oct 22; resistance;
Insulin deficiency; Hyperlipidemia; under the corresponding
274(43): 30864-30873). Hyperketonemia; Non-insulin dependent
Diabetes Construct ID number Mellitus (NIDDM); Insulin-dependent
Diabetes Mellitus (IDDM); A Condition Associated With Diabetes
Including, But Not Limited To Obesity, Heart Disease,
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic
Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
Interferon alfa (Interferon Stimulates glucose Interferon activity
may be assayed Hyperglycemia, Diabetes; Diabetes Insipidus;
Diabetes *See TABLE 2 for the 2249, 2343, 2366, alfa-2b;
recombinant; uptake; increases insulin in vitro using a
[3-H]-glucose mellitus; Type 1 diabetes; Type 2 diabetes; Insulin
Therapeutic protein description 2381, 2382, 2410, Interferon
alfa-n1; Interferon sensitivity. uptake assay. (J Biol Chem 1999
resistance; Insulin deficiency; Hyperlipidemia; under the
corresponding and 3165. alfa-n3; Peginterferon alpha- Oct 22;
274(43): 30864-30873). Hyperketonemia; Non-insulin dependent
Diabetes Construct ID number 2b; Ribavirin and interferon Mellitus
(NIDDM); Insulin-dependent Diabetes alfa-2b; Interferon alfacon-1;
Mellitus (IDDM); A Condition Associated With interferon consensus;
YM Diabetes Including, But Not Limited To Obesity, Heart 643; CIFN;
interferon-alpha Disease, Hyperglycemia, Infections, Retinopathy,
consensus; recombinant And/Or Ulcers; Metabolic Disorders; Immune
methionyl consensus Disorders; Obesity; Vascular Disorders;
Suppression of interferon; recombinant Body Weight; Suppression of
Appetite; Syndrome X. consensus interferon; CGP 35269; RO 253036;
RO 258310; INTRON A; PEG- INTRON; OIF; OMNIFERON; PEG- OMNIFERON;
VELDONA; PEG-REBETRON; ROFERON A; WELLFERON; ALFERON N/LDO;
REBETRON; ALTEMOL; VIRAFERONPEG; PEGASYS; VIRAFERON; VIRAFON;
AMPLIGEN; INFERGEN; INFAREX; ORAGEN) HCE1P80 Stimulates glucose
HCE1P80 activity may be assayed Hyperglycemia; Diabetes; Diabetes
Insipidus; Diabetes *See TABLE 2 for the 2908, 3049, 3050, uptake;
increases insulin in vitro using a [3-H]-glucose mellitus; Type 1
diabetes; Type 2 diabetes; Insulin Therapeutic protein description
3051, 3052. sensitivity. uptake assay. (J Biol Chem 1999
resistance; Insulin deficiency; Hyperlipidemia; under the
corresponding Oct 22; 274(43): 30864-30873). Hyperketonemia;
Non-insulin dependent Diabetes Construct ID number Mellitus
(NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A Condition
Associated With Diabetes Including, But Not Limited To Obesity,
Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or
Ulcers; Metabolic Disorders; Immune Disorders; Obesity; Vascular
Disorders; Suppression of Body Weight; Suppression of Appetite;
Syndrome X. HDRMI82 Stimulates glucose HDRMI82 activity may be
assayed Hyperglycemia; Diabetes; Diabetes Insipidus; Diabetes *See
TABLE 2 for the 2909. uptake; increases insulin in vitro using a
[3-H]-glucose mellitus; Type 1 diabetes; Type 2 diabetes; Insulin
Therapeutic protein description sensitivity. uptake assay. (J Biol
Chem 1999 resistance; Insulin deficiency; Hyperlipidemia; under the
corresponding Oct 22; 274(43): 30864-30873). Hyperketonemia;
Non-insulin dependent Diabetes Construct ID number Mellitus
(NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A Condition
Associated With Diabetes Including, But Not Limited To Obesity,
Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or
Ulcers; Metabolic Disorders; Immune Disorders; Obesity; Vascular
Disorders; Suppression of Body Weight; Suppression of Appetite;
Syndrome X. PYY (Peptide YY) including Decreases appetite; Appetite
and food intake can be can Most preferred: Treatment of Obesity;
treatment of *See TABLE 2 for the 3108, 3109, 3281, PYY.sub.3-36
increases satiety; be measured by methods known in Diabetes;
suppression of body weight gain; suppression Therapeutic protein
description 3117, 3118, 3282. (amino acid residues 31-64 decreases
food intake. the art (Batterham et
al. Nature of appetite. under the corresponding of full length PYY,
amino 2002; 418: 650654); Activity may Also Preferred:
Hyperglycemia; Diabetes; Diabetes Construct ID number acid residues
3-36 of mature also be assayed in vitro using a [3- Insipidus;
Diabetes mellitus; Type 1 diabetes; Type 2 PYY) H]-glucose uptake
assay. (J Biol diabetes; Insulin resistance; Insulin deficiency;
Chem 1999 Oct 22; 274(43): 30864-30873). Hyperlipidemia;
Hyperketonemia; Non-insulin dependent Diabetes Mellitus (NIDDM);
Insulin- dependent Diabetes Mellitus (IDDM); A Condition Associated
With Diabetes Including, But Not Limited To Obesity, Heart Disease,
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic
Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
Other indications for antibodies, antagonists: treatment of weight
loss; treatment of AIDS wasting; appetite stimulant; treatment of
cachexia. HCBOG68 (enteric Controls proliferation/ Activation of
cAMP-mediated Treatment of Obesity; treatment of Diabetes; *See
TABLE 2 for the 3106, 3270 adipokine; Fat SID; proline
differentiation or transcription in adipocytes can be suppression
of body weight gain; suppression of Therapeutic protein description
rich acidic protein) metabolism/ assayed using methods known in
appetite. Hyperglycemia; Diabetes; Diabetes Insipidus; under the
corresponding physiology/pathology/of the art (Berger et al., Gene
66: 1-10 Diabetes mellitus; Type 1 diabetes; Type 2 diabetes;
Construct ID number adipocytes and adipose (1998); Cullen and Malm,
Methods Insulin resistance; Insulin deficiency; Hyperlipidemia;
tissue in response to in Enzymol 216: 362-368 (1992);
Hyperketonemia; Non-insulin dependent Diabetes dietary conditions.
Henthorn et al., Proc Natl Acad Sci Mellitus (NIDDM);
Insulin-dependent Diabetes USA 85: 6342-6346 (1988); Reusch
Mellitus (IDDM); A Condition Associated With et al., Mol Cell Biol
20(3): 1008-1020 Diabetes Including, But Not Limited To Obesity,
Heart (2000); and Klemm et al., J Disease, Hyperglycemia,
Infections, Retinopathy, Biol Chem 273: 917-923 (1998)); And/Or
Ulcers; Metabolic Disorders; Immune Activity may also be assayed in
Disorders; Obesity; Vascular Disorders; Suppression of vitro using
a [3-H]-glucose uptake Body Weight; Suppression of Appetite;
Syndrome X. assay. (J Biol Chem 1999 Oct 22; Other indications for
antibodies and/or antagonists 274(43): 30864-30873). include
treatment of weight loss; treatment of AIDS wasting; appetite
stimulant; treatment of cachexia. F-992 Vasopressin agonist
Activity may be assayed in vitro Hyperglycemia; Diabetes; Diabetes
Insipidus; Diabetes Vasopressin (Genbank (Ferring Pharmaceuticals)
using a [3-H]-glucose uptake assay. mellitus; Type 1 diabetes; Type
2 diabetes; Insulin Accession AAB86629) and/or (J Biol Chem 1999
Oct 22; resistance; Insulin deficiency; Hyperlipidemia; fragments
or variants thereof 274(43): 30864-30873). Hyperketonemia;
Non-insulin dependent Diabetes including but not limited to [D-
Mellitus (NIDDM); Insulin-dependent Diabetes
Phe.sup.2-Thi.sup.3-alpha-Me-Abu.sup.4- Mellitus (IDDM); A
Condition Associated With
Hyp.sup.7-D-Arg.sup.8]-dC.sup.1-vasopressin. Diabetes Including,
But Not Limited To Obesity, Heart Disease, Hyperglycemia,
Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune
Disorders; Obesity; Vascular Disorders; Suppression of Body Weight;
Suppression of Appetite; Syndrome X. INGAP peptide Stimulates the
synthesis Activity may be assayed in vitro Hyperglycemia; Diabetes;
Diabetes Insipidus; Diabetes SEQ ID NO: 840 (See Genbank (GMP
Companies) and release of insulin; using a [3-H]-glucose uptake
assay. mellitus; Type 1 diabetes; Type 2 diabetes; Insulin
Accession No. U41737) and/or enhances the sensitivity (J Biol Chem
1999 Oct 22; resistance; Insulin deficiency; Hyperlipidemia;
fragments or variants thereof. of adipose, muscle, and 274(43):
30864-30873). Hyperketonemia; Non-insulin dependent Diabetes liver
tissues towards Mellitus (NIDDM); Insulin-dependent Diabetes
insulin; stimulates Mellitus (IDDM); A Condition Associated With
glucose uptake; slows Diabetes Including, But Not Limited To
Obesity, Heart the digestive process; Disease, Hyperglycemia,
Infections, Retinopathy, suppresses appetite; And/Or Ulcers;
Metabolic Disorders; Immune blocks the secretion of Disorders;
Obesity; Vascular Disorders; Suppression of glucagons; involves
islet Body Weight; Suppression of Appetite; Syndrome X. neogenesis
associated protein Chrysalin Thrombin mimetic; Activity may be
assayed in vitro Most preferred indications include: Diabetic foot
ulcer; SEQ ID NO: 841 (Chrysalis Biotechnology) chemotactic for
using a [3-H]-glucose uptake assay. bone fractures; chronic wound
healing; heart (508-530 of mature neutrophils; enhance (J Biol Chem
1999 Oct 22; angiogenesis; cartilage repair and spine surgery
(spinal prothrombin); or collagen accumulation in 274(43):
30864-30873). fusion repair); and a condition associated with
diabetes SEQ ID NO: 842 (508-530 of wounds; enhance including but
not limited to obesity, heart disease, preprothrombin, see Genbank
revascularization of hyperglycemia, infections, and as otherwise
described Accession No. P00734); and/or wounds; accelerate herein.
fragments or variants thereof. healing of incisional and Also
preferred are: Hyperglycemia; Diabetes; Diabetes open wounds in
normal Insipidus; Diabetes mellitus; Type 1 diabetes; Type 2
animals and in animals diabetes; Insulin resistance; Insulin
deficiency; with impaired healing Hyperlipidemia; Hyperketonemia;
Non-insulin dependent Diabetes Mellitus (NIDDM); Insulin- dependent
Diabetes Mellitus (IDDM); A Condition Associated With Diabetes
Including, But Not Limited To Obesity, Heart Disease,
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic
Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
NBI-6024 Therapeutic vaccine to Activity may be assayed in vitro
Hyperglycemia; Diabetes; Diabetes Insipidus; Diabetes SEQ ID NO:
843 and/or (Neurocrine Biosciences) elicit a potentially using a
[3-H]-glucose uptake assay. mellitus; Type 1 diabetes; Type 2
diabetes; Insulin fragments or variants thereof protective immune
(J Biol Chem 1999 Oct 22; resistance; Insulin deficiency;
Hyperlipidemia; response to slow islet 274(43): 30864-30873).
Hyperketonemia; Non-insulin dependent Diabetes cell destruction
Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A
Condition Associated With Diabetes Including. But Not Limited To
Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy,
And/Or Ulcers; Metabolic Disorders; Immune Disorders; Obesity;
Vascular Disorders; Suppression of Body Weight; Suppression of
Appetite; Syndrome X. SPM-933 C-peptide analog Activity may be
assayed in vitro Hyperglycemia; Diabetes; Diabetes Insipidus;
Diabetes SEQ ID NO: 844 (see Genbank (Schwarz Pharma AG and using a
[3-H]-glucose uptake assay. mellitus; Type 1 diabetes; Type 2
diabetes; Insulin Accession No. P01308) and/or Creative Peptides)
(J Biol Chem 1999 Oct 22; resistance; Insulin deficiency;
Hyperlipidemia; fragments or variants thereof. 274(43):
30864-30873). Hyperketonemia; Non-insulin dependent Diabetes
Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A
Condition Associated With Diabetes Including, But Not Limited To
Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy,
And/Or Ulcers; Late-stage diabetic complications of Type 1 IDDM
(e.g., nephropathy, neuropathy, and retinopathy; Metabolic
Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
Octreotide; SMS 995, Growth hormone Activity may be assayed in
vitro Proliferative diabetic retinopathy; prevention of obesity;
SEQ ID NO: 845 and/or Sandostatin LAR antagonist (somatostatin
using a [3-H]-glucose uptake assay. metastatic carcinoid syndrome
and acromegaly; control fragments or variants thereof (Novartis)
analog) (J Biol Chem 1999 Oct 22; diarrhea due to vasoactive
intestinal peptide-secreting including but not limited to [R-
274(43): 30864-30873). tumours; prevention of variceal bleeds;
psoriasis; viral (R*, R*)]-D-Phe-L-Cys-L-Phe- infection;
Alzheimer's disease and migraine. D-Trp-L-Lys-L-Thr-N-[2- Also
preferred include: Hyperglycemia; Diabetes; hydroxy-1- Diabetes
Insipidus; Diabetes mellitus; Type 1 diabetes;
(hydrozymethyl)propyl]-L- Type 2 diabetes; Insulin resistance;
Insulin deficiency; cysteinamide cyclic (2-7)- Hyperlipidemia;
Hyperketonemia; Non-insulin disulfide. dependent Diabetes Mellitus
(NIDDM); Insulin- dependent Diabetes Mellitus (IDDM); A Condition
Associated With Diabetes Including, But Not Limited To Obesity,
Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or
Ulcers; Late-stage diabetic complications of Type I IDDM (e.g.,
nephropathy, neuropathy, and retinopathy; Metabolic Disorders;
Immune Disorders; Obesity; Vascular Disorders; Suppression of Body
Weight; Suppression of Appetite; Syndrome X; Late-stage diabetic
complications of Type I IDDM (e.g., nephropathy, neuropathy, and
retinopathy Pexiganan Acetate Anti-infective; Disrupts Pexiganan
acetate activity can be Most preferred include: Diabetic foot
ulcer; wound Lysinamide (SEQ ID NO: 846) (CAS-172820-23-4)
bacterial membranes. assessed using in vitro antibacterial healing;
diabetic foot ulcer infections; bacterial acetate and/or fragments
or assays known in the art. (Zasloff et infection; impetigo; burns;
and a condition associated variants thereof. al., Antimicrobial
Agents and with diabetes including but not limited to obesity,
heart Chemotherapy 1999, 43: 782-788); disease, hyperglycemia,
infections, and as otherwise Activity may be also assayed in
described herein. vitro using a [3-H]-glucose uptake Also preferred
include: Hyperglycemia; Diabetes; assay. (J Biol Chem 1999 Oct 22;
Diabetes Insipidus; Diabetes mellitus; Type 1 diabetes; 274(43):
30864-30873).. Type 2 diabetes; Insulin resistance; Insulin
deficiency; Hyperlipidemia; Hyperketonemia; Non-insulin dependent
Diabetes Mellitus (NIDDM); Insulin- dependent Diabetes Mellitus
(IDDM); A Condition Associated With Diabetes Including, But Not
Limited To Obesity, Heart Disease, Hyperglycemia, Infections,
Retinopathy, And/Or Ulcers; Late-stage diabetic complications of
Type I IDDM (e.g., nephropathy, neuropathy, and retinopathy;
Metabolic Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
Pramlintide (Amylin) Amylin analog; Appetite and food intake can be
can Treatment of Obesity; treatment of Diabetes; SEQ ID NO: 847,
with a C- (CAS-151126-32-8) improves glycemic be measured by
methods known in suppression of body weight gain; suppression of
termnal L-tyrosinamide cyclic control; reduces the art (Batterham
et al. Nature appetite; treatment of endocrine disorders;
(2-7)-disulfide and/or fragments postprandial blood 2002; 418:
650654); Activity may be Hyperglycemia; Diabetes; Diabetes
Insipidus; Diabetes or variants thereof. glucose peaks; flattens
also assayed in vitro using a [3-H]- mellitus; Type 1 diabetes;
Type 2 diabetes; Insulin glucose peaks and glucose uptake assay. (J
Biol Chem resistance; Insulin deficiency; Hyperlipidemia; troughs
observed in 1999 Oct 22; 274(43): 30864-30873). Hyperketonemia;
Non-insulin dependent Diabetes diabetic patients; Slows Mellitus
(NIDDM); Insulin-dependent Diabetes gastric emptying; Mellitus
(IDDM); A Condition Associated With decreases food intake. Diabetes
Including, But Not Limited To Obesity, Heart Disease,
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic
Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
Other indications for antibodies, antagonists: treatment of weight
loss; treatment of AIDS wasting; appetite stimulant; treatment of
cachexia HP-228 Cytokine release Activity may be assayed in vitro
Diabetes mellitus; obesity; inflammation; pain; SEQ ID NO: 848
and/or (LION Biosciences) modulator; melanocortin using a
[3-H]-glucose uptake assay. chemotherapy-induced emesis; toxicity;
and a condition fragments or variants thereof modulator; lowers (J
Biol Chem 1999 Oct 22; associated with diabetes including but not
limited to including but not limited to N- plasma insulin and
274(43): 30864-30873). obesity, heart disease, hyperglycemia,
infections, and as acetyl-L-norleucyl-L-Gln-L-His- glucose levels
otherwise described herein. D-Phe-L-Arg-D-Trp- Also preferred
include: Hyperglycemia; Diabetes; Glycinamide. Diabetes Insipidus;
Diabetes mellitus; Type 1 diabetes; Type 2 diabetes; Insulin
resistance; Insulin deficiency; Hyperlipidemia; Hyperketonemia;
Non-insulin
dependent Diabetes Mellitus (NIDDM); Insulin- dependent Diabetes
Mellitus (IDDM); A Condition Associated With Diabetes Including,
But Not Limited To Obesity, Heart Disease, Hyperglycemia,
Infections, Retinopathy, And/Or Ulcers; Late-stage diabetic
complications of Type I IDDM (e.g., nephropathy, neuropathy, and
retinopathy; Metabolic Disorders; Immune Disorders; Obesity;
Vascular Disorders; Suppression of Body Weight; Suppression of
Appetite; Syndrome X. Neurulin Activity may be assayed in vitro
Most preferred: Diabetic Neuropathy; and a condition (Cortecs
International Ltd) using a [3-H]-glucose uptake assay. associated
with diabetes including but not limited to (J Biol Chem 1999 Oct
22; obesity, heart disease, hyperglycemia, infections, and as
274(43): 30864-30873).. otherwise described herein. Also preferred:
Hyperglycemia; Diabetes; Diabetes Insipidus; Diabetes mellitus;
Type 1 diabetes; Type 2 diabetes; Insulin resistance; Insulin
deficiency; Hyperlipidemia; Hyperketonemia; Non-insulin dependent
Diabetes Mellitus (NIDDM); Insulin- dependent Diabetes Mellitus
(IDDM); A Condition Associated With Diabetes Including, But Not
Limited To Obesity, Heart Disease, Hyperglycemia, Infections,
Retinopathy, And/Or Ulcers; Late-stage diabetic complications of
Type I IDDM (e.g., nephropathy, neuropathy, and retinopathy;
Metabolic Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
AC-253 GLP-1 agonist; appetite Activity may be assayed in vitro
Most preferred: Type II NIDDM; obesity; (Amylin Pharmaceuticals)
suppressant; using a [3-H]-glucose uptake assay. hypertension; and
a condition associated with diabetes hypoglycemic agent; (J Biol
Chem 1999 Oct 22; including but not limited to obesity, heart
disease, amylin antagonist 274(43): 30864-30873). hyperglycemia,
infections, and as otherwise described herein. Also preferred:
Hyperglycemia; Diabetes; Diabetes Insipidus; Diabetes mellitus;
Type 1 diabetes; Type 2 diabetes; Insulin resistance; Insulin
deficiency; Hyperlipidemia; Hyperketonemia; Non-insulin dependent
Diabetes Mellitus (NIDDM); Insulin- dependent Diabetes Mellitus
(IDDM); A Condition Associated With Diabetes Including, But Not
Limited To Obesity, Heart Disease, Hyperglycemia, Infections,
Retinopathy, And/Or Ulcers; Late-stage diabetic complications of
Type I IDDM (e.g., nephropathy, neuropathy, and retinopathy;
Metabolic Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
ALT-300 Glucagon antagonist; Activity may be assayed in vitro Most
preferred: Type I IDDM and Type II NIDDM; (Alteon) lowers plasma
glucose using a [3-H]-glucose uptake assay. acidosis; and a
condition associated with diabetes levels (J Biol Chem 1999 Oct 22;
including but not limited to obesity, heart disease, 274(43):
30864-30873). hyperglycemia, infections, and as otherwise described
herein. Also preferred: Hyperglycemia; Diabetes; Diabetes
Insipidus; Diabetes mellitus; Type 1 diabetes; Type 2 diabetes;
Insulin resistance; Insulin deficiency; Hyperlipidemia;
Hyperketonemia; Non-insulin dependent Diabetes Mellitus (NIDDM);
Insulin- dependent Diabetes Mellitus (IDDM); A Condition Associated
With Diabetes Including, But Not Limited To Obesity, Heart Disease,
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Late-stage
diabetic complications of Type I IDDM (e.g., nephropathy,
neuropathy, and retinopathy; Metabolic Disorders; Immune Disorders;
Obesity; Vascular Disorders; Suppression of Body Weight;
Suppression of Appetite; Syndrome X. ANERVAX.DB Peptide vaccine;
delay Activity may be assayed in vitro Hyperglycemia; Diabetes;
Diabetes Insipidus; Diabetes (Corixa Corp) and suppress the using a
[3-H]-glucose uptake assay. mellitus; Type 1 diabetes; Type 2
diabetes; Insulin development of Type I (J Biol Chem 1999 Oct 22;
resistance; Insulin deficiency; Hyperlipidemia; IDDM 274(43):
30864-30873). Hyperketonemia; Non-insulin dependent Diabetes
Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A
Condition Associated With Diabetes Including, But Not Limited To
Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy,
And/Or Ulcers; Late-stage diabetic complications of Type I IDDM
(e.g., nephropathy, neuropathy, and retinopathy; Metabolic
Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
AZM-140 Amylase inhibitor; Activity may be assayed in vitro
Hyperglycemia; Diabetes; Diabetes Insipidus; Diabetes (Alizyme)
hypoglycemic agent using a [3-H]-glucose uptake assay. mellitus;
Type 1 diabetes; Type 2 diabetes; Insulin (J Biol Chem 1999 Oct 22;
resistance; Insulin deficiency; Hyperlipidemia; 274(43):
30864-30873). Hyperketonemia; Non-insulin dependent Diabetes
Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A
Condition Associated With Diabetes Including, But Not Limited To
Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy,
And/Or Ulcers; Late-stage diabetic complications of Type I IDDM
(e.g., nephropathy, neuropathy, and retinopathy; Metabolic
Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
Interferon beta (Interferon Modulates MHC antigen Activity may be
assayed in vitro Hyperglycemia; Diabetes; Diabetes Insipidus;
Diabetes See Table 2, SEQ ID NO: Z for 1778, 1779, 2011, beta-1a;
Interferon beta 1b; expression, NK cell using a [3-H]-glucose
uptake assay. mellitus; Type 1 diabetes; Type 2 diabetes; Insulin
particular construct. 2013, 2053, 2054, Interferon-beta-serine; SH
activity and IFNg (J Biol Chem 1999 Oct 22; resistance; Insulin
deficiency; Hyperlipidemia; 2492, 2580, 2795, 579; ZK 157046; BCDF;
production and IL12 274(43): 30864-30873). Hyperketonemia;
Non-insulin dependent Diabetes 2796, 2797 beta-2 IF;
Interferon-beta-2; production in Mellitus (NIDDM);
Insulin-dependent Diabetes rhIL-6; SJ0031; DL 8234; monocytes.
Mellitus (IDDM); A Condition Associated With FERON; IFNbeta;
Diabetes Including, But Not Limited To Obesity, Heart BETASERON;
AVONEX; Disease, Hyperglycemia, Infections, Retinopathy, REBIF;
BETAFERON; And/Or Ulcers; Metabolic Disorders; Immune SIGOSIX)
Disorders; Obesity; Vascular Disorders; Suppression of Body Weight;
Suppression of Appetite; Syndrome X. TR6 (DcR3; Decoy Receptor
Inhibits Fas Ligand and Cellular apoptosis can be measured Renal
failure; insulin dependent diabetes mellitus; See Table 2, SEQ ID
NO: Z for 1520, 1537, 1545, 3; FASTR) AIM-2 (TL5, LIGHT) by annexin
staining, TUNEL staining, rheumatoid arthritis; inflammatory bowel
disease; particular construct. 1546, 1568, 1570, mediated
apoptosis. measurement of caspase levels. autoimmune disease; toxic
epidermal necrolysis; 1622, 1623, 1645, Inhibition of cell growth
can also be multiple sclerosis. 1700, 1702, 1703, directly
measured, for example by Also preferred: Hyperglycemia; Diabetes;
Diabetes 1704, 1891, 1892, ALOMAR Blue staining. Assay refs:
Insipidus; Diabetes mellitus; Type 1 diabetes; Type 2 1912, and
1913. cytotoxicity assay on human diabetes; Insulin resistance;
Insulin deficiency; fibrosarcoma (Epsevik and Nissen-
Hyperlipidemia; Hyperketonemia; Non-insulin Meyer, 1986, J.
Immunol. methods); dependent Diabetes Mellitus (NIDDM); Insulin-
Activity may be assayed in vitro using dependent Diabetes Mellitus
(IDDM); A Condition a [3-H]-glucose uptake assay. (J Biol
Associated With Diabetes Including, But Not Limited Chem 1999 Oct
22; 274(43): 30864-30873). To Obesity, Heart Disease,
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Late-stage
diabetic complications of Type I IDDM (e.g., nephropathy,
neuropathy, and retinopathy; Metabolic Disorders; Immune Disorders;
Obesity; Vascular Disorders; Suppression of Body Weight;
Suppression of Appetite; Syndrome X. TNFR2 (p75) (ENBREL) Binds
both TNFa and T-cell proliferation can be measured Hyperglycemia;
Diabetes; Diabetes Insipidus; Diabetes See Table 2, SEQ ID NO: Z
for 1777 and 1784. TNFb; mediates T-cell using assays known in the
art. For mellitus; Type 1 diabetes; Type 2 diabetes; Insulin
particular construct. proliferation by TNF; example, "Lymphocytes:
a practical resistance; Insulin deficiency; Hyperlipidemia; reduces
signs and approach" edited by: SL Rowland, AJ Hyperketonemia;
Non-insulin dependent Diabetes structural damage in McMichael--ch.
6, pages 138-160 Mellitus (NIDDM); Insulin-dependent Diabetes
patients with moderately Oxford University Press (2000); and
Mellitus (IDDM); A Condition Associated With to severly active
"Current Protocols on CD-ROM" Diabetes Including, But Not Limited
To Obesity, Heart rheumatoid arthritis section 3.12 Proliferation
Assays for Disease, Hyperglycemia, Infections, Retinopathy, (RA).
T-cell Function John Wiley & Soncs, And/Or Ulcers; Late-stage
diabetic complications of Inc. (1999); Activity may be assayed Type
I IDDM (e.g., nephropathy, neuropathy, and in vitro using a
[3-H]-glucose uptake retinopathy; Metabolic Disorders; Immune
Disorders; assay. (J Biol Chem 1999 Oct 22; Obesity; Vascular
Disorders; Suppression of Body 274(43): 30864-30873). Weight;
Suppression of Appetite; Syndrome X. ACE2 inhibitor (DX512)
Inhibits production of Inhibition of angiotensin can be
Hyperglycemia; Diabetes; Diabetes Insipidus; Diabetes See Table 2,
SEQ ID NO: Z for 1989, 2000, 2001, angiotensin II which determined
using assays known in the mellitus; Type 1 diabetes; Type 2
diabetes; Insulin particular construct. and 2002. induces
aldosterone art. For example, in vitro using a resistance; Insulin
deficiency; Hyperlipidemia; production, arteriolar proliferation
assay with rat cardiac Hyperketonemia; Non-insulin dependent
Diabetes smooth muscle fibroblasts as described in Naunyn Mellitus
(NIDDM); Insulin-dependent Diabetes vasoconstriction, and
Schmiedebergs Arch Pharmacol 1999 Mellitus (IDDM); A Condition
Associated With proliferation of cardiac May; 359(5): 394-9;
Activity may be Diabetes Including, But Not Limited To Obesity,
Heart fibroblasts, Induces assayed in vitro using a [3-H]-glucose
Disease, Hyperglycemia, Infections, Retinopathy, angiogenesis; an
enzyme uptake assay. (J Biol Chem 1999 Oct And/Or Ulcers;
Late-stage diabetic complications of that converts angiotensin 22;
274(43): 30864-30873). Type I IDDM (e.g., nephropathy, neuropathy,
and I to angiotensin1-9; also retinopathy; Metabolic Disorders;
Immune Disorders; cleaves des-Arg, Obesity; Vascular Disorders;
Suppression of Body bradykinin and Weight; Suppression of Appetite;
Syndrome X: neurotensin. Proteinuria; Renal disorders, congestive
heart failure. Interferon Hybrids, Confers a range of Activity may
be assayed in vitro Hyperglycemia; Diabetes; Diabetes Insipidus;
Diabetes See Table 2, SEQ ID NO: Z for 2875, 2872, 2876,
specifically preferred: cellular responses using a [3-H]-glucose
uptake assay. mellitus: Type 1 diabetes: Type 2 diabetes; Insulin
particular construct. 2874, 2873. IFNalpha A/D hybrid (BgIII
including antiviral, (J Biol Chem 1999 Oct 22; resistance; Insulin
deficiency; Hyperlipidemia; version) antiproliferative, 274(43):
30864-30873). Hyperketonemia; Non-insulin dependent Diabetes
IFNalpha A/D hybrid (PvuII antitumor and Mellitus (NIDDM);
Insulin-dependent Diabetes version) immunomodulatory Mellitus
(IDDM): A Condition Associated With IFNalpha A/F hybrid activities;
stimulate Diabetes Including, But Not Limited To Obesity, Heart
IFNalpha A/B hybrid production of two Disease, Hyperglycemia,
Infections, Retinopathy, IFNbeta 1/alpha D hybrid enzymes: a
protein And/Or Ulcers; Metabolic Disorders; Immune
(IFNbeta-1/alpha-1 hybrid) kinase and an Disorders; Obesity;
Vascular Disorders; Suppression of IFNalpha/beta hybrid
oligoadenylate Body Weight; Suppression of Appetite; Syndrome X.
synthetase. Also, modulates MHC antigen expression, NK cell
activity and IFNg production and IL12 production in monocytes.
HWHGZ51 (CD59; Stimulates an immune The ability to affect
chondrocyte Hyperglycemia, Diabetes; Diabetes Insipidus; Diabetes
See Table 2, SEQ ID NO: Z for 2407, 2408 Metastasis-associated GPI-
response and induces differentiation can be measured mellitus; Type
1 diabetes; Type 2 diabetes; Insulin particular construct. adhered
protein homolog) inflammation by using methods known in the art,
resistance; Insulin deficiency; Hyperlipidemia;
inducing mononuclear such as described in Bone (1995)
Hyperketonemia; Non-insulin dependent Diabetes cell, eosinophil and
Sep: 17(3): 279-86; Activity may be Mellitus (NIDDM);
Insulin-dependent Diabetes PMN infiltration; assayed in vitro using
a [3-H]- Mellitus (IDDM); A Condition Associated With Inhibits
growth of breast glucose uptake assay. (J Biol Chem Diabetes
Including, But Not Limited To Obesity, Heart cancer, ovarian
cancer, 1999 Oct 22; 274(43): 30864-30873). Disease, Hyperglycemia,
Infections, Retinopathy, leukemia, and And/Or Ulcers; Metabolic
Disorders; Immune melanoma; Disorders; Obesity; Vascular Disorders;
Suppression of Overexpressed in colon, Body Weight; Suppression of
Appetite; Syndrome X; lung, breast and rectal Kidney disorders;
Hyperinsulinemia; Hypoinsulinemia tumors; Regulates glucose and/or
FFA update by adipocytes and skeletal muscle; Induces
redifferentiation of chondrocytes C17 (cytokine-like protein
Inhibits glucose and/or Proliferation of kidney mesangial Kidney
diseases and disorders; Shonlein-Henoch See Table 2, SEQ ID NO: Z
for 2489, 2490 C17) FFA uptake by cells can be assayed using
purpura, Berger disease, celiac disease, dermatitis particular
construct. adipocytes; Induces techniques described in J. Investig.
herpetiformis, Chron disease; Hyperglycemia, Diabetes;
proliferation of kidney Med. (1998) Aug; 46(6): 297-302; Diabetes
Insipidus; Diabetes mellitus; Type 1 diabetes; mesangial cells;
Activity may be assayed in vitro Type 2 diabetes; Insulin
resistance; Insulin deficiency; Regulation of cytokine using a
[3-H]-glucose uptake assay. Hyperlipidemia; Hyperketonemia;
Non-insulin production and antigen (J Biol Chem 1999 Oct 22;
dependent Diabetes Mellitus (NIDDM); Insulin- presentation 274(43):
30864-30873). dependent Diabetes Mellitus (IDDM); A Condition
Associated With Diabetes Including, But Not Limited To Obesity,
Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or
Ulcers; Metabolic Disorders; Immune Disorders; Obesity; Vascular
Disorders; Suppression of Body Weight; Suppression of Appetite;
Syndrome X; Kidney disorders; Hyperinsulinemia; Hypoinsulinemia
Somatostatin (Octreotide; Inhibits growth Inhibition of growth
hormone Hyperglycemia, Diabetes; Diabetes Insipidus; Diabetes See
Table 2, SEQ ID NO: Z for 2798, 2825, 2830, octreotide acetate;
hormone, glucagons and release in humans by somatostatin mellitus;
Type 1 diabetes; Type 2 diabetes; Insulin particular construct.
2831, 2902 Sandostating LAR .RTM.) insulin; Suppresses LF can be
measured as described in J. resistance; Insulin deficiency;
Hyperlipidemia; response to GnRH; Clin. Endocrinol. Metab. (1973)
Hyperketonemia; Non-insulin dependent Diabetes Decreases splanchnic
Oct; 37(4): 632-4. Mellitus (NIDDM); Insulin-dependent Diabetes
blood flow; Inhibits Inhibition of insulin secretion by Mellitus
(IDDM); A Condition Associated With release of serotonin,
somatostatin can be measured as Diabetes Including, But Not Limited
To Obesity, Heart gastrin, vasoactive described in the Lancet
(1973) Dec. Disease, Hyperglycemia, Infections, Retinopathy,
intestinal peptide, 8; 2(7841): 1299-1301; Activity may And/Or
Ulcers; Metabolic Disorders; Immune secretin, motilin, and be
assayed in vitro using a [3-H]- Disorders; Obesity; Vascular
Disorders; Suppression of pancreatic polypeptide. glucose uptake
assay. (J Biol Chem Body Weight; Suppression of Appetite; Syndrome
X; 1999 Oct 22; 274(43): 30864-30873). Kidney disorders HDALV07
(adiponectin; Modulates insulin action HDALV07 activity may be
assayed Hyperglycemia, Diabetes; Diabetes Insipidus; Diabetes See
Table 2, SEQ ID NO: Z for 3053, 3055, 3056 gelatin-binding 28 k
protein in vitro using a [3-H]-glucose mellitus; Type 1 diabetes;
Type 2 diabetes; Insulin particular construct. precurson; adipose
most uptake assay. (J Biol Chem 1999 resistance; Insulin
deficiency; Hyperlipidemia; abundant gene transcript; Oct 22;
274(43): 30864-30873). Hyperketonemia; Non-insulin dependent
Diabetes APM-1; GBP28; ACRP30; Mellitus (NIDDM); Insulin-dependent
Diabetes ADIPOQ) Mellitus (IDDM); A Condition Associated With
Diabetes Including, But Not Limited To Obesity, Heart Disease,
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic
Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X;
Hyperglycemia; Familial combined hyperlipidemia; Metabolic syndrome
C Peptide An insulin precursor C-peptide concentrations can be
Hyperglycemia, Diabetes; Diabetes Insipidus; Diabetes See Table 2,
SEQ ID NO: Z for 3088, 3149 involved in insulin measured using
assays well known mellitus; Type 1 diabetes; Type 2 diabetes;
Insulin particular construct. regulation in the art, such as the
one described resistance; Insulin deficiency; Hyperlipidemia; in
PNAS (1970) Sep; 67(1): 148-55; Hyperketonemia; Non-insulin
dependent Diabetes Activity may be assayed in vitro Mellitus
(NIDDM); Insulin-dependent Diabetes using a [3-H]-glucose uptake
assay. Mellitus (IDDM); A Condition Associated With (J Biol Chem
1999 Oct 22; Diabetes Including, But Not Limited To Obesity, Heart
274(43): 30864-30873). Disease, Hyperglycemia, Infections,
Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;
Obesity; Vascular Disorders; Suppression of Body Weight;
Suppression of Appetite; Syndrome X; Hyperglycemia; Familial
combined hyperlipidemia; Metabolic syndrome WNT10b Inhibits
adipogenesis. WNT10b activity can be measured Most preferred;
Treatment of Obesity; suppression of See Table 2, SEQ ID NO: Z for
3141 using adipogenesis inhibition assays body weight gain;
suppression of appetite. particular construct. (Ross et al.,
Science 2000; Other indications: Hyperglycemia; Diabetes; Diabetes
289(5481): 950-953; Activity may Insipidus; Diabetes mellitus; Type
1 diabetes; Type 2 be assayed in vitro using a [3-H]- diabetes;
Insulin resistance; Insulin deficiency; glucose uptake assay. (J
Biol Chem Hyperlipidemia; Hyperketonemia; Non-insulin 1999 Oct 22;
274(43): 30864-30873). dependent Diabetes Mellitus (NIDDM);
Insulin- dependent Diabetes Mellitus (IDDM). Also preferred:
Hyperglycemia; Diabetes; Diabetes Insipidus; Diabetes mellitus;
Type 1 diabetes; Type 2 diabetes; Insulin resistance; Insulin
deficiency; Hyperlipidemia; Hyperketonemia; Non-insulin dependent
Diabetes Mellitus (NIDDM); Insulin- dependent Diabetes Mellitus
(IDDM); A Condition Associated With Diabetes Including, But Not
Limited To Obesity, Heart Disease, Hyperglycemia, Infections,
Retinopathy, And/Or Ulcers; Late-stage diabetic complications of
Type 1 IDDM (e.g., nephropathy, neuropathy, and retinopathy;
Metabolic Disorders; Immune Disorders; Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X.
CART (Cocaine-and Inhibits food intact and Appetite and food intake
can be can Most preferred: Treatment of Obesity; suppression of See
Table 2, SEQ ID NO: Z for 3232 Amphetamine-Regulated fat storage;
promotes be measured by methods known in body weight gain;
suppression of appetite. particular construct. Transcript) lipid
oxidation, the art (Batterham et al. Nature Other indications:
Hyperglycemia; Diabetes; Diabetes 2002; 418: 650654); Activity may
be Insipidus; Diabetes mellitus; Type 1 diabetes; Type 2 assayed in
vitro using a [3-H]- diabetes; Insulin resistance; Insulin
deficiency; glucose uptake assay. (J Biol Chem Hyperlipidemia;
Hyperketonemia; Non-insulin 1999 Oct 22; 274(43): 30864-30873).
dependent Diabetes Mellitus (NIDDM); Insulin- dependent Diabetes
Mellitus (IDDM); A Condition Associated With Diabetes Including,
But Not Limited To Obesity, Heart Disease, Hyperglycemia,
Infections, Retinopathy, And/Or Ulcers; Late-stage diabetic
complications of Type 1 IDDM (e.g., nephropathy, neuropathy, and
retinopathy; Metabolic Disorders; Immune Disorders; Obesity;
Vascular Disorders: Suppression of Body Weight; Suppression of
Appetite; Syndrome X.
TABLE-US-00002 TABLE 2 Fusion Expression SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID Leader No. Construct ID Construct Name Description Vector
NO: Y NO: X NO: Z NO: A NO: B Sequence 1 1520 pC4:HSA/TR6.V30-H1300
Amino acids V30 to H300 of TR6 (fragment shown pC4 70 1 139 208 209
HSA as V1 to H271 of SEQ ID NO: 139) fused downstream of HSA. 2
1537 pYPG:HSA.TR6coV30-E294 Amino acids V30 to E294 of TR6
(fragment shown pYPGaf 71 2 140 210 211 HSA as V1 to E265 of SEQ ID
NO: 140) fused downstream of HSA. DNA encoding TR6 has been codon
optimized. 3 1545 pYPG:HSA.TR6coV30-L288 Amino acids V30 to L288 of
TR6 (fragment shown pYPGaf 72 3 141 212 213 HSA as V1 to L259 of
SEQ ID NO: 141) fused downstream of HSA. DNA encoding TR6 has been
codon optimized. 4 1546 pYPG:HSA.TR6coV30-R284 Amino acids V30 to
R284 of TR6 (fragment shown pYPGaf 73 4 142 214 215 HSA as V1 to
R255 of SEQ ID NO: 142) fused downstream of HSA. DNA encoding TR6
has been codon optimized. 5 1568 pSAC35:HSA-yTR6 TR6 fused
downstream of HSA. DNA encoding pSAC35 74 5 143 216 217 HSA/kex2
TR6 has been codon optimized. 6 1570 pSAC35:TR6-HSA Mature TR6
fused downstream of the HSA/kex2 pSAC35 75 6 144 218 219 HSA/kex2
leader and upstream of the mature HSA. 7 1622
pC4:synTR6.M1-H300.HSA Synthetic TR6 fused upstream of mature HSA,
with pC4 76 7 145 220 221 Native TR6 2 extra amino acids between
the TR6 and HSA portions. 8 1623 pC4:HSA.synTR6.V30-H300 Synthetic
mature TR6 fused downstream of FL pC4 77 8 146 222 223 HSA HSA.
Last amino acid HSA sequence is missing at BSU36I site. 9 1645
pSAC35:yTR6(N173Q).HSA Mutant mature TR6 cloned upstream of mature
HSA pSAC35 78 9 147 224 225 HSA/kex2 and downstream of the HSA/kex2
leader sequence. 10 1700 pSAC35:HSA-yTR6(N173Q) Mutant TR6 fused
downstream of mature HSA with pSAC35 79 10 148 226 227 HSA/kex2
HSA/kex2 leader sequence. 11 1702 pYPG:HSA.ek.TR6coV30-L288 Amino
acids V30 to L288 of TR6 (fragment shown pYPGaf 80 11 149 228 229
HSA as V1 to L259 of SEQ ID NO: 149) fused downstream of FL HSA
with an enterokinase site in between. DNA encoding TR6 has been
codon optimized. 12 1703 pYPG:HSA.ek.TR6coV30-R284 Amino acids V30
to R284 of TR6 (fragment shown pYPGaf 81 12 150 230 231 HSA as V1
to R255 of SEQ ID NO: 150) fused downstream of HSA with an
enterokinase site in between. DNA encoding TR6 has been codon
optimized. 13 1704 pYPG:HSA.TR6.V30-E294 Amino acids V30 to E294 of
TR6 fused downstream pYPGaf 82 13 151 232 233 HSA of HSA. Two
additional amino acids (Leu, Glu) are in between HSA and TR6. 14
1777 pSAC35:TNFR2.L23-D257.HSA Mature TNFR2 fused downstream of the
HSA/kex2 pSAC35 83 14 152 234 235 HSA/kex2 signal and upstream of
mature HSA. 15 1778 pSAC35:IFN.beta..M22-N187:HSA Residues M22-N187
of full-length IFNb (shown as pSAC35 84 15 153 236 237 HSA/kex2 M1
to N166 of SEQ ID NO: 153) fused upstream of mature HSA and
downstream of HSA/kex2 leader sequence. 16 1779
pSAC35:HSA:IFN.beta..M22-N187 Residues M22-N187 of full-length IFNb
(shown as pSAC35 85 16 154 HSA/kex2 M1 to N166 of SEQ ID NO: 154)
fused downstream of HSA with HSA/kex2 leader sequence. 17 1784
pSAC35:HSA.TNFR2.L23-D257 Mature TNFR2 fused downstream of FL HSA.
pSAC35 86 17 155 238 239 HSA 18 1891 pEE12:HSA.sTR6 Soluble mature
TR6 fused downstream of HSA. pEE12.1 87 18 156 240 241 HSA 19 1892
pEE12:sTR6.HSA Synthetic full length TR6 fused upstream of mature
pEE12.1 88 19 157 242 243 TR6 HSA. 20 1912 pC4:sTR6.HSA Synthetic
full length TR6 fused upstream of mature pC4 89 20 158 244 245
Native TR6 HSA. leader 21 1913 pC4:HSA.synTR6.V30-H300(seamless)
Amino acids V30 to H300 of synthetic TR6 (shown pC4 90 21 159 246
247 HSA as V1 to H271 of SEQ ID NO: 159) fused downstream of
full-length HSA. 22 1989 pSAC35:activeAC2inhibitor:HSA Active
inhibitor of ACE2 (DX512) fused upstream pSAC35 91 22 160 248 249
HSA/kex2 of mature HSA and downstream of HSA/kex2 leader sequence.
23 2000 pSAC35:HSA:activeAC2inhibitor Active inhibitor of ACE2
(DX512) fused pSAC35 92 23 161 250 251 HSA downstream of HSA. 24
2001 pSAC35:inactiveAC2inhibitor:HSA Inactive inhibitor of ACE2
(DX510) fused upstream pSAC35 93 24 162 252 253 HSA/kex2 of mature
HSA and downstream of HSA/kex2 leader sequence. 25 2002
pSAC35:HSA.inactiveAC2inhibitor Inactive inhibitor of ACE2 (DX510)
fused pSAC35 94 25 163 254 255 HSA downstream of HSA. 26 2011
pC4:IFNb-HSA Full length IFNb fused upstream of mature HSA. pC4 95
26 164 256 257 Native IFNb leader 27 2013 pC4:HSA-IFNb.M22-N187
Amino acids M22 to N187 of IFNb (fragment shown pC4 96 27 165 HSA
as amino acids M1 to N166 of SEQ ID NO: 165) fused downstream of
HSA. 28 2053 pEE12:IFNb-HSA Full length IFNb fused upstream of
mature HSA. pEE12.1 97 28 166 Native IFNb also named
pEE12.1:IFN.beta.-HSA leader 29 2054 pEE12:HSA-IFNb Mature IFNb
fused downstream of HSA. pEE12.1 98 29 167 HSA 30 2146
pC4:Leptin.HSA Full length Leplin fused upstream of mature HSA. pC4
99 30 168 258 259 Native leptin 31 2184 pC4:HSA.Leptin.V22-C166
Amino acids V22 to C167 of Leptin fused pC4 100 31 169 260 261 HSA
downstream of HSA. 32 2186 pSAC35:Leptin.V22-C166.HSA Amino acids
V22 to C167 of Leptin fused upstream pSAC35 101 32 170 262 263
HSA/kex2 of mature HSA and downstream of HSA/kex2 leader sequence.
33 2187 pSAC35:HSA.Leptin.V22-C166 Amino acids V22 to C167 of
Leptin fused pSAC35 102 33 171 264 265 HSA/kex2 downstream of HSA
with HSA/kex2 leader sequence. 34 2249 pSAC35:IFNa2-HSA Mature
IFNa2 fused upstream of mature HSA and pSAC35 103 34 172 266 267
HSA/kex2 also named: pSAC23:IFN.alpha.2-HSA downstream of HSA/kex2
leader sequence. 35 2250 pSAC35:HSA.INSULIN(GYG) Mature Insulin
wherein the C-peptide is replaced by pSAC35 104 35 173 268 269 HSA
also named: the C-domain of IGF-1 fused downstream of HSA.
pSAC35.HSA.INSULING(GYG).F1-N62 DNA encoding Insulin was codon
optimized. 36 2255 pSAC35:INSULIN(GYG).HSA Mature Insulin wherein
the C-peptide is replaced by pSAC35 105 36 174 271 272 HSA/kex2
also named the C-domain of IGF-1 fused upstream of mature
pSAC35.INSULING(GYG).F1-N62.HSA HSA and downstream of HSA/kex2
leader. DNA encoding Insulin was codon optimized. 37 2276
pSAC35:HSA.INSULIN(GGG) Mature Insulin wherein the C-peptide is
replaced by pSAC35 106 37 175 273 274 HSA also named: a synthetic
linker fused downstream of HSA. DNA pSAC35.HSA.INSULING(GGG).F1-N58
encoding Insulin was codon optimized. 38 2278
pSAC35:insulin(GGG).HSA Mature Insulin wherein the C-peptide is
replaced by pSAC35 107 38 176 275 276 HSA/kex2 a synthetic linker
fused downstream of HSA/kex2 leader and upstream of mature HSA. DNA
encoding Insulin was codon optimized. 39 2295
pSAC35:humanresistin.K19-P108:HSA Amino acids K19 to P108 of
Resistin fused pSAC35 108 39 177 277 278 HSA/kex2 upstream of
mature HSA and downstream of HSA/kex2 leader sequence. 40 2296
pSAC35:HSA:humanresistin.K19-P108 Amino acids K19 to P108 of
Resistin fused pSAC35 109 40 178 279 280 HSA downstream of HSA. 41
2297 pSAC35:humanresistin.K19-P108.stop:HSA Amino acids K19 to P108
of Resistin fused pSAC35 110 41 179 281 282 HSA/kex2 upstream of
mature HSA and downstream of HSA/kex2 leader sequence. Includes two
stops at 3' end for termination of translation before the HSA. 42
2300 pC4:humanresistin.M1-P108:HSA Amino acids M1 to P108 of
Resistin fused upstream pC4 111 42 180 283 284 Native resistin of
mature HSA. 43 2309 pEE12.1:humanresistin.M1-P108:HSA Amino acids
M1 to P108 of Resistin fused upstream pEE12.1 112 43 181 285 Native
resistin of mature HSA. 44 2328 pC4:HLDOU18.K23-R429.HSA Amino
acids K23 to R429 of HLDOU18 fused pC4 113 44 182 286 287 HSA
upstream of mature HSA and downstream of native HSA leader
sequence. 45 2340 pC4:HSA.HLDOU18.K23-R429 Amino acids K23 to R429
of HLDOU18 fused pC4 114 45 183 288 289 HSA downstream of HSA. 46
2343 pSAC35.INV-IFNA2.HSA Mature Interferon alpha2 fused upstream
of mature pSAC35 115 46 184 290 291 invertase HSA and downstream of
invertase signal peptide. 47 2350 pC4:MPIFsp.HLDOU18(S320-R429).HSA
Amino acids S320 to R429 of HLDOLU18 fused pC4 116 47 185 292 293
MPIF upstream of mature HSA and downstream of MPIF leader sequence.
48 2351 pC4:HSA.HLDOU18(S320-R429) Amino acids S320 to R429 of
HLDOU18 fused pC4 117 48 186 294 295 HSA downstream of HSA. 49 2359
pEE12:HLDOU18.K23-R429.HSA Amino acids K23 to R429 of HLDOU18 fused
pEE12.1 118 49 187 HSA upstream of mature HSA and downstream of
native HSA leader sequence. 50 2362 pEE12:HSA.HLDOU18.K23-R429
Amino acids K23 to R429 of HLDOU18 fused pEE12.1 119 50 188 HSA
downstream of HSA. 51 2366 pSAC35.MAF-IFNa2.HSA Mature IFNa2 fused
upstream of mature HSA and PSAC35 120 51 189 296 297 MF.alpha.-1
downstream of yeast mating factor alpha leader sequence. 52 2367
pEE12.MPIFsp.HLDOU18.S320-R429.HSA Amino acids S320 to R429 of
HLDOU18 fused pEE12.1 121 52 190 298 299 MPIF upstream of mature
HSA and downstream of MPIF leader sequence. 53 2369 pC4:HLDOU18.HSA
Amino acids M1 to R429 of HLDOU18 fused pC4 122 53 191 300 301
Native upstream of mature HSA. HLDOU18 54 2370 pEE12:HLDOU18.HSA
Amino acids M1 to R429 of HLDOU18 fused pEE12.1 123 54 192 Native
upstream of mature HSA. HLDOU18 55 2381 pC4:HSA-IFNa2(C17-E181)
Amino acids C17 to E181 of IFNa2 (fragment pC4 124 55 193 302 303
HSA shown as amino acids C1 to E165 of SEQ ID NO: 193) fused
downstream of HSA. 56 2382 pC4:IFNa2-HSA IFNa2 fused upstream of
mature HSA. pC4 125 56 194 304 305 Native IFN.alpha.2 leader 57
2407 pC4:HWHGZ51.M1-N323.HSA Amino acids M1 to N323 of HWHGZ51
fused pC4 126 57 195 306 307 Native upstream of mature HSA. HWHGZ51
58 2408 pEE12.1:HWHGZ51.M1-N323.HSA Amino acids M1 to N323 of
HWNGZ51 fused pEE12.1 127 58 196 308 309 Native upstream of mature
HSA. HWHGZ51 59 2410 pSAC35INV:IFNa-HSA Mature IFNa2 fused
downstream of the invertase pSAC35 128 59 197 310 311 invertase
signal peptide and upstream of mature HSA. 60 2448
pSAC35:GLP-1(7-36).HSA Amino acids H98 to R127 of preproglucagon
(SEQ pSAC35 129 60 198 312 313 HSA/kex2 ID NO: 198) (hereinafter
this specific domain will be referred to as "GLP-1(7-36)") is fused
upstream of mature HSA and downstream of HSA/kex2 leader sequence.
61 2455 pSAC35:HSA.GLP-1(7-36) GLP-1(7-36) is fused downstream of
mature HSA pSAC35 130 61 199 314 315 HSA/kex2 and HSA/kex2 leader
sequence. 62 2456 pSAC35:GLP-1(7-36(A8G)).HSA Amino acids H98 to
R127 of Preproglucagon (SEQ pSAC35 131 62 200 316 317 HSA/kex2 ID
NO: 200)(also referred to as "GLP-1(7-36)") is mutated at amino
acid 99 of SEQ ID NO: 200 to replace the alanine with a glycine.
This particular GLP-1 mutant will be hereinafter referred to as
"GLP-1(7-36(A8G))" and corresponds to the sequence shown in SEQ ID
NO: 698. GLP-1(7- 36(A8G)) is fused upstream of mature HSA and
downstream of HSA/kex2 leader sequence. 63 2457
pSAC35:HSA.GLP-1(7-36(A8G)) GLP-1(7-36(A8G)) (SEQ ID NO: 698) is
fused pSAC35 132 63 201 318 319 HSA/kex2 downstream of mature HSA
and HSA/kex2 leader sequence. 64 2469 pSAC35:HSA.exendin.H48-S86
Amino acids H48 to S86 of Extendin fused pSAC35 133 64 202 HSA
downstream of full length HSA. 65 2470 pSAC35:Exendin.H48-S86.HSA
Amino acids H48 to S86 of Extendin
fused upstream pSAC35 134 65 203 HSA/kex2 of mature HSA and
downstream of HSA/kex2 leader sequence. 66 2473
pC4.HLDOU18:HSA:S320-R429 M1-R319 of HLDOU18 (containing the furin
site pC4 135 66 204 320 321 Native RRKR) followed by residues `LE`
followed by HLDOU18 mature HSA followed by `LE` and amino acids
S320 through R429 of HLDOU18 (fragment shown as SEQ ID NO: 204). 67
2489 pSAC35:HSA.C17.A20-R136 Amino acids A20 to R136 of C17 fused
downstream pSAC35 136 67 205 322 323 HSA/kex2 of mature HSA with
HSA/kex2 leader sequence. 68 2490 pSAC35:C17.A20-R136.HSA Amino
acids A20 to R136 of C17 fused downstream pSAC35 137 68 206 324 325
HSA/kex2 of the HSA/kex2 leader and upstream of mature HSA. 69 2492
pC4.IFNb(deltaM22).HSA Mutant full length INFbeta fused upstream of
mature pC4 138 69 207 Native IFN.beta. HSA. First residue of
native, mature IFNbeta (M22) leader has been deleted. 70 2580
pC4.IFNb(deltaM22,C38S).HSA IFNb fused upstream of mature HSA. The
IFNb pC4 555 480 632 Native IFN.beta. used in this fusion lacks the
first residue of the mature form of IFNb, which corresponds to M22
of SEQ ID NO: 632. Also amino acid 38 of SEQ ID NO: 632 has been
mutated from Cys to Ser. 71 2623
pC4:(AGVSG,14-18)HSA.HLDOU18.K23-R429 A modified HSA A14 leader
followed by mature pC4 556 481 633 Modified HSA HSA and amino acids
K23 through R429 of (A14) leader HLDOU18. 72 2624
pC4:(SGVSG,14-18)HSA.HLDOU18.K23-R429 Modified HSA S14 leader
followed by mature HSA pC4 557 482 634 Modified HSA and amino acids
K23 to R429 of HLDOU18. (S14) leader 73 2625
pC4:(GGVSG,14-18)HSA.HLDOU18.K23-R429 A modified HSA G14 leader
sequence followed by pC4 558 483 635 Modified HSA mature HSA and
amino acids K23 through R429 of (G14) leader HLDOU18. 74 2631
pEE12.1:(AGVSG,14-18)HSA.HLDOU18.K23-R429 A modified HSA A14 leader
sequence followed by pEE12.1 559 484 636 Modified HSA mature HSA
and amino acids K23 through R429 of (A14) leader HLDOU18. 75 2632
pEE12.1:(SGVSG,14-18)HSA.HLDOU18.K23-R429 Modified HSA S14 leader
followed by mature HSA pEE12.1 560 485 637 Modified HSA and amino
acids K23 to R429 of HLDOU18. (S14) leader 76 2633
pEE12.1:(GGVSG,14-18)HSA.HLDOU18.K23-R429 A modified HSA G14 leader
sequence followed by pEE12.1 561 486 638 Modified HSA mature HSA
and amino acids K23 through R429 of (G14) leader HLDOU18. 77 2656
pSac35:Insulin(KR.GGG.KR).HSA Synthetic gene coding for a
single-chain insulin with pScCHSA 562 487 639 707 708 HSA/kex2 HSA
at C-terminus. Contains a modified loop for processing resulting in
correctly disulfide bonded insulin coupled to HSA. 78 2668
pSac35:HSA.Insulin(KR.GGG.KR) Synthetic gene coding for insulin
with FL HSA at N- pScNHSA 563 488 640 709 710 HSA terminus.
Contains a modified loop for processing resulting in correctly
disulfide bonded insulin coupled to HSA. 79 2669
pSac35:Insulin(GGG.KK).HSA Synthetic gene coding for a single-chain
insulin with pScCHSA 564 489 641 711 712 HSA/kex2 HSA at
C-terminus. Contains a modified loop. 80 2671
pSac35:HSA.Insulin(GGG.KK) Synthetic gene coding for a single-chain
insulin with pScNHSA 565 490 642 713 714 HSA HSA at N-terminus.
Contains a modified loop for greater stability. 81 2726
pSac35.INV.GYGinsulin.HSA Synthetic gene coding for a single-chain
insulin with pSAC35 566 491 643 715 716 Invertase HSA at
C-terminus. The signal peptide of invertase is used for this
construct. 82 2727 pSac35.INV.GYGinsulin(delF1).HSA Synthetic gene
coding for a single-chain insulin with pSAC35 567 492 644 717 718
invertase HSA at C-terminus. Construct uses the invertase signal
peptide and is lacking the first amino acid (F) of mature human
insulin. 83 2784 pSAC35:Insulin(GYG)-HSAcodon optimized Synthetic
gene coding for a single-chain insulin with pSAC35 568 493 645 719
720 invertase HSA at C-terminus. 84 2789
pSAC35:Insulin(GGG).HSA(codon optimized) Synthetic gene coding for
a single-chain insulin with pSAC35 569 494 646 721 722 invertase
HSA at C-terminus. 85 2795 pC4:HSA(A14)-IFNb.M22-N187 The mature
form of IFNb is fused to the C-terminus pC4 570 495 647 Modified
HSA of HSA, which contains an modified signal peptide, (A14)
designed to improve processing and homogeneity. 86 2796
pC4:HSA(S14)-IFNb.M22-N187 The mature form of IFNb is fused to the
C-terminus pC4 571 496 648 Modified HSA of HSA, which contains a
modified signal peptide, (S14) designed to improve processing and
homogeneity. 87 2797 pC4:HSA(G14)-IFNb.M22-N187 The mature form of
IFNb is fused to the C-terminus pC4 572 497 649 Modified HSA of
HSA, which contains an modified signal peptide. (G14) 88 2798
pSAC35:Somatostatin(S14).HSA A 14 amino acid peptide of
Somatostatin fused pScCHSA 573 498 650 723 724 HSA/kex2 downstream
of HSA/kex2 leader and upstream of mature HSA. 89 2802
pSAC35:GLP-1(7-36(A8G)).IP2.HSA GLP-1(7-36(A8G)) (SEQ ID NO: 698)
is fused pScNHSA 574 499 651 HSA/kex2 downstream from the HSA/kex2
leader sequence and upstream from the intervening peptide-2 of
proglucagon peptide and upstream from mature HSA. 90 2803
pSAC35:GLP-1(7-36(A8G))x2.HSA GLP-1(7-36(A8G)) (SEQ ID NO: 698) is
tandemly pScCHSA 419 412 426 433 434 HSA/kex2 repeated and fused
downstream of the HSA/kex2 signal sequence, and upstream of mature
HSA. 91 2804 pSAC35:coGLP-1(7-36(A8G))x2.HSA GLP-1(7-36(A8G)) (SEQ
ID NO: 698) is tandemly pScCHSA 420 413 427 435 436 HSA/kex2
repeated and fused downstream of the HSA/kex2 signal sequence, and
upstream of mature HSA. 92 2821 pSac35.delKex2.Insulin(GYG).HSA
Synthetic gene coding for a single-chain insulin with pScCHSA 575
500 652 Modified HSA at C-terminus. The kex2 site has been deleted
HSA/kex2, from the HSA/KEX2 signal peptide. lacking the Kex2 site.
93 2822 pSac35.alphaMF.Insulin(GYG).HSA Synthetic gene coding for a
single-chain insulin with pSAC35 576 501 653 725 726 MF.alpha.-1
HSA at C-terminus. The signal peptide of alpha mating factor
(MF.alpha.-1) is used for this construct. 94 2825
pSAC35:HSA.Somatostatin(S14) 14 amino acid peptide of Somatostatin
was fused pScNHSA 577 502 654 727 728 HSA/kex2 downstream of
HSA/kex2 leader and mature HSA. 95 2830 pSAC35:S28.HSA 28 amino
acids of somatostatin fused downstream pScCHSA 578 503 655 729 730
HSA/kex2 of HSA/kex2 leader and upstream of mature HSA. 96 2831
pSAC35:HSA.S28 28 amino acids of somatostatin fused downstream
pScNHSA 579 504 656 731 732 HSA/kex2 of HSA/kex2 leader and mature
HSA. 97 2832 pSAC35:Insulin.HSA (yeast Long-acting insulin peptide
fused upstream of pScCHSA 580 505 657 733 734 invertase codon
optimized) mature HSA. 98 2872 pSAC35:HSA.IFNaA(C1-Q91)/D(L93-E166)
This construct contains a hybrid form of IFNaA and pSAC35 455 456
457 458 459 HSA/kex2 IFNaD fused downstream of mature HSA. 99 2873
pSAC35:HSA.IFNaA(C1-Q91)/B(L93-E166) This construct contains a
hybrid form of IFNaA and pSAC35 460 461 462 463 464 HSA/kex2 IFNaB
fused downstream of mature HSA. 100 2874
pSAC35:HSA.IFNaA(C1-Q91)/F(L93-E166) This construct contains a
hybrid form of IFNaA and pSAC35 465 466 467 468 469 HSA/kex2 IFNaF
fused downstream of mature HSA. 101 2875
pSAC35:HSA.IFNaA(C1Q-62)/D(Q64-E166) This construct contains a
hybrid form of IFNaA and pSAC35 470 471 472 473 474 HSA/kex2 IFNaD
fused downstream of mature HSA. 102 2876
pSAC35:HSA.IFNaA(C1-Q91)/D(L93-E166);R23K,A113V This construct
contains a hybrid form of IFNaA and pSAC35 475 476 477 478 479
HSA/kex2 IFNaD fused downstream of mature HSA. 103 2877
pSAC35:KT.Insulin.HSA Killer toxin signal peptide fused to
synthetic gene pScCHSA 581 506 658 735 736 Killer toxin coding for
a single-chain insulin with C-terminal HSA 104 2878
pSAC35:AP.Insulin.HSA Acid phospatase signal peptide fused to
synthetic pSAC35 582 507 659 Acid gene coding for a single-chain
insulin with C- phosphatase terminal HSA. 105 2882
pSac35.alphaMFprepro.Insulin(GYG).HSA MF.alpha.-1 prepro signal
followed by GYG insulin pSAC35 583 508 660 MF.alpha.-1 followed by
mature HSA. 106 2885 pSac35.alphaMFpreproEEA.Insulin(GYG).HSA Yeast
MF.alpha.-1 prepro signal followed by GYG insulin pSAC35 584 509
661 Yeast follwed by mature HSA. MF.alpha.-1 107 2891
pGAP.alphaMF.Insulin(GYG).HSA Synthetic gene coding for a
single-chain insulin with pYPGaf 585 510 662 737 738 HSA/kex2 HSA
at C-terminus. The signal peptide of HSA/kex2 is used for this
construct. 108 2897 pGAP.Insulin(KR.GGG.KR).HSA Long-acting insulin
analog using a synthetic gene pYPGaf 586 511 663 739 740 HSA/kex2
coding for a single-chain insulin with HSA at C- terminus. Contains
a modified loop for processing resulting in correctly disulfide
bonded insulin coupled to HSA 109 2900 pSAC:GLP-1(7-36)x2.HSA
GLP-1(7-36) is tandemly repeated and then fused pScCHSA 421 414 428
437 438 HSA/kex2 downstream of the HSA/kex2 signal sequence and
upstream of mature HSA. 110 2901 pSAC35:IL22.A18-P202.HSA Amino
acids A18-P202 of IL22 fused downstream pSAC35 587 512 664 741 742
HSA/kex2 of HSA/kex2 leader and upstream of mature HSA. 111 2902
pSAC35:Somatostatin(S14(A-G)).HSA A 14 amino acid peptide of
Somatostatin, an pScCHSA 588 513 665 743 744 HSA/kex2 inhibitor of
growth hormone, synthesized as a C- terminal HSA fusion.
Somatostatin has an alanine to glycine change at amino acid 1 of
SEQ ID NO: 665. 112 2903 pSAC35:HSA.A18-P202.IL22 Amino acids
A18-P202 of IL22 fused downstream pSAC35 589 514 666 745 746 HSA of
full length HSA. 113 2904 pSAC35:GLP-1(9-36).GLP-1(7-36).HSA Amino
acids E100 to R127 of preproglucagon (SEQ pScCHSA 422 415 429 439
440 HSA/kex2 ID NO: 429) (hereinafter, this particular mutant is
referred to as GLP-1(9-36)) is fused downstream from the HSA/kex2
signal sequence and upstream from GLP-1(7-36), and mature HSA. 114
2908 pSAC35:HSA.HCE1P80 Mature HSA fused downstream of the HSA/kex2
pSAC35 590 515 667 747 748 HSA/kex2 leader and upstream of HCE1P80.
115 2909 pSAC35:HSA.HDRMI82 Mature HSA fused downstream of the
HSA/kex2 pSAC35 591 516 668 749 750 HSA/kex2 leader sequence and
upstream of HDRMI82. 116 2910 pSAC35:HSA.RegIV Mature HSA fused
downstream of the HSA/kex2 pSAC35 592 517 669 751 752 HSA/kex2
leader sequence and upstream of RegIV. 117 2930
pC4.MPIF.Insulin(GYG).HSA Insulin is downstream of an MPIF signal
peptide pC4 593 518 670 753 754 MPIF and upstream of mature HSA.
118 2931 pC4.HSA.Insulin(GYG) Synthetic gene coding for a mature
single-chain pC4 594 519 671 755 756 Modified HSA insulin fused
downstream of the modified HSA A14 (A14) leader leader and mature
HSA. 119 2942 pSac35.TA57.Insulin(GYG).HSA The TA57 Propeptide
fused to a single chain insulin pScNHSA 595 520 672 757 758 TA57
(GYG), and then mature HSA. propeptide 120 2945
pSAC35:GLP-1(7-36(A8S)).GLP-1(7-36).HSA Amino acids H98 to R127 of
preproglucagon (SEQ pScCHSA 423 416 430 441 442 HSA/kex2 ID NO:
430) is mutated at position 99 from alanine to serine (hereinafter,
this particalar mutant is referred to as GLP-1(7-36(A8S)), which is
fused downstream from the HSA/kex2 signal sequence and upstream
from GLP-1(7-36), and mature HSA. 121 2964 pSAC35:GLP-1(7-36)x2.HSA
GLP-1(7-36) is tandemly repeated as a dimer and pSAC35 424 417 431
443 444 HSA/kex2 fused downstream from the HSA/kex2 leader sequence
and upstream from mature HSA. 122 2982
pSAC35:GLP-1(7-36(A8G).GLP-1(7-36).HSA GLP-1(7-36(A8G)) (SEQ ID NO:
698) is fused pScCHSA 425 418 432 445 446 HSA/kex2 downstream from
the HSA/kex2 signal sequence and upstream from GLP-1(7-36) and
mature HSA. 123 2986 pSac35.y3SP.TA57PP.Insulin(GYG).HSA The TA57
Propeptide fused to a single chain insulin pScCHSA 596 521 673 759
760 TA57 (GYG), and then mature HSA. propeptide 124 3025
pSAC35:INU.Insulin.HSA Inulinase signal peptide is fused upstream
of single pScCHSA 597 522 674 761 762 inulinase chain insulin (GYG)
and HSA. 125 3027 pSAC35:INV.GLP-1(7-36A8G)x2.HSA Invertase signal
peptide followed by GLP-1(7- pSAC35 598 523 675 763 764 invertase
36(A8G)) (SEQ ID NO: 698) tandemly repeated as a dimer, followed by
mature HSA. 126 3028 pSAC35:INV.GLP-1(7-36(A8G)).GLP-1(7-36).HSA
Invertase signal peptide followed by GLP-1(7- pSAC35 599 524 676
765 766 invertase 36(A8G)) (SEQ ID NO: 698), then GLP-1(7-
36(A8G)), and then mature HSA. 127 3045
pSAC35:DeltaKex.GLP-1(7-36A8G)x2.HSA HSA/kex2 signal sequence,
minus the last six amino pSAC35 600 524 676 767 768 HSA/Kex2 last
acids of the leader, infused to GLP-1(7-36(A8G)) six amino acids
(SEQ ID NO: 698) which is tandemly repeated as a dimer, followed by
mature HSA. 128 3046 pSAC35:Delta
Kex.GLP-1(7-36A8G).GLP-1(7-36).HSA HSA/kex2 signal sequence, minus
the last six amino pSAC35 601 524 676 769 770 HSA/kex2 last acids
of the leader, is fused to GLP-1(7-36(A8G)) six amino acids (SEQ ID
NO: 698), GLP-1(7-36), and mature HSA. 129 3049
pC4.HSA.HCE1P80.D92-L229 Amino acids D92 to L229 of HCE1P80 are
fused pC4 602 525 677 771 772 HSA
downstream of the full length HSA. 130 3050
pC4.HSA.HCE1P80.A20-L229 Amino acids A20-L229 of HCE1P80 are fused
pC4 603 526 678 773 774 HSA downstream of the full length human HSA
131 3051 pSAC35.HSA.HCE1P80.D92-L229 Amino acids D92 to L229 of
HCE1P80, a member pSAC35 604 527 679 775 776 HSA of the C1q family
of proteins, are fused downstream of the full length human HSA 132
3052 pSAC35.HSA.HCE1P80.A20-L229 Amino acids A20-L229 of HCE1P80
are fused pSAC35 605 528 680 777 778 HSA downstream of the full
length human HSA 133 3053 pC4.HSA.HDALV07.K101-N244 The globular
domain of adiponectin (amino acids pC4 606 529 681 779 780 HSA
K101-N244) has been inserted downstream of full length human HSA
134 3055 pSAC35.HSA.HDALV07(GD) Full length HSA followed by amino
acids K101-N244 pSAC35 607 530 682 781 782 HSA of
HDALV07(GD)/Adiponectin. 135 3056 pSAC35.HSA.HDALV07.MP Full length
HSA followed by amino acids Q18 to pSAC35 608 531 683 783 784 HSA
N244 of HDALV07. 136 3069 pSAC35:INU.GLP-1(7-36(A8G))x2.HSA The
inulinase signal sequence is fused to GLP-1(7- pSAC35 609 532 684
785 786 inulinase 36(A8G)) (SEQ ID NO: 698), which is tandemly
repeated as a dimer and fused to mature HSA. 137 3070
pSAC35:KT.GLP-1(7-36(A8G))x2.HSA GLP-1(7-36(A8G)) (SEQ ID NO: 698)
is tandemly pSAC35 447 448 449 450 451 Killer toxin repeated as a
dimer and fused upstream from mature HSA and downstream from the
killer toxin signal sequence. 138 3071
pSAC35:MAF.GLP-1(7-36(A8G))x2.HSA The yeast mating factor .alpha.-1
(hereinafter MF.alpha.-1) pSAC35 610 533 685 787 788 MF.alpha.-1
signal sequence is fused to tandemly repeated copies of
GLP-1(7-36(A8G)) (SEQ ID NO: 698), which are fused to mature HSA.
139 3072 pSAC35:AP.GLP-1(7-36(A8G))x2.HSA The acid phosphatase
signal sequence is fused to pSAC35 611 534 686 789 790 Acid
tandemly repeated copies of GLP-1(7-36(A8G)) phosphatase (SEQ ID
NO: 698), which are fused to mature HSA. 140 3085
pSAC35:MAF.GLP-1(7-36(A8G)).GLP-1(7-36).HSA The yeast mating factor
.alpha.-1 (hereinafter MF.alpha.-1) pSAC35 612 535 687 791 792
MF.alpha.-1 signal sequence is fused to GLP-1(7-36(A8G)) (SEQ ID
NO: 698), GLP-1(7-36), and mature HSA. 141 3086
pSAC35:INU.GLP-1(7-36(A8G)).GLP-1(7-36).HSA The inulinase signal
sequence is fused to GLP-1(7- pSAC35 613 536 688 793 794 inulinase
36(A8G)) (SEQ ID NO: 698), GLP-1(7-36), and mature HSA. 142 3087
pSAC35:AP.GLP-1(7-36(A8G)).GLP-1(7-36).HSA The acid phosphatase
signal sequence is fused to pSAC35 614 537 689 795 796 Acid
GLP-1(7-36(A8G)) (SEQ ID NO: 698), GLP-1(7- phosphatase 36), and
mature HSA. 143 3088 pSAC35.HSA.C-Peptide HSA/kex2 signal peptide,
followed by HSA, pSAC35 615 538 690 797 798 HSA/kex2 followed by
the C-Peptide sequence. 144 3106 pSACHSA.HCBOG68 mature HCBOG68
fused downstream of mature pSAC35 616 539 691 HSA/kex2 HSA and the
HSA/kex2 leader sequence. 145 3108 pSAC35HSA.PYY Mature PYY fused
downstream of mature HSA and pSAC35 617 540 692 HSA/kex2 the
HSA/kex2 leader. 146 3109 pSAC35HSA.PYY3-36 HSA/kex2 leader
followed by mature HSA and then pSAC35 618 541 693 HSA/kex2 PYY3-36
(SEQ ID NO: 693). 147 3117 pC4:PYY3-36/HSA HSA leader followed by
PYY3-36 (SEQ ID pC4 619 542 694 799 800 HSA NO: 694) and mature
HSA. 148 3118 pSAC35:PYY3-36/HSA HSA/kex2 leader followed by
PYY3-36 (SEQ ID pSAC35 620 543 695 801 802 HSA/kex2 NO: 695) and
mature HSA. 149 3133 pSac35.ySP.TA57PP.Insulin(GYG).HSA Variant
TA57 propeptide leader followed by single pSAC35 621 544 696 803
804 TA57 variant 1 chain insulin, followed by mature HSA. 150 3134
pSac35.ySP.TA57PP + S.Insulin(GYG).HSA Variant TA57 propeptide
leader followed by single pSAC35 622 545 697 805 806 TA57 variant 2
chain insulin, followed by mature HSA. 151 3140
pSAC35:GLP1(mut)DAHK.HSA GLP-1(7-36(A8G)) (SEQ ID NO: 698) is
linked to pSAC35 623 546 698 807 808 HSA/kes2 mature HSA by a 16
amino acid linker derived from the N-terminus of HSA. The HSA/kex2
signal sequence is used. 152 3141 pSAC35:Wnt10b/HSA HSA/kex2 leader
followed by amino acids N29 to pSAC35 624 547 699 809 810 HSA/kex2
K389 of Wnt10b followed by mature HSA. 153 3149
pSAC35.HSA.C-peptide Full length HSA fused to amino acids E7 to Q37
of pSAC35 625 548 700 811 812 HSA tandem SEQ ID NO: 700, tandemly
repeated. 154 3165 pSAC35:HSA.IFNa HSA fused upstream of IFN.alpha.
and downstream of the pSAC35 452 453 454 HSA/kex2 also named CID
3165,pSAC35:HSA.INF.alpha. HSA/kex2 leader. 155 3197
pC4.MPIF.Insulin(EAE).HSA A single-chain insulin is downstream of
the MPIF pC4 626 549 701 MPIF signal peptide and upstream of mature
human HSA. 156 3198 pSac35.INV.insulin(EAE).HSA Single-chain
insulin is downstream of the invertase pSAC35 627 550 702 invertase
signal peptide and upstream of mature human HSA 157 3232
pSAC35:CART/HSA HSA/kex2 leader followed by processed active pSAC35
628 551 703 813 814 HSA/kex2 cocaine-amphetamine regulated
transcript (CART) (amino acids V69 through L116) followed by mature
HSA. 158 3270 pSAC35:adipokine/HSA HSA/kex2 leader followed by
adipokine followed by pSAC35 629 552 704 815 816 HSA/kex2 mature
HSA. 159 3281 pSAC35.PY3-36(x2)/HSA PYY3-36 tandem repeat (x2)
fused upstream of pSAC35 630 553 705 817 818 HSA/kex2 HSA and
downstream of the HSA/kex2 signal peptide. 160 3282
pSAC35:HSA/PYY3-36(x2) PYY3-36 tandem repeat (x2) fused downstream
of pSAC35 631 554 706 819 820 HSA/kex2 mature HSA and HSA/kex2
leader. 161 3309 pSAC:KT.GLP-1(7-36(A8G))x2.MSA.E25-A608 Killer
toxin leader sequence followed by GLP-1(7- pSAC35 834 833 835 836
837 Killer toxin 36(A8G) followed by mature mouse serum
albumin.
[0073] Table 2 provides a non-exhaustive list of polynucleotides
which encode an albumin fusion protein of the invention. The first
column, "Fusion No." assigns a fusion number to each
polynucleotide. Column 2, "Construct ID" provides a unique
numerical identifier for the corresponding polynucleotide of the
invention. The Construct Ids (or CIDs) may be used to refer to
polynucleotides which encode albumin fusion proteins comprising a
Therapeutic protein portion corresponding to Therapeutic Protein:X
identified in the corresponding row of Table 1. The "Construct
Name" column (column 3) provides the name of a given albumin fusion
construct.)
[0074] The fourth column in Table 2, "Description" provides a
general description of a given albumin fusion construct, and the
fifth column, "Expression Vector" lists the vector into which the
polynucleotide corresponding to a nucleic acid molecule encoding a
given albumin fusion protein was cloned. Vectors are known in the
art, and are available commercially or described elsewhere. For
example, as described in the Examples, an "expression cassette"
comprising, or alternatively consisting of, one or more of (1) a
polynucleotide encoding a given albumin fusion protein, (2) a
leader sequence, (3) a promoter region, and (4) a transcriptional
terminator, may be assembled in a convenient cloning vector and
subsequently be moved into an alternative vector, such as, for
example, an expression vector including, for example, a yeast
expression vector or a mammalian expression vector. In one
embodiment, for expression in S. cervisiae, an expression cassette
comprising, or alternatively consisting of, a nucleic acid molecule
encoding an albumin fusion protein is cloned into pSAC35. In
another embodiment, for expression in CHO cells, an expression
cassette comprising, or alternatively consisting of, a nucleic acid
molecule encoding an albumin fusion protein is cloned into pC4. In
a further embodiment, a polynucleotide comprising or alternatively
consisting of a nucleic acid molecule encoding the Therapeutic pi
Nein portion of an albumin fusion protein is cloned into pC4:HSA.
In a still further embodiment, for expression in NS0 cells, an
expression cassette comprising, or alternatively consisting of, a
nucleic acid molecule encoding an albumin fusion protein is cloned
into pEE12. Other useful cloning and/or expression vectors will be
known to the skilled artisan and are within the scope of the
invention.
[0075] Column 6, "SEQ ID NO:Y," provides the full length amino acid
sequence of representative albumin fusion proteins of the
invention. In most instances, SEQ ID NO: Y shows the unprocessed
form of the albumin fusion protein encoded in other words, SEQ ID
NO: Y shows the signal sequence, a HSA portion, and a therapeutic
portion all encoded by a particular construct. Specifically
contemplated by the present invention are all polynucleotides that
encode SEQ ID NO: Y. When these polynucleotides are used to express
the encoded protein from a cell, the cell's natural secretion and
processing steps produces a protein that lacks the signal sequence
listed in column 4 and/or 11 of Table 2. The specific amino acid
sequence of the listed signal sequence is shown later in the
specification or is well known in the art. Thus, most preferred
embodiments of the present invention include the albumin fusion
protein produced by a cell (which would lack the leader sequence
shown in column 4 and/or 11 Table 2). Also most preferred are
polypeptides comprising SEQ ID NO:Y without the specific leader
sequence listed in column 4 and/or 11 of Table 2. Compositions
comprising these two preferred embodiments, including
pharmaceutical compositions, are also preferred. Moreover, it is
well within the ability of the skilled artisan to replace the
signal sequence listed in column 4 and/or 11 of Table 2 with a
different signal sequence, such as those described later in the
specification to facilitate secretion of the processed albumin
fusion protein.
[0076] The seventh column, "SEQ ID NO:X," provides the parent
nucleic acid sequence from which a polynucleotide encoding a
Therapeutic protein portion of a given albumin fusion protein may
be derived. In one embodiment, the parent nucleic acid sequence
from which a polynucleotide encoding a Therapeutic protein portion
of an albumin fusion protein may be derived comprises the wild type
gene sequence encoding a Therapeutic protein shown in Table 1. In
an alternative embodiment, the parent nucleic acid sequence from
which a polynucleotide encoding a Therapeutic protein portion of an
albumin fusion protein may be derived comprises a variant or
derivative of a wild type gene sequence encoding a Therapeutic
protein shown in Table 1, such as, for example, a synthetic codon
optimized variant of a wild type gene sequence encoding a
Therapeutic protein.
[0077] The eighth column, "SEQ ID NO:Z," provides a predicted
translation of the parent nucleic acid sequence (SEQ ID NO:X). This
parent sequence can be a full length parent protein used to derive
the particular construct, the mature portion of a parent protein, a
variant or fragment of a wild type protein, or an artificial
sequence that can be used to create the described construct. One of
skill in the art can use this amino acid sequence shown in SEQ ID
NO:Z to determine which amino acid residues of an albumin fusion
protein encoded by a given construct are provided by the
therapeutic protein. Moreover, it is well within the ability of the
skilled artisan to use the sequence shown as SEQ ID NO:Z to derive
the construct described in the same row, For example, if SEQ ID
NO:Z corresponds to a full length protein, but only a portion of
that protein is used to generate the specific CID, it is within the
skill of the art to rely on molecular biology techniques, such as
PCR, to amplify the specific fragment and clone it into the
appropriate vector.
[0078] Amplification primers provided in columns 9 and 10, "SEQ ID
NO:A" and "SEQ ID NO:B" respectively, are exemplary primers used to
generate a polynucleotide comprising or alternatively consisting of
a nucleic acid molecule encoding the Therapeutic protein portion of
a given albumin fusion protein, in one embodiment of the invention,
oligonucleotide primers having the sequences shown in columns 9
and/or 10 (SEQ ID NOS:A and/or B) are used to PCR amplify a
polynucleotide encoding the Therapeutic protein portion of an
albumin fusion protein using a nucleic acid molecule comprising or
alternatively consisting of the nucleotide sequence provided in
column 7 (SEQ ID NO:X) of the corresponding row as the template
DNA. PCR methods are well-established in the art. Additional useful
primer sequences could readily be envisioned and utilized by those
of ordinary skill in the art.
[0079] As shown in Table 3, certain albumin fusion constructs
disclosed in this application have been deposited with the
ATCC.RTM.. The ATCC is located at 10803 University Boulevard,
Manassas, Va. 20110-2209, USA. The ATCC deposits were made pursuant
to the terms of the Budapest Treaty on the international
recognition of the deposit of microorganisms for the purposes of
patent procedure.
TABLE-US-00003 TABLE 3 Construct ID Construct Name ATCC Deposit
No./Date 2053 pEE12:IFNb-HSA PTA-3764 also named
pEE12.1:IFN.beta.-HSA Oct. 4, 2001 2054 pEE12:HSA-IFNb PTA-3941
Dec. 19, 2001 2249 pSAC35:IFNa2-HSA PTA-3763 also named
pSAC23:IFN.alpha.2-HSA Oct. 4, 2001 2250 pSAC35:HSA.INSULIN(GYG)
PTA-3916 also named pSAC35.HSA.INSULING(GYG).F1-N62 Dec. 07, 2001
2255 pSAC35:INSULIN(GYG).HSA PTA-3917 also named
pSAC35.INSULING(GYG).F1-N62.HSA Dec. 07, 2001 2276
pSAC35:HSA.INSULIN(GGG) PTA-3918 also named
pSAC35.HSA.INSULING(GGG).F1-N58 Dec. 07, 2001 2343
pSAC35.INV-IFNA2.HSA PTA-3940 Dec. 19, 2001 2381
pC4:HSA-IFNa2(C17-E181) PTA-3942 Dec. 19, 2001 2382 pC4:IFNa2-HSA
PTA-3939 Dec. 19, 2001 2492 pC4.IFNb(deltaM22).HSA PTA-3943 Dec.
19, 2001 3070 pSAC35:KT.GLP-1(7-36(AtG))x2.HSA PTA-4671 Sept. 16,
2002 3165 pSAC35:HSA.IFNa PTA-4670 also named CID 3165,
pSAC35:HSA.INF.alpha. Sept. 16, 2002
[0080] Albumin fusion constructs may routinely be isolated from the
deposit by techniques known in the art and described elsewhere
herein.
[0081] "Expression cassettes" containing one or more of (1) a
polynucleotide encoding a given albumin fusion protein, (2) a
leader sequence, (3) a promoter region, and (4) a transcriptional
terminator can routinely be moved or "subcloned" from one vector
into another. Fragments to be subcloned may be generated by methods
known in the art, such as, for example, per amplification (e.g.,
using oligonucleotide primers having the sequence shown in SEQ ID
NO:A or B), and/or restriction enzyme digestion.
[0082] In preferred embodiments, the albumin fusion proteins of the
invention are capable of a therapeutic activity and/or biological
activity corresponding to the therapeutic activity and/or
biological activity of the therapeutic protein corresponding to the
therapeutic protein portion of the albumin fusion protein listed in
the corresponding row of Table 1. In further preferred embodiments,
the therapeutically active protein portions of the albumin fusion
proteins of the invention are fragments or variants of the protein
encoded by the sequence shown in SEQ ID NO:X column of Table 2, and
are capable of the therapeutic activity and/or biologic activity of
the corresponding therapeutic protein.
[0083] Polypeptide and Polynucleotide Fragments and Variants
[0084] Fragments
[0085] The present invention is further directed to fragments of
the therapeutic proteins described in Table 1, albumin proteins,
and/or albumin fusion proteins of the invention.
[0086] The present invention is also directed so polynucleotides
encoding fragments of the Therapeutic proteins described in Table
1, albumin proteins, and/or albumin fusion proteins of the
invention.
[0087] Even if deletion of one or more amino acids from the
N-terminus of a protein results in modification or loss of one or
more biological functions of the Therapeutic protein, albumin
protein, and/or albumin fusion protein of the invention, other
Therapeutic activities and/or functional activities (e.g., a
biological activity (e.g., as provided in Table 1, column 2, for
the corresponding therapeutic protein), ability to multimerize,
ability to bind a ligand) may still be retained. For example, the
ability of polypeptides with N-terminal deletions to induce and/or
bind to antibodies which recognize the complete or mature forms of
the polypeptides generally will be retained when less than the
majority of the residues of the complete polypeptide are removed
from the N-terminus. Whether a particular polypeptide lacking
N-terminal residues of a complete polypeptide retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art. It is not unlikely
that a mutein with a large number of deleted N-terminal amino acid
residues may retain some biological or immunogenic activities. In
fact, peptides composed of as few as six amino acid residues may
often evoke an immune response.
[0088] Accordingly, fragments of a Therapeutic protein
corresponding to a Therapeutic protein portion of an albumin fusion
protein of the invention, include the full length protein as well
as polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the reference polypeptide
(i.e., a Therapeutic protein referred to in Table 1, or a
Therapeutic protein portion of an albumin fusion protein encoded by
a polynucleotide or albumin fusion construct described in Table 2).
In particular, N-terminal deletions may be described by the general
formula m-q, where q is a whole integer representing the total
number of amino acid residues in a reference polypeptide (e.g., a
Therapeutic protein referred to in Table 1, or a Therapeutic
protein portion of an albumin fusion protein of the invention, or a
Therapeutic protein portion of an albumin fusion protein encoded by
a polynucleotide or albumin fusion construct described in Table 2),
and to is defined as any integer ranging from 2 to q-6.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0089] In addition, fragments of serum albumin polypeptides
corresponding to an albumin protein portion of an albumin fusion
protein of the invention, include the full length protein as well
as polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the reference polypeptide
(i.e., serum albumin, or a serum albumin portion of an albumin
fusion protein encoded by a polynucleotide or albumin fusion
construct described in Table 2). In particular, N-terminal
deletions may be described by the general formula m-585, where 585
it a whole integer representing the total number of amino acid
residues in serum albumin (SEQ ID NO:327), and in is defined as any
integer ranging from 2 to 579. Polynucleotides encoding these
polypeptides are also encompassed by the invention. In additional
embodiments, N-terminal deletions may be described by the general
formula m to 609, where 609 is a whole integer representing the
total number of amino acid residues in full length human serum
albumin (SEQ ID NO:379), and m is defined as any integer ranging
from 2 to 603. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0090] Moreover, fragments of albumin fusion proteins of the
invention, include the full length albumin fusion protein as well
as polypeptides having one or more residues deleted from the amino
terminus of the albumin fusion protein (e.g., an albumin fusion
protein encoded by a polynucleotide or albumin fusion construct
described in Table 2; or an albumin fusion protein having the amino
acid sequence disclosed in column 6 of Table 2). In particular,
N-terminal deletions may be described by the general formula m-q,
where q is a whole integer representing the total number of amino
acid residues in the albumin fusion protein, and at is defined as
any integer ranging from 2 to q-6. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0091] Also as mentioned above, even if deletion of one or more
amino acids from the N-terminus or C-terminus of a reference
polypeptide (e.g., a Therapeutic protein; serum albumin protein; or
albumin fusion protein of the invention) results in modification or
loss of one or more biological functions of the protein, other
functional activities (e.g., a biological activity (e.g., as
provided in Table column 2, for the corresponding therapeutic
protein), ability to multimerize, ability to bind a ligand) and/or
Therapeutic activities may still be retained. For example the
ability of polypeptides with C-terminal deletions to induce and/or
bind to antibodies which recognize the complete or mature forms of
the polypeptide generally will be retained when less than the
majority of the residues of the complete or mature polypeptide are
removed from the C-terminus. Whether a particular polypeptide
lacking the N-terminal and/or C-terminal residues of a reference
polypeptide retains Therapeutic activity can readily be determined
by routine methods described herein and/or otherwise known in the
art.
[0092] The present invention further provides polypeptides having
one or more residues deleted from the carboxy terminus of the amino
acid sequence of a Therapeutic protein corresponding to a
Therapeutic protein portion of an albumin fusion protein of the
invention (e.g., a Therapeutic protein referred to in Table 1, or a
Therapeutic protein portion of an albumin fusion protein encoded by
a polynucleotide or albumin fusion construct described in Table 2).
In particular, C-terminal deletions may be described by the general
formula l-n, where n is any whole integer ranging from 6 to q-1,
and where q is a whole integer representing the total number of
amino acid residues in a reference polypeptide (e.g., a Therapeutic
protein referred to in Table 1, or a Therapeutic protein portion of
an albumin fusion protein encoded by a polynucleotide or albumin
fusion construct described in Table 2). Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0093] In addition, the present invention provides polypeptides
having one or more residues deleted from the carboxy terminus of
the amino acid sequence of an albumin protein corresponding to an
albumin protein portion of an albumin fusion protein of the
invention (e.g., serum albumin or an albumin protein portion of an
albumin fusion protein encoded by a polynucleotide or albumin
fusion construct described in Table 2). In particular, C-terminal
deletions may be described by the general formula l-n, where n is
any whole integer ranging from 6 to 584, where 584 is the whole
integer representing the total number of amino acid residues in
serum albumin (SEQ ID NO:327) minus 1. Polynucleotides encoding
these polypeptides are also encompassed by the invention. In
particular, C-terminal deletions may be described by the general
formula l to n, where n is any whole integer ranging from 6 to 608,
where 608 is the whole integer representing the total number of
amino acid residues in serum albumin (SEQ ID NO:379) minus 1.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0094] Moreover, the present invention provides polypeptides having
one or more residues deleted from the carboxy terminus of an
albumin fusion protein of the invention. In particular, C-terminal
deletions may be described by the general formula l-n, where n is
any whole integer ranging from 6 to q-1, and where q is a whole
integer representing the total number of amino acid residues in an
albumin fusion protein of the invention. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0095] In addition, any of the above described N- or C-terminal
deletions can be combined to produce a N- and C-terminal deleted
reference polypeptide. The invention also provides polypeptides
having one or more amino acids deleted from both the amino and the
carboxyl termini, which may be described generally as having
residues m-n of a reference polypeptide (e.g., a Therapeutic
protein referred to in Table 1, or a Therapeutic protein portion of
an albumin fusion protein of the invention, or a Therapeutic
protein portion encoded by a polynucleotide or albumin fusion
construct described in Table 2, or serum albumin (e.g., SEQ ID
NO:327), or an albumin protein portion of an albumin fusion protein
of the invention, or an albumin protein portion encoded by a
polynucleotide or albumin fusion construct described in Table 2, or
an albumin fusion protein, or an albumin fusion protein encoded by
a polynucleotide or albumin fusion construct of the invention)
where n and m are integers as described above. Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0096] The present application is also directed to proteins
containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%
or 99% identical to a reference polypeptide sequence (e.g., a
Therapeutic protein referred to in Table 1, or a Therapeutic
protein portion of an albumin fusion protein of the invention, or a
Therapeutic protein portion encoded by a polynucleotide or albumin
fusion construct described in Table 2, or serum albumin (e.g., SEQ
ID NO:327), or an albumin protein portion of an albumin fusion
protein of the invention, or an albumin protein portion encoded by
a polynucleotide or albumin fusion construct described in Table 2,
or an albumin fusion protein, or an albumin fusion protein encoded
by a polynucleotide or albumin fusion construct of the invention)
set forth herein, or fragments thereof. In preferred embodiments,
the application is directed to proteins comprising polypeptides at
least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
reference polypeptides having the amino acid sequence of N- and
C-terminal deletions as described above. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0097] Preferred polypeptide fragments of the invention are
fragments comprising, or alternatively, consisting of an amino acid
sequence that displays a Therapeutic activity and/or functional
activity (e.g. a biological activity such as that provided in Table
1, column 2, for the corresponding therapeutic protein) of the
polypeptide sequence of the Therapeutic protein or serum albumin
protein of which the amino acid sequence is a fragment.
[0098] Other preferred polypeptide fragments are biologically
active fragments. Biologically active fragments are those
exhibiting activity similar, but not necessarily identical, to an
activity of the polypeptide of the present invention. The
biological activity of the fragments may include an improved
desired activity, or a decreased undesirable activity.
[0099] Variants
[0100] "Variant" refers to a polynucleotide or nucleic acid
differing from a reference nucleic acid or polypeptide, but
retaining essential properties thereof. Generally, variants are
overall closely similar, and, in many regions, identical to the
reference nucleic acid or polypeptide.
[0101] As used herein, "variant", refers to a Therapeutic protein
portion of an albumin fusion protein of the invention, albumin
portion of an albumin fusion protein of the invention, or albumin
fusion protein of the invention differing in sequence from a
Therapeutic protein (e.g. see "therapeutic" column of Table 1),
albumin protein, and/or albumin fusion protein, respectively, but
retaining at least one functional and/or therapeutic property
thereof as described elsewhere herein or otherwise known in the
art. Generally, variants are overall very similar, and, in many
regions, identical to the amino acid sequence of the Therapeutic
protein corresponding to a Therapeutic protein portion of an
albumin fusion protein, albumin protein corresponding to an albumin
protein portion of an albumin fusion protein, and/or albumin fusion
protein. Nucleic acids encoding these variants are also encompassed
by the invention.
[0102] The present invention is also directed to proteins which
comprise, or alternatively consist of, an amino acid sequence which
is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%,
identical to, for example, the amino acid sequence of a Therapeutic
protein corresponding to a Therapeutic protein portion of an
albumin fusion protein of the invention (e.g., the amino acid
sequence of a Therapeutic protein:X disclosed in Table 1; or the
amino acid sequence of a Therapeutic protein portion of an albumin
fusion protein encoded by a polynucleotide or albumin fusion
construct described in Table 1 and 2, or fragments or variants
thereof), albumin proteins corresponding to an albumin protein
portion of an albumin fusion protein of the invention (e.g., the
amino acid sequence of an albumin protein portion of an albumin
fusion protein encoded by a polynucleotide or albumin fusion
construct described in Table 1 and 2; the amino acid sequence shown
in SEQ ID NO:327; or fragments or variants thereof), and/or albumin
fusion proteins. Fragments of these polypeptides are also provided
(e.g., those fragments described herein). Further polypeptides
encompassed by the invention are polypeptides encoded by
polynucleotides which hybridize to the complement of a nucleic acid
molecule encoding an albumin fusion protein of the invention under
stringent hybridization conditions (e.g., hybridization to filter
bound DNA in 6.times. Sodium chloride/Sodium citrate (SSC) at about
45 degrees Celsius, followed by one or more washes in
0.2.times.SSC, 0.1% SDS at about 50-65 degrees Celsius), under
highly stringent conditions (e.g., hybridization to filter bound
DNA in 6.times. sodium chloride/Sodium citrate (SSC) at about 45
degrees Celsius, followed by one or more washes in 0.1.times.SSC,
0.2% SDS at about 68 degrees Celsius), or under other stringent
hybridization conditions which are known to those of skill in the
an (see, for example, Ausubel, F. M. et al., eds., 1989 Current
protocol in Molecular Biology, Green publishing associates, Inc.,
and John Wiley & Sons Inc., New York, at pages 6.3.1-6.3.6 and
2.10.3). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0103] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence, it is
intended that the amino acid sequence of the subject polypeptide is
identical to the query sequence except that the subject polypeptide
sequence may include up to five amino acid alterations per each 100
amino acids of the query amino acid sequence. In other words, to
obtain a polypeptide having an amino acid sequence at least 95%
identical to a query amino acid sequence, up to 5% of the amino
acid residues in the subject sequence may be inserted, deleted, or
substituted with another amino acid. These alterations of the
reference sequence may occur at the amino- or carboxy-terminal
positions of the reference amino acid sequence or anywhere between
those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0104] As a practical matter, whether any particular polypeptide is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the amino acid sequence of an albumin fusion protein of
the invention or a fragment thereof (such as a Therapeutic protein
portion of the albumin fusion protein or an albumin portion of the
albumin fusion protein), can be determined conventionally using
known computer programs. A preferred method for determining the
best overall match between a query sequence (a sequence of the
present invention) and a subject sequence, also referred to as a
global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al, (Comp.
App. Biosci. 6.237-245 (1990)). In a sequence alignment the query
and subject sequences are either both nucleotide sequences or both
amino acid sequences. The result of said global sequence alignment
is expressed as percent identity. Preferred parameters used in a
FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff
Score=-1, Window Size, sequence length, Gap Penalty=5, Gap Size
Penalty=0.05, Window Size=500 or the length of the subject amino
acid sequence, whichever is shorter.
[0105] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of rite FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[0106] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity setae calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are to made for the purposes of the
present invention.
[0107] The variant will usually have at least 75% (preferably at
least about 80%, 90%, 95% or 99%) sequence identity with a length
of normal HA or Therapeutic protein which is the same length as the
variant. Homology or identity at the nucleotide or amino acid
sequence level is determined by BLAST (Basic Local Alignment Search
Tool) analysis using the algorithm employed by the programs blastp,
blastn, blastx, tblastn and tblastx (Karlin et al., Proc. Natl.
Acad. Ser. USA 87: 2264-2268 (1990) and Altschul, J. Mol. Evol. 36:
290-300 (1993), fully incorporated by reference) which are tailored
for sequence similarity searching.
[0108] The approach used by the BLAST program is to first consider
similar segments between a query sequence and a database sequence,
then to evaluate the statistical significance of all matches that
are identified and finally to summarize only those matches which
satisfy a preselected threshold of significance. For a discussion
of basic issues in similarity searching of sequence databases, see
Altschul et al, (Nature Genetics 6: 119-129 (1994)) which is fully
incorporated by reference. The search parameters for histogram,
descriptions, alignments, expect (i.e., the statistical
significance threshold for reporting matches against database
sequences), cutoff, matrix and filter are at the default settings.
The default scoring matrix used by hlastp, blastx, tblastn, and
tblastx is the BLOSUM62 matrix (Henikoff et Proc. Natl. Acad. Sci.
USA 89: 10915-10919 (1992), fully incorporated by reference). For
blastp, the scoring matrix is set by the ratios of M (i.e., the
reward score for a pair of matching residues) to N (i.e., the
penalty score for mismatching residues), wherein the default values
for M and N are 5 and -4, respectively, Four btastn parameters may
be adjusted as follows: Q=10 (gap erection penalty); R=10 (gap
extension penalty); wink=1 (generates word hits at every
wink.sup.th position along the query); and gapw=1.6 (sets the
window width within which gapped alignments are generated). The
equivalent Blastp parameter settings were Q=9; R=2; wink=1; and
gapw=32. A Bestfit comparison between sequences, available in the
COG package version 10.0, uses DNA parameters GAP=50 (gap creation
penalty) and LEN=3 (gap extension penalty) and the equivalent
settings in protein comparisons are GAP=8 and LEN=2.
[0109] The polynucleotide variants of the invention may contain
alterations in the coding regions, non-coding regions, or both.
Especially preferred are polynucleotide variants containing
alterations which produce silent substitutions, additions, or
deletions, but do not alter the properties or activities of the
encoded polypeptide. Nucleotide variants produced by silent
substitutions due to the degeneracy of the genetic code are
preferred. Moreover, polypeptide variants in which less than 50,
less than 40, less than 30, less than 20, less than 10, or 5-50,
5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or
added in any combination are also preferred. Polynucleotide
variants can be produced for a variety of reasons, e.g., to
optimize codon expression for a particular host (change codons in
the human mRNA to those preferred by a bacterial host, such as,
yeast or E. coli).
[0110] Functional Activity
[0111] "A polypeptide having functional activity" refers to a
polypeptide capable of displaying one or more known functional
activities associated with the full-length, pro-protein, and/or
mature form of a Therapeutic protein. Such functional activities
include, but are not limited to, biological activity (e.g., a
biological activity as provided in Table 1, column 2, for the
corresponding therapeutic protein), antigenicity [ability to bind
(or compete with a polypeptide for binding) to an anti-polypeptide
antibody], immunogenicity (ability to generate antibody which binds
to a specific polypeptide of the invention), ability to form
multimers with polypeptides of the invention, and ability to bind
to a receptor or ligand for a polypeptide.
[0112] "A polypeptide having biological activity" refers to a
polypeptide exhibiting activity similar to, but not necessarily
identical to, an activity of a Therapeutic protein of the present
invention, including mature forms, as measured in a particular
biological assay, with or without dose dependency. In the case
where dose dependency does exist, it need not be identical to that
of the polypeptide, but rather substantially similar to the
dose-dependence in a given activity as compared to the polypeptide
of the present invention (i.e., the candidate polypeptide will
exhibit greater activity or not more than about 25-fold less and,
preferably, not more than about tenfold less activity, and most
preferably, not more than about three-fold less activity relative
to the polypeptide of the present invention).
[0113] In preferred embodiments, an albumin fusion protein of the
invention has at least one biological and/or therapeutic activity
associated with the Therapeutic protein portion (or fragment or
variant thereof) when it is not fused to albumin.
[0114] The albumin fusion proteins of the invention can be assayed
for functional activity (e.g., biological activity) using or
routinely modifying assays known in the art, as well as assays
described herein. Additionally, one of skill in the art may
routinely assay fragments of a Therapeutic protein corresponding to
a Therapeutic protein portion of an albumin fusion protein, for
activity using assays referenced in its corresponding row of Table
1. Further, one of skill in the art may routinely assay fragments
of an albumin protein corresponding to an albumin protein portion
of an albumin fusion protein, for activity using assays known in
the an and/or as described in the Examples section below.
[0115] For example, in one embodiment where one is assaying for the
ability of an albumin fusion protein to bind or compete with a
Therapeutic protein for binding to an and Therapeutic polypeptide
antibody and/or anti-albumin antibody, various immunoassays known
in the art can be used, including but not limited to, competitive
and non-competitive assay systems using techniques such as
radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitation reactions, immunodiffusion assays, in situ
immunoassays (using colloidal gold, enzyme or radioisotope labels,
for example), western blots, precipitation reactions, agglutination
assays (e.g., gel agglutination assays, hemagglutination assays),
complement fixation assays, immunofluorescence assays, protein A
assays, and immunoelectrophoresis assays, etc. In one embodiment,
antibody binding is detected by detecting a label on the primary
antibody. In another embodiment, the primary antibody is detected
by detecting binding of a secondary antibody or reagent to the
primary antibody. In a further embodiment, the secondary antibody
is labeled. Many means are known in the an for detecting binding in
an immunoassay and are within the scope of the present
invention.
[0116] In a preferred embodiment, where a binding partner (e.g., a
receptor or a ligand) of a Therapeutic protein is identified,
binding to that binding partner by an albumin fusion protein which
comprises that Therapeutic protein as the Therapeutic protein
portion of the fusion can be assayed, e.g., by means well-known in
the art, such as, for example, reducing and non-reducing gel
chromatography, protein affinity chromatography, and affinity
blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123
(1995). In another embodiment, the ability of physiological
correlates of an albumin fusion protein to bind to a substrate(s)
of the Therapeutic polypeptide corresponding to the Therapeutic
protein portion of the fusion can be routinely assayed using
techniques known in the art.
[0117] In an alternative embodiment, where the ability of an
albumin fusion protein to multimerize is being evaluated,
association with other components of the multimer can be assayed,
e.g., by means well-known in the art, such as, for example,
reducing and non-reducing gel chromatography, protein affinity
chromatography, and affinity blotting. See generally, Phizicky et
al., supra.
[0118] In preferred embodiments, an albumin fusion protein
comprising all or a portion of an antibody that binds a Therapeutic
protein, has at least one biological and/or therapeutic activity
(e.g., to specifically bind a polypeptide or epitope) associated
with the antibody that binds a Therapeutic protein (or fragment or
variant thereof) when it is not fused to albumin. In other
preferred embodiments, the biological activity and/or therapeutic
activity of an albumin fusion protein comprising all or a portion
of an antibody that binds a Therapeutic protein is the inhibition
(i.e., antagonism) or activation (i.e., agonism) of one or More of
the biological activities and/or therapeutic activities associated
with the polypeptide that is specifically bound by antibody that
binds a Therapeutic protein.
[0119] Albumin fusion proteins comprising at least a fragment or
variant of an antibody that binds a Therapeutic protein may be
characterized in a variety of ways. In particular, albumin fusion
proteins comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein may be assayed for the ability to
specifically bind to the same antigens specifically hound by the
antibody that binds a Therapeutic protein corresponding to the
Therapeutic protein portion of the albumin fusion protein using
techniques described herein or routinely modifying techniques known
in the art.
[0120] Assays for the ability of the albumin fusion proteins (e.g.,
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein) to (specifically) bind a specific protein or
epitope may be performed in solution (e.g., Houghten,
Bio/Techniques 13:412-421 (1992), on beads (e.g., Lam, Nature
354:82-84 (1991)), on chips (e.g. Fodor, Nature 364:555-556
(1993)), on bacteria (e.g., U.S. Pat. No. 5,223,409), on spores
(e.g., U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on
plasmids (e.g., Cull et al., Proc. Natl. Acad. Sci. USA
89:1865-1869 (1492)) or on phage (e.g., Scott and Smith, Science
249:386-390 (1990); Devlin, Science 249:404.406 (1990); Cwirla et
al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); and Felici, J.
Mol. Biol. 222:301-310 (1991)) (each of these references is
incorporated herein in its entirety by reference). Albumin fusion
proteins comprising at least a fragment or variant of a Therapeutic
antibody may also be assayed for their specificity and affinity for
a specific protein or epitope using or routinely modifying
techniques described herein or otherwise known in the art.
[0121] The albumin fusion proteins comprising at least a fragment
or variant of an antibody that binds a Therapeutic protein may be
assayed for cross-reactivity with other antigens (e.g., molecules
that have sequence/structure conservation with the molecule(s)
specifically bound by the antibody that binds a Therapeutic protein
(or fragment or variant thereof) corresponding to the Therapeutic
protein portion of the albumin fusion protein of the invention) by
any method known in the art.
[0122] Immunoassays which can be used to analyze (immunospecific)
binding and cross-reactivity include, but are not limited to,
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, and
protein A immunoassays, to name but a few. Such assays are routine
and well known in the art (see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology: Vol. 1, John Wiley &
Sons, Inc., New York, which is incorporated by reference herein in
its entirety). Exemplary immunoassays are described briefly below
(but are not intended by way of limitation).
[0123] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SOS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the albumin fusion protein of
the invention (e.g., comprising at least a fragment or variant of
an antibody that binds a Therapeutic protein) to the cell lysate,
incubating for a period of time 1 to 4 hours) at 40 degrees C.
adding sepharose beads coupled to an anti-albumin antibody, for
example, to the cell lysate, incubating for about an hour or more
at 40 degrees C., washing the beads in lysis buffer and
resuspending the beads in SOS/sample buffer. The ability of the
albumin fusion protein to immunoprecipitate a particular antigen
can be assessed by, e.g., western blot analysis. One of skill in
the art would be knowledgeable as to the parameters that can be
modified to increase the binding of the albumin fusion protein to
an antigen and decrease the background (e.g., pre-clearing the cell
lysate with sepharose beads). For further discussion regarding
immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York at 10.16.1.
[0124] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%.sup., 20% SOS-PAGE depending on the molecular weight
of the antigen), transferring the protein sample from the
polyacrylamide gel to a membrane such as nitrocellulose, PVDF or
nylon, blocking the membrane in blocking solution (e.g., PBS with
3% BSA or non-fat milk), washing the membrane in washing buffer
(e.g., PBS-Tween 20), applying the albumin fusion protein of the
invention (diluted in blocking buffer) to the membrane, washing the
membrane in washing buffer, applying a secondary antibody (which
recognizes the albumin fusion protein, e.g., an anti-human serum
albumin antibody) conjugated to an enzymatic substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) or radioactive
molecule (e.g., .sup.32P or .sup.125I) diluted in blocking buffer,
washing the membrane in wash buffer, and detecting the presence of
the antigen. One of skill in the art would be knowledgeable as to
the parameters that can be modified to increase the signal detected
and to reduce the background noise. For further discussion
regarding western blot protocols see, e.g., Ausubel et al, eds,
1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley
& Sons, Inc., New York at 10.8.1.
[0125] ELISAs comprise preparing antigen, coating the well of a
96-well microliter plate with the antigen, washing away antigen
that did not bind the wells, adding the albumin fusion protein
(e.g., comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein) of the invention conjugated to a
detectable compound such as an enzymatic substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) to the wells and
incubating for a period of time, washing away unbound or
non-specifically bound albumin fusion proteins, and detecting the
presence of the albumin fusion proteins specifically bound to the
antigen coating the well. In ELISAs the albumin fusion protein does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes albumin fusion protein)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the albumin
fusion protein may be coated to the well. In this case, the
detectable molecule could be the antigen conjugated to a detectable
compound such as an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase). One of skill in the art would
be knowledgeable as to the parameters that can be modified to
increase the signal detected as well as other variations of ELISAs
known in the art. For further discussion regarding ELISAs see,
e.g., Ausubel at al, eds, 1994, Current Protocols in Molecular
Biology, Vol. 1, John Wiley & Sons, Inc., New York at
11.2.1.
[0126] The binding affinity of an albumin fusion protein to a
protein, antigen, or epitope and the off-rate of an albumin fusion
protein-protein/antigen/epitope interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., .sup.3H or .sup.125I) with the albumin fusion
protein of the invention in the presence of increasing amounts of
unlabeled antigen, and the detection of the antibody bound to the
labeled antigen. The affinity of the albumin fusion protein for a
specific protein, antigen, or epitope and the binding off rates can
be determined from the data by Scatchard plot analysis. Competition
with a second protein that binds the same protein, antigen or
epitope as the albumin fusion protein, ran also be determined using
radioimmunoassays. In this case, the protein, antigen or epitope is
incubated with an albumin fusion protein conjugated to a labeled
compound (e.g., .sup.3H or .sup.125I) in the presence of increasing
amounts of an unlabeled second protein that binds the same protein,
antigen, or epitope as the albumin fusion protein of the
invention.
[0127] In a preferred embodiment, BIAcore kinetic analysis is used
to determine the binding on and off rates of albumin fusion
proteins of the invention to a protein, antigen or epitope. BIAcore
kinetic analysis comprises analyzing the binding and dissociation
of albumin fusion proteins, or specific polypeptides, antigens or
epitopes from chips with immobilized specific polypeptides,
antigens or epitopes or albumin fusion proteins, respectively, on
their surface.
[0128] Antibodies that bind a Therapeutic protein corresponding to
the Therapeutic protein portion of an albumin fusion protein may
also be described or specified in terms of their binding affinity
for a given protein or antigen, preferably the antigen which they
specifically bind. Preferred binding affinities include those with
a dissociation constant or Kid less than 5.times.10.sup.-2 M,
10.sup.-2 M, 5.times.10.sup.-3 M, M, 5.times.10.sup.-6 M, 10.sup.-4
M. More preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-5 M,
10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6M, 5.times.10.sup.-7 M,
10.sup.7 M, 5.times.M.sup.-8 M or 10.sup.-8 M. Even more preferred
binding affinities include those with a dissociation constant or KA
less than 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M,
10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-11 M,
5.times.10.sup.-12 M, .sup.10-12 M, 5.times.10.sup.-13 M,
10.sup.-13 M, 5.times.10.sup.-14 M, 10.sup.-14 M,
5.times.10.sup.-15 M, or 10.sup.-15 M. In preferred embodiments,
albumin fusion proteins comprising at least a fragment or variant
of an antibody that binds a Therapeutic protein, has an affinity
for a given protein or epitope similar to that of the corresponding
antibody (not fused to albumin) that binds a Therapeutic protein,
taking into account the valency of the albumin fusion protein
(comprising at least a fragment or variant of an antibody that
binds a Therapeutic protein) and the valency of the corresponding
antibody. In addition, assays described herein (see Examples and
Table 1) and otherwise known in the art may routinely be applied to
measure the ability of albumin fusion proteins and fragments,
variants and derivatives thereof to elicit biological activity
and/or Therapeutic activity (either in vitro or in vivo) related to
either the Therapeutic protein portion and/or albumin portion of
the albumin fusion protein. Other methods will be known to the
skilled artisan and are within the scope of the invention.
[0129] Albumin
[0130] As described above, an albumin fusion protein of the
invention comprises at least a fragment or variant of a Therapeutic
protein and at least a fragment or variant of human serum albumin,
which are associated with one another, preferably by genetic fusion
or chemical conjugation.
[0131] The terms, human serum albumin (HSA) and human albumin (HA)
are used interchangeably herein. The terms, "albumin and "serum
albumin" are broader, and encompass human serum albumin (and
fragments and variants thereof) as well as albumin from other
species (and fragments and variants thereof).
[0132] As used herein, "albumin" refers collectively to albumin
protein or amino acid sequence, or an albumin fragment or variant,
having one or more functional activities (e.g., biological
activities) of albumin. In particular, "albumin" refers to human
albumin or fragments thereof (see EP 701 239, EP 322 094 WO
97/24445, WO95/23857) especially the mature form of human albumin
as shown in FIG. 5A-D and SEQ ID NO:327, or albumin froth other
vertebrates or fragments thereof, or analogs or variants of these
molecules or fragments thereof.
[0133] In preferred embodiments, the human serum albumin protein
used in the albumin fusion proteins oldie invention contains one or
both of the following sets of point mutations with reference to SEQ
ID NO:327: Leu-407 to Ala, Leu-408 to Val, Val-409 to Ala, and
Arg-410 to Ala; or Arg-410 to A, Lys-413 to Gln, and Lys-414 to Gin
(see, e.g., International Publication No. WO95/23857, hereby
incorporated in its entirety by reference herein). In even more
preferred embodiments, albumin fusion proteins of the invention
that contain one or both of above-described sets of point mutations
have improved stability/resistance to yeast Yap3p proteolytic
cleavage, allowing increased production of recombinant albumin
fusion proteins expressed in yeast host cells.
[0134] As used herein, a portion of albumin sufficient to prolong
the therapeutic activity or shelf-life of the Therapeutic protein
refers to a portion of albumin sufficient in length or structure to
stabilize or prolong the therapeutic activity, of the protein so
that the shelf life of the Therapeutic protein portion of the
albumin fusion protein is prolonged or extended compared to the
shelf-life in the non-fusion state. The albumin portion of the
albumin fusion proteins may comprise the full length of the HA
sequence as described above or as shown in FIG. 5A-D, or may
include One or more fragments thereof that are capable of
stabilizing or prolonging the therapeutic activity. Such fragments
may be of 10 or more amino acids in length or may include about 15,
20, 25, 30, 50, or more contiguous amino acids from the HA sequence
or may include part or all of specific domains of HA. For instance,
one or more fragments of HA spanning the first two
immunoglobulin-like domains may be used.
[0135] The albumin portion of the albumin fusion proteins of the
invention may be a variant of normal HA. The Therapeutic protein
portion of the albumin fusion proteins of the invention may also be
variants of the Therapeutic proteins as described herein. The term
"variants" includes insertions, deletions and substitutions, either
conservative or non conservative, where such changes do not
substantially alter one or more of the oncotic, useful
ligand-binding and non-immunogenic properties of albumin, or the
active site, or active domain which confers the therapeutic
activities of the Therapeutic proteins.
[0136] In particular, the albumin fusion proteins of the invention
may include naturally occurring polymorphic variants of human
albumin and fragments of human albumin, for example those fragments
disclosed in EP 322 094 (namely HA (Pn), where it is 369 to 419).
The albumin may be derived from any vertebrate, especially any
mammal, for example human, cow, sheep, or pig. Non-mammalian
albumins include, but are not limited to, hen and salmon. The
albumin portion of the albumin fusion protein may be from a
different animal than the Therapeutic protein portion.
[0137] Generally speaking, an HA fragment or variant will be at
least 100 amino acids long, preferably at least 150 amino acids
long. The HA variant may consist of or alternatively comprise at
least one whole domain of HA, for example domains 1 (amino acids
1-194 of SEQ ID NO:327). 2 (amino acids 195-387 of SEQ ID NO:327),
3 (amino acids 388-585 of SEQ ID NO:327), 1+2 (1-387 of SEQ ID
NO:327), 2+3 (195-585 of SEQ ID NO:327) or 1+3 (amino acids 1-194
of SEQ ID NO:327+amino acids 388-585 of SEQ ID NO:327). Each domain
is itself made up of two homologous subdomains namely 1-105,
120-194, 195-291, 316-387, 388-491 and 512-585, with flexible
inter-subdomain linker regions comprising residues Lys106 to
Glu119, Glu292 to Val315 and Glu492 to Ala511.
[0138] Preferably, the albumin portion of an albumin fusion protein
of the invention comprises at least one subdomain or domain of HA
or conservative modifications thereof. If the fusion is based on
subdomains, some or all of the adjacent linker is preferably used
to link to the Therapeutic protein moiety.
[0139] Antibodies that Specifically Bind Therapeutic Proteins are
also Therapeutic Proteins
[0140] The present invention also encompasses albumin fusion
proteins that comprise at least a fragment or variant of an
antibody that specifically binds a Therapeutic protein disclosed in
Table 1. It is specifically contemplated that the term "Therapeutic
protein" encompasses antibodies that bind a Therapeutic protein
(e.g., as Described in column 1 of Table 1) and fragments and
variants thereof. Thus an albumin fusion protein of the invention
may contain at least a fragment or variant of a Therapeutic
protein, and/or at least a fragment or variant of an antibody that
binds a Therapeutic protein.
[0141] Antibody Structure and Background
[0142] The basic antibody structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Human light chains are
classified as kappa and lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
See generally, Fundamental Immunology Chapters 3-5 (Paul. W., ed.,
4th ed. Raven Press. N.Y. (1998)) (incorporated by reference in its
entirety for all purposes). The variable regions of each
light/heavy chain pair form the antibody binding site.
[0143] Thus, an intact IgG antibody has two binding sites. Except
in bifunctional or bispecific antibodies, the two binding sites are
the same.
[0144] The chains all exhibit the same general structure of
relatively conserved framework regions (FR) joined by three
hypervariable regions, also called complementarity determining
regions or CDRs. The CDR regions, in general, are the portions of
the antibody which make contact with the antigen and determine its
specificity. The CDRs front the heavy and the light chains of each
pair are aligned by the framework regions, enabling binding to a
specific epitope. From N-terminal to C-terminal, both light and
heavy chains variable regions comprise the domains FR1, CDR1, FR2,
CDR2, FR3. CDR3 and FR4. The variable regions are connected to the
heavy or light chain constant region. The assignment of amino acids
to each domain is in accordance with the definitions of Kabat
Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia
& Lesk J Mol Biol. 196:901-917 (1987); Chothia et al. Nature
342:878-883 (1989).
[0145] As used herein, "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
that specifically binds an antigen (e.g., a molecule containing one
or more CDR regions of an antibody). Antibodies that may correspond
to a Therapeutic protein portion of an albumin fusion protein
include, but arc not limited to, monoclonal, multispecific, human,
humanized or chimeric antibodies, single chain antibodies (e.g.,
single chain Fvs), Fab fragments, F(ab') fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies (including, e.g., anti-Id antibodies specific to
antibodies of the invention), and epitope-binding fragments of any
of the above (e.g., VH domains, VL domains, or one or more CDR
regions).
[0146] Antibodies that Bind Therapeutic Proteins
[0147] The present invention encompasses albumin fusion proteins
that comprise at least a fragment or variant of an antibody that
binds a Therapeutic Protein (e.g., as disclosed in Table 1) or
fragment or variant thereof.
[0148] Antibodies that bind a Therapeutic protein (or fragment or
variant thereof) may be from any animal origin, including birds and
mammals. Preferably, the antibodies are human, marine (e.g., mouse
and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or
chicken antibodies. Most preferably, the antibodies are human
antibodies. As used herein, "human" antibodies include antibodies
having the amino acid sequence of a human immunoglobulin and
include antibodies isolated from human immunoglobulin libraries and
xenomice or other organisms that have been genetically engineered
to produce human antibodies.
[0149] The antibody molecules that bind to a Therapeutic protein
and that may correspond to a Therapeutic protein portion of an
albumin fusion protein of the invention can be of any type (e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3,
IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In
preferred embodiments, the antibody molecules that bind to a
Therapeutic protein and that may correspond to a Therapeutic
protein portion of an albumin fusion protein are IgG1. In other
preferred embodiments, the immunoglobulin molecules that bind to a
Therapeutic protein and that may correspond to a Therapeutic
protein portion of an albumin fusion protein are IgG2. In other
preferred embodiments, the immunoglobulin molecules that bind to a
Therapeutic protein and that may correspond to a Therapeutic
protein portion of an albumin fusion protein are IgG4.
[0150] Most preferably the antibodies that bind to a Therapeutic
protein and that may correspond to a Therapeutic protein portion of
an albumin fusion protein are human antigen-binding antibody
fragments of the present invention and include, but are not limited
to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the
variable region(s) alone or in combination with the entirety or a
portion of the following: hinge region, CH1, CH2, and CH3
domains.
[0151] The antibodies that bind to a Therapeutic protein and that
may correspond to a Therapeutic protein portion of an albumin
fusion protein may be monospecific, bispecific, trispecific or of
greater multispecificity. Multispecific antibodies may be specific
for different epitopes of a Therapeutic protein or may be specific
for both a Therapeutic protein as well as for a heterologous
epitope, such as a heterologous polypeptide or solid support
material, See, e.g., PCT publications WO 93/17715: WO 92/08802; WO
91/00360; WO 92/05793; Tun, et al., J. Immunol. 147:60-69 (1991);
U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[0152] Antibodies that bind a Therapeutic protein (or fragment or
variant thereof) may be bispecific or bifunctional which means that
the antibody is an artificial hybrid antibody having two different
heavy/light chain pairs and two different binding sites. Bispecific
antibodies can be produced by a variety of methods including fusion
of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai
& Lachmann Clin. Rap. Immunol. 79: 315-321 (1990), Kostelny et
al. J Immunol. 148:1547 1553 (1992). In addition, bispecific
antibodies may be formed as "diabodies" (Holliger et al.
"Diabodies': small bivalent and bispecific antibody fragments" PNAS
USA 90:6414-6448 (1993)) or "Janusins" (Traunecker et al.
"Bispecific single chain molecules (Janusins) target cytotoxic
lymphocytes on HIV infected cells" EMBO J 10:3655-3659 (1991) and
Traunecker et al. "Janusin: new molecular design for bispecific
reagents" Int J Cancer Suppl 7:51-52 (1992)).
[0153] The present invention also provides albumin fusion proteins
that comprise, fragments or variants (including derivatives) of an
antibody described herein or known elsewhere in the art. Standard
techniques known to those of skill in the art can be used to
introduce mutations in the nucleotide sequence encoding a molecule
of the invention, including, for example, site-directed mutagenesis
and PCR-mediated mutagenesis which result in amino acid
substitutions. Preferably, the variants (including derivatives)
encode less than 50 amino acid substitutions, less than 40 amino
acid substitutions, less than 30 amino acid substitutions, less
than 25 amino acid substitutions, less than 20 amino acid
substitutions, less than 15 amino acid substitutions, less than 30
amino acid substitutions, less than 5 amino acid substitutions,
less than 4 amino acid substitutions, less than 3 amino acid
substitutions, or less than 2 amino acid substitutions relative to
the reference VH domain. VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1,
VLCDR2, or VLCDR3. In specific embodiments, the variants encode
substitutions of VHCDR3. In a preferred embodiment, the variants
have conservative amino acid substitutions at one or more predicted
non essential amino acid residues.
[0154] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein may be described or specified in terms of the epitope(s) or
portion(s) of a Therapeutic protein which they recognize or
specifically bind. Antibodies which specifically bind a Therapeutic
protein or a specific epitope of a Therapeutic protein may also be
excluded. Therefore, the present invention encompasses antibodies
that specifically bind Therapeutic proteins, and allows for the
exclusion of the same. In preferred embodiments, albumin fusion
proteins comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein, hinds the same epitopes as the
unfused fragment or variant of that antibody.
[0155] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein may also be described or specified in terms of their
cross-reactivity. Antibodies that do not hind any other analog,
ortholog, or homolog of a Therapeutic protein are included.
Antibodies that bind polypeptides with at least 95%, at least 90%,
at least 85%, at least 80%, at least 75%, at least 70%, at least
65%, at least 60%, at least 55%, and at least 50% sequence identity
(as calculated using methods known in the art and described herein)
to a Therapeutic protein are also included in the present
invention. In specific embodiments, antibodies that bind to a
Therapeutic protein and that may correspond to a Therapeutic
protein pen inn of an albumin fusion protein cross-react with
murine, rat and/or rabbit homologs of human proteins and the
corresponding epitopes thereof. Antibodies that do not bind
polypeptides with less than 95%, less than 90%, less than 85%, less
than 80%, less than 75%, less than 70%, less than 65%, less than
60%, legs than 55%, and less than 50% sequence identity (as
calculated using methods known in the an and described herein) to a
Therapeutic protein are also included in the present invention. In
a specific embodiment, the above-described cross-reactivity is with
respect to any single specific antigenic or immunogenic
polypeptide, or combination(s) of 2, 3, 4, 5, or inure of the
specific antigenic and/or immunogenic polypeptides disclosed
herein. In preferred embodiments, albumin fusion proteins
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein, has similar or substantially identical cross
reactivity characteristics compared to that particular
antibody.
[0156] Further included in the present invention ate antibodies
which bind polypeptides encoded by polynucleotides which hybridize
to a polynucleotide encoding a Therapeutic protein under stringent
hybridization conditions (as described herein). Antibodies that
bind to a Therapeutic protein and that may correspond to a
Therapeutic protein portion of an albumin fusion protein of the
invention may also be described or specified in terms of their
binding affinity to a polypeptide of the invention. Preferred
binding affinities include those with a dissociation constant or Kd
less than 5.times.10.sup.-2 M, 10.sup.-2 M, 5.times.10.sup.-3 M,
10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M. More preferred
binding affinities include those with a dissociation constant or Kd
less than 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6M, 5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M
or 10.sup.-8 M. Even more preferred binding affinities include
those with a dissociation constant or Kd less than
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
.sup.10-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M. In preferred embodiments, albumin fusion proteins
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein, has an affinity for a given protein or
epitope similar to that of the corresponding antibody (not fisted
to albumin) that binds a Therapeutic protein, taking into account
the valency of the albumin fusion protein (comprising at least a
fragment or variant of an antibody that binds a Therapeutic
protein) and the valency of the corresponding antibody.
[0157] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of a Therapeutic
protein as determined by any method known in the art for
determining competitive binding, for example, the immunoassays
described herein. In preferred embodiments, the antibody
competitively inhibits binding to the epitope by at least 95%, at
least 90%, at least 85%, at least 80%, at least 75%, at least 70%,
at least 60%, or at least 50%. In preferred embodiments, albumin
fusion proteins comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein, competitively inhibits
binding of a second antibody to an epitope of a Therapeutic protein
as well as the fragment or variant of the antibody comprised by the
fusion protein competitively inhibits binding of said second
antibody to an epitope of a Therapeutic protein. In other preferred
embodiments, albumin fusion proteins comprising at least a fragment
or variant of an antibody that binds a Therapeutic protein,
competitively inhibits binding of a second antibody to an epitope
of a Therapeutic protein by at least 95%, at least 90%, at least
85%, at least 30%, at least 75%, at least 70%, at least 60%, or at
least 50%.
[0158] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein of the invention may act as agonists or antagonists of the
Therapeutic protein. For example, the present invention includes
antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the invention either partially or fully. The
invention features both receptor-specific antibodies and
ligand-specific antibodies. The invention also features
receptor-specific antibodies which do not prevent ligand binding
but prevent receptor activation. Receptor activation (i.e.,
signaling) may be determined by techniques described herein or
otherwise known in the art. For example, receptor activation can be
determined by detecting the phosphorylation (e.g., tyrosine or
seine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example,
as described supra). In specific embodiments, antibodies are
provided that inhibit ligand activity or receptor activity by at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody. In preferred embodiments, albumin fusion
proteins comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein, has similar or substantially
similar characteristics with regard to preventing ligand binding
and/or preventing receptor activation compared to an un-fused
fragment or variant of the antibody that binds the Therapeutic
protein.
[0159] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralising antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation, for example, by inducing
dimerization of the receptor. The antibodies may be specified as
agonists, antagonists or inverse agonists for biological activities
comprising the specific biological activities of the Therapeutic
proteins (e.g. as disclosed in Table 1). The above antibody
agonists can be made using methods known in the art. See, e.g., PCT
publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al.,
Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214
(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et
al., 0.1. Cell. Sci. 111(Pt 2):237-247 (1998); Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liatuard et al., Cytokine
9(4):233-241 (1997); Carlson et al., J. Biol. Chem.
272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762
(1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et
al., Cytokine 8(1):14-20 (1996) (which are all incorporated by
reference herein in their entireties). In preferred embodiments,
albumin fusion proteins comprising at least a fragment or variant
of an antibody that binds a Therapeutic protein, have similar or
substantially identical agonist or antagonist properties as an
un-fused fragment or variant of the antibody that binds the
Therapeutic protein.
[0160] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein of the invention may be used, for example, to purify,
detect, and target Therapeutic proteins, including both in ire
vitro and in vivo diagnostic and therapeutic methods. For example,
the antibodies have utility in immunoassays for qualitatively and
quantitatively measuring levels of the Therapeutic protein in
biological samples. See, e.g., Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); incorporated by reference herein in its entirety. Likewise,
albumin fusion proteins comprising at least a fragment or variant
of an antibody that binds a Therapeutic protein, may be used, for
example, to purify, detect, and target Therapeutic proteins,
including both in in vitro and ire vivo diagnostic and therapeutic
methods.
[0161] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein include derivatives that are modified, i.e., by the
covalent attachment of any type of molecule to the antibody. For
example, but not by way of limitation, the antibody derivatives
include antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation, phosphylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids. Albumin fusion proteins of the invention may also be
modified as described above.
[0162] Methods of Producing Antibodies that Hind Therapeutic
Proteins
[0163] The antibodies that bind to a Therapeutic protein and that
may correspond to a Therapeutic protein portion of an albumin
fusion protein of the invention may be generated by any suitable
method known in the art. Polyclonal antibodies to an
antigen-of-interest can be produced by various procedures well
known in the art. For example, a Therapeutic protein may be
administered to various host animals including, but not limited to,
rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include but are not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as HCG (bacille Calmette-Guerin) and corynebacterium parvum.
Such adjuvants are also well known in the art.
[0164] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited so antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0165] Methods for producing and screening for specific antibodies
using Hybridoma technology are routine and well known in the art.
In a non-limiting example, mice can be immunized with a Therapeutic
protein or fragment or variant thereof or a cell expressing such a
Therapeutic protein or fragment or variant thereof. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC. Hybridomas are selected and
cloned by limited dilution. The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies
capable of binding a polypeptide of the invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0166] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody wherein, preferably, the hybridoma is generated by fusing
splenocytes isolated from a mouse immunized with an antigen of the
invention with myeloma cells and then screening the hybridomas
resulting from the fusion for hybridoma clones that secrete an
antibody able to bind a polypeptide of the invention.
[0167] Another well known method for producing both polyclonal and
monoclonal human B cell lines is transformation using Epstein Barr
Virus (EBV). Protocols for generating EBV-transformed ft cell lines
are commonly known in the art, such as, for example, the protocol
outlined in Chapter 7.22 of Current Protocols in Immunology,
Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is
hereby incorporated in its entirety by reference. The source of B
cells for transformation is commonly human peripheral blood, but B
cells for transformation may also be derived from other sources
including, but not limited to, lymph nodes, tonsil, spleen, tumor
tissue, and infected tissues. Tissues are generally made into
single cell suspensions prior to EBV transformation. Additionally,
steps may be taken to either physically remove or inactivate T
cells (e.g., by treatment with cyclosporin A) in B cell containing
samples, because T cells from individuals seropositive for anti-EBV
antibodies can suppress B cell immortalization by EBV.
[0168] In general, the sample containing human B cells is
innoculated with EBV, and cultured for 3-4 weeks. A typical source
of EBV is the culture supernatant of the B95-8 cell line (ATCC
#VR-1492). Physical signs of EBV transformation can generally be
seen towards the end of the 3-4 week culture period. By
phase-contrast microscopy, transformed cells may appear large,
clear, hairy and tend to aggregate in tight clusters of cells.
Initially. EBV lines are generally polyclonal. However, over
prolonged periods of cell cultures, EBV lines may become monoclonal
or polyclonal as a result of the selective outgrowth of particular
B cell clones. Alternatively, polyclonal EBV transformed lines may
be subcloned (e.g., by limiting dilution culture) or fused with a
suitable fusion partner and plated at limiting dilution to obtain
monoclonal B cell lines. Suitable fusion partners for EBV
transformed cell lines include mouse myeloma cell lines (e.g.,
SP2/0, X63-Ag8.653), heteromyeloma cell lines (human.times.mouse;
e.g., SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM
1508, SKO-007, RPMI 8226, and KR-4). Thus, the present invention
also provides a method of generating polyclonal or monoclonal human
antibodies against polypeptides of the invention or fragments
thereof, comprising EBV-transformation of human B cells.
[0169] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0170] For example, antibodies that bind to a Therapeutic protein
can also be generated using various phage display methods known in
the art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular embodiment,
such phage can be utilized to display antigen binding domains
expressed from a repertoire or combinatorial antibody library
(e.g., human or marine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labeled antigen or antigen bound or
captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 binding
domains expressed from phage with Fab, Fv or disulfide stabilized
Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII protein. Examples of phage display methods that
can be used to make antibodies that bind to a Therapeutic protein
include those disclosed in Brinkman et al., J. Immunol, Methods
182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186
(1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994);
Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in
Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134;
PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.
5,698,426; 5,223409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;
5,733,743 and 5,469,108; each of which is incorporated herein by
reference in its entirety.
[0171] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax at al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[0172] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in viva use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gullies et al., (1989) J.
Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816397, which are incorporated herein by reference in their
entirety. Humanized antibodies are antibody molecules from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and a framework regions from a human
immunoglobulin molecule. Often, framework residues in the human
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089;
Ricchmann et al., Nature 332:323 (1988), which are incorporated
herein by reference in their entireties.) Antibodies can be
humanized using a variety of techniques known in the art including,
for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967;
U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology
28(4/5):489-498 (1991); Studnicka et al, Protein Engineering
7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)), and
chain shuffling (U.S. Pat. No. 5,565,332).
[0173] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety.
[0174] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/14096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which
are incorporated by reference herein in their entirety. In
addition, companies such as Abgenix, Inc. (Freemont, Calif.) and
Genpharm (San Jose, Calif.) can be engaged to provide human
antibodies directed against a selected antigen using technology
similar to that described above.
[0175] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al Bio/technology 12:899-903 (1988)).
[0176] Polynucleotides Encoding Antibodies
[0177] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody and fragments thereof. The
invention also encompasses polynucleotides that hybridize under
stringent or alternatively, under lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically hinds to a Therapeutic
protein, and more preferably, an antibody that binds to a
polypeptide having the amino acid sequence of a "Therapeutic
protein:X" as disclosed in the "SEQ ID NO: Z" column of Table
2.
[0178] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0179] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody) by PCR amplification using synthetic primers
hybridizable to the 3' and 5' ends of the sequence or by cloning
using an oligonucleotide probe specific for the particular gene
sequence to identify, e.g., a cDNA clone from a cDNA library that
encodes the antibody. Amplified nucleic acids generated by PCR may
then be cloned into replicable cloning vectors using any method
well known in the art (See Example 46).
[0180] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel at al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0181] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0182] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81.851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0183] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are farmed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra at al., Science
242:1038-1041 (1988)).
[0184] Recombinant Expression of Antibodies
[0185] Recombinant expression of an antibody, or fragment,
derivative or analog thereof, (e.g., a heavy or light chain of an
antibody or a single chain antibody), requires construction of an
expression vector containing a polynucleotide that encodes the
antibody. Once a polynucleotide encoding an antibody molecule or a
heavy or light chain of an antibody, or portion thereof (preferably
containing the heavy or light chain variable domain), of the
invention has been obtained, the vector for the production of the
antibody molecule may be produced by recombinant DNA technology
using techniques well known in the art. Thus, methods for preparing
a protein by expressing a polynucleotide containing an antibody
encoding nucleotide sequence are described herein. Methods which
are well known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in viva genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[0186] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody. Thus, the
invention includes host cells containing a polynucleotide encoding
an antibody of the invention, or a heavy or light chain thereof, or
a single chain antibody, operably linked to a heterologous
promoter. In preferred embodiments for the expression of double
chained antibodies, vectors encoding both the heavy and light
chains may be co-expressed in the host cell for expression of the
entire immunoglobulin molecule, as detailed below.
[0187] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; 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 (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter),
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic especially for the expression of whole
recombinant antibody molecule, are used for the expression of a
recombinant antibody molecule. For example, mammalian cells such as
Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2 (1990)).
[0188] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable, Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Rusher et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.,
24:5503-55119 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione-agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0189] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[0190] In mammalian here cells, a number of viral-based expression
systems may be utilized. In cases where all adenovirus is used as
an expression vector, the antibody coding sequence of interest may
be ligated to an adenovirus transcription/translation control
complex, e.g., the late promoter and a tripartite leader sequence.
This chimeric gene may then be inserted in the adenovirus genome by
in vitro or in vivo recombination. Insertion in a non-essential
region of the viral genome (e.g., region E1 or E3) will result in a
recombinant sires that is viable and capable of expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
roust be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al. Methods in Enzymol.
153:51-544 (1987)).
[0191] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MUCK, 293, 3T3, WI38, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T471),
and normal mammary gland cell line such as, for example, CRL7030
and Hs578Bst.
[0192] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0193] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
can be employed in tk-, hgprt- or aprt- cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); ilea, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu et al.,
Biotherapy 3:87-95 (1999); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:7191-217 (1993);
May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers
resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).
Methods commonly known in the art of recombinant DNA technology may
be routinely applied to select the desired recombinant clone, and
such methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression. A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al, (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their
entireties.
[0194] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentachel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York. 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
[0195] Vectors which use glutamine synthase (GS) or DHFR as the
selectable markers can be amplified in the presence of the drugs
methionine sulphoximine or methotrexate, respectively. An advantage
of glutamine synthase based vectors are the availability of cell
lines (e.g., the murine myeloma cell line, NS0) which are glutamine
synthase negative. Glutamine synthase expression systems can also
function in glutamine synthase expressing cells (e.g. Chinese
Hamster Ovary (CHO) cells) by providing additional inhibitor to
prevent the functioning of the endogenous gene. A glutamine
synthase expression system and components thereof are detailed in
PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404:
and WO91/06657 which are incorporated in their entireties by
reference herein. Additionally, glutamine synthase expression
vectors that may be used according to the present invention are
commercially available from suppliers, including, for example Lonza
Biologics, Inc. (Portsmouth, N.H.). Expression and production of
monoclonal antibodies using a CS expression system in murine
myeloma cells is described in Bebbington at al., Bio/technology
10:169 (1992) and in Biblia and Robinson Biotechnol. Prog. 11:1
(1995) which are incorporated in then entireties by reference
herein.
[0196] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing, both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy
and light chains may comprise cDNA or germane DNA.
[0197] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized, or recombinantly expressed,
it may be purified by any method known in the art for purification
of an immunoglobulin molecule, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies that bind to a Therapeutic protein and
that may correspond to a Therapeutic protein portion of an albumin
fusion protein of the invention or fragments thereof can be fused
to heterologous polypeptide sequences described herein or otherwise
known in the art, to facilitate purification.
[0198] Modifications of Antibodies
[0199] Antibodies that bind a Therapeutic protein or fragments or
variants can be fused to marker sequences, such as a peptide to
facilitate purification. In preferred embodiments, the marker amino
acid sequence is a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311), among others, many of which are commercially
available. As described in Gentz et al. Proc. Natl. Acad. Sci. USA
86:821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. Other peptide tags
useful for purification include, but are not limited to, the "HA"
tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
"flag" tag.
[0200] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically to, for example, monitor
the development or progression of a tumor as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. The detectable substance may be coupled or conjugated
either directly to the antibody (or fragment thereof) or
indirectly, through an intermediate (such as, for example, a linker
known in the an) using techniques known in the art. See, for
example, U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include 125I, 131I, 111In or 99Tc. Other examples of
detectable substances have been described elsewhere herein.
[0201] Further, an antibody of the invention may be conjugated to a
therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are nut limited to,
antimembolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5 fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine f CCNU), cyclothosphamide,
busulfan, dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0202] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as minor
necrosis factor, alpha-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), a thrombotic agent or
an anti-angiogenic agent, e.g., angiostatin or endostatin; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor
("CM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0203] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0204] Techniques for conjugating such therapeutic moiety to
antibodies are well known. See, for example, Amon et al.,
"Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer
Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et
al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd
Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc.
1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer
Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62; 119-58 (1982).
[0205] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0206] An antibody, with or without a therapeutic moiety conjugated
to it, administered alone or in combination with cytotoxic
factor(s) and/or cytokine(s) can be used as a therapeutic.
[0207] Antibody-Albumin Fusion
[0208] Antibodies that bind to a Therapeutic protein and that may
correspond to a Therapeutic protein portion of an albumin fusion
protein of the invention include, but ale not limited to,
antibodies that bind a Therapeutic protein disclosed in the
"Therapeutic Protein X" column of Table 1, or a fragment or variant
thereof.
[0209] In specific embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, the VH
domain. In other embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, one, two or
three VH CDRs. In other embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, the VH
CDR1. In other embodiments, the fragment or variant of an antibody
that immunospecifically binds a Therapeutic protein and that
corresponds to a Therapeutic protein portion of an albumin fusion
protein comprises, or alternatively consists of, the VH CDR2. In
other embodiments, the fragment or variant of an antibody that
immunospecifically binds a Therapeutic protein and that corresponds
to a Therapeutic protein portion of an albumin fusion protein
comprises, or alternatively consists of, the VH CDR3.
[0210] In specific embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, the VL
domain. In other embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, one, two or
three VL CDRs. In other embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, the VL
CDR1. In other embodiments, the fragment or variant of an antibody
that immunospecifically binds a Therapeutic protein and that
corresponds to a Therapeutic protein portion of an albumin fusion
protein comprises, or alternatively consists of, the VL CDR2. In
other embodiments, die fragment or variant of an antibody that
immunospecifically binds a Therapeutic protein and that corresponds
to a Therapeutic protein portion of an albumin fusion protein
comprises, or alternatively consists of, the VL CDR3.
[0211] In other embodiments, the fragment or variant of an antibody
that immunospecifically binds a Therapeutic protein and that
corresponds to a Therapeutic protein portion of an albumin fusion
protein comprises, or alternatively consists of, one, two, three,
four, five, or six and/or VL CDRs.
[0212] In preferred embodiments, the fragment or variant of an
antibody that immunospecifically binds a Therapeutic protein and
that corresponds to a Therapeutic protein portion of an albumin
fusion protein comprises, or alternatively consists of, an scFv
comprising the VH domain of the Therapeutic antibody, linked to the
VL domain of the therapeutic antibody by a peptide linker such as
(Gly.sub.4Ser).sub.3 (SEQ ID NO:378).
[0213] Immunophenotyping
[0214] The antibodies of the invention or albumin fusion proteins
of the invention comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein (or fragment or variant
thereof) may be utilized for immunophenotyping of cell lines and
biological samples. Therapeutic proteins of the present invention
may be useful as cell-specific markers, or more specifically as
cellular markers that are differentially expressed at various
stages of differentiation and/or maturation of particular cell
types. Monoclonal antibodies (or albumin fusion proteins comprising
at least a fragment or variant of an antibody that binds a
Therapeutic protein) directed against a specific epitope, or
combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be
utilized using monoclonal antibodies (or albumin fusion proteins
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein) to screen for cellular populations
expressing the marker(s), and include magnetic separation using
antibody-coated magnetic beads, "panning" with antibody attached to
a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S.
Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49
(1999)).
[0215] These techniques allow for the screening of particular
populations of cells, such as might be found with hematological
malignancies (i.e., minimal residual disease (BIRD) in acute
leukemic patients) and "non-self" cells in transplantations to
prevent Graft-versus-Host Disease (GVHD). Alternatively, these
techniques allow for the screening of hematopoietic stem and
progenitor cells capable of undergoing proliferation and/or
differentiation, as might be found in human umbilical cord
blood.
[0216] Characterizing Antibodies that Bind a Therapeutic Protein
and Albumin Fusion Proteins Comprising a Fragment or Variant of an
Antibody that Binds a Therapeutic Protein
[0217] The antibodies of the invention or albumin fusion proteins
of the invention comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein (or fragment or variant
thereof) may be characterized in a variety of ways. In particular,
Albumin fusion proteins of the invention comprising at least a
fragment or variant of an antibody that binds a Therapeutic protein
may be assayed for the ability to specifically bind to the same
antigens specifically bound by the antibody that binds a
Therapeutic protein corresponding to the antibody that binds a
Therapeutic protein portion of the albumin fusion protein using
techniques described herein or routinely modifying techniques known
in the art.
[0218] Assays for the ability of the antibodies of the invention or
albumin fusion proteins of the invention comprising at least a
fragment or variant of an antibody that binds a Therapeutic protein
(or fragment or variant thereof) to (specifically) bind a specific
protein or epitope may be performed in solution (e.g., Houghten,
Bio/Techniques 13:412-421 (1992)), on beads (e.g., Lam, Nature
354:82-84 (1991)), on chips (e.g., Fodor, Nature 364:555-556
(1993)), on bacteria (e.g., U.S. Pat. No. 5,223,409), on spores
(e.g., U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on
plasmids (e.g., Cull at al., Proc. Nat. Acad. Sci. USA 89:1865-1869
(1992)) or on phage (e.g., Scott and Smith, Science 249:386-390
(1990); Devlin, Science 249:404-406 (1990); Cwirla et al., Proc.
Natl., Acad. Sci. USA 87:6378-6382 (1990); and Felici, J. Mol.
Biol. 222:301-310 (1991)) (each of these references is incorporated
herein in its entirety by reference). The antibodies of the
invention or albumin fusion proteins of the invention comprising at
least a fragment or variant of an antibody that binds a Therapeutic
protein (or fragment or variant thereof) may also be assayed for
their specificity and affinity for a specific protein or epitope
using or routinely modifying techniques described herein or
otherwise known in the art.
[0219] The albumin fusion proteins of the invention comprising at
least a fragment or variant of an antibody that binds a Therapeutic
protein may be assayed for cross-reactivity with other antigens
(e.g., molecules that have sequence/structure conservation with the
molecule(s) specifically bound by the antibody that binds a
Therapeutic protein (or fragment or variant thereof) corresponding
to the Therapeutic protein portion of the albumin fusion protein of
the invention) by any method known in the art.
[0220] Immunoassays which can be used to analyze (immunospecific)
binding and cross-reactivity include, but are not limited to,
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, and
protein A immunoassays, to name but a few. Such assays are routine
and well known in the art (see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York, which is incorporated by reference herein in
its entirety). Exemplary immunoassays are described briefly below
(but are not intended by way of limitation).
[0221] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SOS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding an antibody of the invention Of
albumin fusion protein of the invention comprising at least a
fragment or variant of an antibody that binds a Therapeutic protein
(or fragment or variant thereof) to the cell lysate, incubating for
a period of time (e.g., 1 to 4 hours) at 40 degrees C., adding
protein A and/or protein G sepharose beads (or beads coated with an
appropriate anti-idiotypic antibody or anti-albumin antibody in the
case when an albumin fusion protein comprising at least a fragment
or variant of a Therapeutic antibody) to the cell lysate,
incubating for about an hour or more at 40 degrees C., washing the
beads in lysis buffer and resuspending the beads in SDS/sample
buffer. The ability of the antibody or albumin fusion protein of
the invention to immunoprecipitate a particular antigen can be
assessed by, e.g., western blot analysis. One of skill in the art
would be knowledgeable as to the parameters that can be modified to
increase the binding of the antibody or albumin fusion protein to
an antigen and decrease the background (e.g., pre-clearing the cell
lysate with sepharose beads). For further discussion regarding
immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York at 10.16.1.
[0222] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
applying the antibody or albumin fusion protein of the invention
(diluted in blocking buffer) to the membrane, washing the membrane
in washing buffer, applying a secondary antibody (which recognizes
the albumin fusion protein, e.g., an anti-human serum albumin
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
.sup.32P or .sup.125I) diluted in blocking buffer, washing the
membrane in wash buffer, and detecting the presence of the antigen.
One of skill in the art would be knowledgeable as to the parameters
that can be modified to increase the signal detected and to reduce
the background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0223] ELISAs comprise preparing antigen, coating the well of a
96-well microtiter plate with the antigen, washing away antigen
that did not bind the wells, adding the antibody or albumin fusion
protein (comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein) of the invention conjugated to a
detectable compound such as an enzymatic substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) to the wells and
incubating for a period of time, washing away unbound or
non-specifically bound albumin fusion proteins, and detecting the
presence of the antibody or albumin fusion proteins specifically
bound to the antigen coating the well. In ELISAs the antibody or
albumin fusion protein does not have to be conjugated to a
detectable compound; instead, a second antibody (which recognizes
the antibody or albumin fusion protein, respectively) conjugated to
a detectable compound may be added to the well. Further, instead of
coating the well with the antigen, antibody or the albumin fusion
protein may be coated to the well. In this case, the detectable
molecule could be the antigen conjugated to a detectable compound
such as an enzymatic substrate (e.g.; horseradish peroxidase or
alkaline phosphatase). One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the signal detected as well as other variations of ELISAs known in
the art. For further discussion regarding ELISAs see, e.g., Ausubel
et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1,
John Wiley & Sons, Inc., New York at 11.2.1.
[0224] The binding affinity of an albumin fusion protein to a
protein, antigen, or epitope and the off-rate of an antibody- or
albumin fusion protein-protein/antigen/epitope interaction can be
determined by competitive binding assays. One example of a
competitive binding assay is a radioimmunoassay comprising the
incubation of labeled antigen (e.g., .sup.3H or .sup.125I) with the
antibody or albumin fusion protein of the invention in the presence
of increasing amounts of unlabeled antigen, and the detection of
the antibody bound to the labeled antigen. The affinity of the
antibody or albumin fusion protein of the invention for a specific
protein, antigen, or epitope and the binding off-rates can be
determined from the data by Scatchard plot analysis. Competition
with a second protein that binds the same protein, antigen or
epitope as the antibody or albumin fusion protein, can also be
determined using radioimmunoassays. In this case, the protein,
antigen or epitope is incubated with an antibody or albumin fusion
protein of the invention conjugated to a labeled compound (e.g.,
.sup.3H or .sup.125I) in the presence of increasing amounts of an
unlabeled second protein that binds the same protein, antigen, or
epitope as the albumin fusion protein of the invention.
[0225] In a preferred embodiment, BIAcore kinetic analysis is used
to determine the binding on and off rates of antibody or albumin
fusion proteins of the invention to a protein, antigen or epitope.
BIAcore kinetic analysis comprises analyzing the binding and
dissociation of antibodies, albumin fusion proteins, or specific
polypeptides, antigens or epitopes from chips with immobilized
specific polypeptides, antigens or epitopes, antibodies or albumin
fusion proteins, respectively, on their surface.
[0226] Therapeutic Uses
[0227] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
or albumin fusion proteins of the invention comprising at least a
fragment or variant of an antibody that binds a Therapeutic protein
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the disclosed diseases,
disorders, or conditions. Therapeutic compounds of the invention
include, but are not limited to, antibodies of the invention
(including fragments, analogs and derivatives thereof as described
herein), nucleic acids encoding antibodies of the invention
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein), albumin fusion
proteins of the invention comprising at least a fragment or variant
of an antibody that binds a Therapeutic protein, and nucleic acids
encoding such albumin fusion proteins. The antibodies of the
invention or albumin fusion proteins of the invention comprising at
least a fragment or variant of an antibody that binds a Therapeutic
protein can be used to wean inhibit or prevent diseases, disorders
or conditions associated with aberrant expression and/or activity
of a Therapeutic protein, including, but not limited to, any one or
more of the diseases, disorders, or conditions described herein.
The treatment and/or prevention of diseases, disorders, or
conditions associated with aberrant expression and/or activity of a
Therapeutic protein includes, but is not limited to, alleviating
symptoms associated with those diseases, disorders or conditions,
antibodies of the invention or albumin fusion proteins of the
invention comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[0228] In a specific and preferred embodiment, the present
invention is directed to antibody-based therapies which involve
administering antibodies of the invention or albumin fusion
proteins of the invention comprising at least a fragment or variant
of an antibody that binds a Therapeutic protein to an animal,
preferably a mammal, and most preferably a human, patient for
treating one or more diseases, disorders, or conditions, including
but not limited to: neural disorders, immune system disorders,
muscular disorders, reproductive disorders, gastrointestinal
disorders, pulmonary disorders, cardiovascular disorders, renal
disorders, proliferative disorders, and/or cancerous diseases and
conditions, and/or as described elsewhere herein. Therapeutic
compounds of the invention include, but are not limited to,
antibodies of the invention (e.g., antibodies directed to the full
length protein expressed on the cell surface of a mammalian cell;
antibodies directed to an epitope of a Therapeutic protein and
nucleic acids encoding antibodies of the invention (including
fragments, analogs and derivatives thereof and anti-idiotypic
antibodies as described herein). The antibodies of the invention
can be used to treat, inhibit or prevent diseases, disorders Of
conditions associated with aberrant expression and/or activity of a
Therapeutic protein, including, but not limited to, any one or more
of the diseases, disorders, or conditions described herein. The
treatment and/or prevention of diseases, disorders, or conditions
associated with aberrant expression and/or activity of a
Therapeutic protein includes, but is not limited to, alleviating
symptoms associated with those diseases, disorders or conditions.
Antibodies of the invention or albumin fusion proteins of the
invention comprising at least a fragment or variant of an antibody
that binds a Therapeutic protein may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[0229] A summary of the ways in which the antibodies of the
invention or albumin fusion proteins of the invention comprising at
least a fragment or variant of an antibody that binds a Therapeutic
protein may be used therapeutically includes binding Therapeutic
proteins locally or systemically in the body or by direct
cytotoxicity of the antibody, e.g. as mediated by complement (CDC)
or by effector cells (ADCC). Some of these approaches are described
in more detail below. Aimed with the teachings provided herein, one
of ordinary skill in the art will know how to use the antibodies of
the invention or albumin fusion proteins of the invention
comprising at least a fragment or variant of an antibody that binds
a Therapeutic protein for diagnostic, monitoring or therapeutic
purposes without undue experimentation.
[0230] The antibodies of the invention or albumin fusion proteins
of the invention comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors
(such as, e.g., L-2, Il-3 and IL-7), for example, which serve to
increase the number or activity of effector cells which interact
with the antibodies.
[0231] The antibodies of the invention or albumin fusion proteins
of the invention comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein may be administered alone
or in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). Generally, administration of products of a
species origin or species reactivity (in the case of antibodies)
that is the same species as that of the patient is preferred. Thus,
in a preferred embodiment, human antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to is human patient for
therapy or prophylaxis.
[0232] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against Therapeutic
proteins, fragments or regions thereof, (or the albumin fusion
protein correlate of such an antibody) for both immunoassays
directed to and therapy of disorders related to polynucleotides or
polypeptides, including fragments thereof, of the present
invention. Such antibodies, fragments, or regions, will preferably
have an affinity for polynucleotides or polypeptides of the
invention, including fragments thereof. Preferred binding
affinities include dissociation constants or Kd's less than
5.times.10.sup.-2 2 M, 10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3
M, 5.times.10.sup.-4 M, 10.sup.-4 M. More preferred binding
affinities include those with a dissociation constant or Kd less
than 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.7 M, 5.times.10.sup.-8 M
or 10.sup.-8 M. Even more preferred binding affinities include
those with is dissociation constant or Kd less than
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 5.times.10.sup.-12 M, .sup.10-12 M,
5.times.10.sup.-13 M, 10.sup.-13 M, 5.times.10.sup.-14 M,
10.sup.-14 M, 5.times.10.sup.-15 M, or 10.sup.-15 M.
[0233] Gene Therapy
[0234] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies that bind therapeutic proteins or albumin
fusion proteins comprising at least a fragment or variant of an
antibody that binds a Therapeutic protein are administered to
treat, inhibit or prevent a disease or disorder associated with
aberrant expression and/or activity of a Therapeutic protein, by
way of gene therapy. Gene therapy refers to therapy performed by
the administration to a subject of an expressed or expressible
nucleic acid. In this embodiment of the invention, the nucleic
acids produce their encoded protein that mediates a therapeutic
effect.
[0235] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described in more detail elsewhere in this application.
[0236] Demonstration of Therapeutic or Prophylactic Activity
[0237] The compounds or pharmaceutical compositions of the
invention are preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, in vitro assays to demonstrate the therapeutic
or prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of stall in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
[0238] Therapeutic/Prophylactic Administration and Composition
[0239] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention,
preferably an antibody. In a preferred embodiment, the compound is
substantially purified (e.g., substantially free from substances
that limit its effect or produce undesired side-effects). The
subject is preferably an animal, including but not limited to
animals such as cows, pigs, horses, chickens, cats, dogs, etc., and
is preferably a mammal, and most preferably human.
[0240] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid or an
immunoglobulin are described above; additional appropriate
formulations and routes of administration can be selected from
among those described herein below.
[0241] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu et al., L Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to,
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The compounds
or compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0242] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as silastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[0243] In another embodiment, the compound or composition 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.)
[0244] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be 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 (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger et al., J. Macromol. Sci Rev. Macromol. Chem. 23:61 (1983);
see also Levy et al., Science 228:190 (1985); During et al., Ann.
Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).
In yet another embodiment, a controlled release system can be
placed in proximity of the therapeutic target, e.g., 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)).
[0245] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0246] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in viva to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0247] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline, solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0248] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0249] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0250] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a Therapeutic protein can be determined by standard clinical
techniques. In addition, in vitro assays may optionally be employed
to help identify optimal dosage ranges. The precise dose to be
employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0251] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[0252] Diagnosis and Imaging
[0253] Labeled antibodies and derivatives and analogs thereof that
bind a Therapeutic protein (or fragment or variant thereof)
(including albumin fusion proteins comprising at least it fragment
or variant of an antibody that binds a Therapeutic protein), can be
used for diagnostic purposes to detect, diagnose, or monitor
diseases, disorders, and/or conditions associated with the aberrant
expression and/or activity of the Therapeutic protein or fragments
or variants thereof. The invention provides for the detection of
aberrant expression of a Therapeutic protein, comprising (a)
assaying the expression of the Therapeutic protein in cells or body
fluid of an individual using one or mole antibodies specific to the
polypeptide interest and (b) comparing the level of gene expression
with a standard gene expression level, whereby an increase or
decrease in the assayed Therapeutic protein expression level
compared to the standard expression level is indicative of aberrant
expression.
[0254] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of the Therapeutic
protein in cells or body fluid of an individual using one or more
antibodies specific to the Therapeutic protein or albumin fusion
proteins comprising at least a fragment of variant of an antibody
specific to a Therapeutic protein, and (b) comparing the level of
gene expression with a standard gene expression level, whereby an
increase or decrease in the assayed Therapeutic protein gene
expression level compared to the standard expression level is
indicative of a particular disorder. With respect to cancer, the
presence of a relatively high amount of transcript in biopsied
tissue front an individual may indicate a predisposition for the
development of the disease, or may provide a means for detecting
the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[0255] Antibodies of the invention or albumin fusion proteins
comprising at least a fragment of variant of an antibody specific
to a Therapeutic protein can be used to assay protein levels in a
biological sample using classical immunohistological methods known
to those of skill in the art (e.g., see Jalkanen et al., J. Cell.
Biol. 101:976-985 (1985); Jalkanen et al., J. Cell Biol.
105:3087-3096 (1987)). Other antibody-based methods useful for
detecting protein gene expression include immunoassays, such as the
enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay
(RIA). Suitable antibody assay labels are known in the art and
include enzyme labels, such as, glucose oxidase; radioisotopes,
such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium
(3H), indium (112In), and technetium (99Tc); luminescent labels,
such as luminol; and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
[0256] One facet of the invention is the detection and diagnosis of
a disease or disorder associated with alien ant expression of a
Therapeutic protein or fragment or variant thereof in an animal,
preferably a mammal and most preferably a human. In one embodiment,
diagnosis comprises: (a) administering (for example, parenterally,
subcutaneously, or intraperitoneally) to a subject an effective
amount of a labeled molecule which specifically binds to the
polypeptide of interest; (b) waiting for a time interval following
the administering for permitting the labeled molecule to
preferentially concentrate at sites in the subject where the
Therapeutic protein is expressed (and for unbound labeled molecule
to be cleared to background level); (c) determining background
level; and (d) detecting the labeled molecule in the subject, such
that detection of labeled molecule above the background level
indicates that the subject has a particular disease or disorder
associated with aberrant expression of the therapeutic protein.
Background level can be determined by various methods including,
comparing the amount of labeled molecule detected to a standard
value previously determined for a particular system.
[0257] It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of 99mTc. The labeled antibody or antibody fragment, or
albumin fusion protein comprising at least a fragment of variant of
an antibody that binds a Therapeutic potent will then
preferentially accumulate at the location of cells which contain
the specific Therapeutic protein. In vivo tumor imaging is
described in S. W. Burchiel at al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor
Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and
B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
[0258] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[0259] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[0260] Presence of the labeled molecule can be detected in the
patient using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0261] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patent using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI). Antibodies that specifically detect the albumin fusion
protein but not albumin or the therapeutic protein alone are a
preferred embodiment. These can be used to detect the albumin
fusion protein as described throughout the specification.
[0262] Kits
[0263] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody,
preferably a purified antibody, in one or more containers. In a
specific embodiment, the kits of the present invention contain a
substantially isolated polypeptide comprising an epitope which is
specifically immunoreactive with an antibody included in the kit.
Preferably, the kits of the present invention further comprise a
control antibody which does not react with the polypeptide of
interest. In another specific embodiment, the kits of the present
invention contain a means for detecting the binding of an antibody
to a polypeptide of interest (e.g., the antibody may be conjugated
to a detectable substrate such as a fluorescent compound, an
enzymatic substrate, a radioactive compound or a luminescent
compound, or a second antibody which recognizes the first antibody
may be conjugated to a detectable substrate).
[0264] In another specific embodiment of the present invention, the
kit is a diagnostic kit for use in screening serum containing
antibodies specific against proliferative and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control
antibody that does not react with the polypeptide of interest. Such
a kit may include a substantially isolated polypeptide antigen
comprising an epitope which is specifically immunoreactive with at
least one anti-polypeptide antigen antibody. Further, such a kit
includes means for detecting the binding of said antibody to the
antigen (e.g., the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[0265] In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which said
polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can
be detected by binding of the said reporter-labeled antibody.
[0266] In an additional embodiment, the invention includes a
diagnostic kit for use in screening sewn) containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0267] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or calorimetric substrate (Sigma, St.
Louis, Mo.).
[0268] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0269] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-antigen antibody.
[0270] Albumin Fusion Proteins
[0271] The present invention relates generally to albumin fusion
proteins and methods of treating (e.g., ameliorating) or preventing
a metabolic/endocrine disease or disorder. In other embodiments,
the present invention relates to albumin fusion proteins and
methods of treating (e.g., ameliorating) or preventing a disease,
disorder, and/or condition associated with aberrant insulin
secretion and/or action. In a preferred embodiment, the present
invention relates to albumin fusion proteins and methods of
treating (e.g., ameliorating) or preventing a disease, disorder,
and/or condition associated with diabetes. In a highly preferred
embodiment, the present invention relates to albumin fusion
proteins and methods of treating (e.g., ameliorating) or preventing
Type II Non-Insulin-Dependent Diabetes Mellitus (NIDDM) and/or a
condition associated with NIDDM. In another highly preferred
embodiment, the present invention relates to albumin fusion
proteins and methods of treating (e.g., ameliorating) or preventing
Type I Insulin-Dependent Diabetes Mellitus (IDDM) and/or a
condition associated with IDDM.
[0272] In further embodiments, the present invention relates to
albumin fusion proteins and methods of treating (e.g.,
ameliorating) or preventing a condition including, but not limited
to, insulin resistance, insulin sensitivity, hyperglycemia,
hyperinsulinemia, hyperlipidemia, obesity, hyperkotonuria,
retinopathy (e.g., diabetic retinopathy), mononeuropathy,
polyneuropathy, atherosclerosis, ulcers, heart disease, stroke,
anemia, gangrene (e.g., of the feel and hands), impotence,
infection, cataract, poor kidney function, malfunctioning of the
autonomic nervous system, impaired white blood cell function,
Carpal tunnel syndrome, Dupuytren's contracture, and diabetic
ketoacidosis.
[0273] As used herein, "albumin fusion protein" refers to a protein
formed by the fusion of at least one molecule of albumin (or a
fragment or variant thereof) to at least one molecule of a
Therapeutic protein (or fragment or variant thereof). An albumin
fusion protein of the invention comprises at least a fragment or
variant of a Therapeutic protein and at least a fragment or variant
of human serum albumin, which are associated with one another,
preferably by genetic fusion (i.e., the albumin fusion protein is
generated by translation of a nucleic acid in which a
polynucleotide encoding all or a portion of a Therapeutic protein
is joined in-frame with a polynucleotide encoding all or a portion
of albumin). The Therapeutic protein and albumin protein, once part
of the albumin fusion protein, may each be referred to as a
"portion", "region" or "moiety" of the albumin fusion protein.
[0274] In a preferred embodiment, the invention provides an albumin
fusion protein encoded by a polynucleotide or albumin fusion
construct described in Table 1 or Table 2-Polynucleotides encoding
these albumin fusion proteins are also encompassed by the
invention.
[0275] Preferred albumin fusion proteins of the invention, include,
but are not limited to, albumin fusion proteins encoded by a
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide encoding at least one molecule of albumin (or a
fragment or variant thereof) joined in frame to at least one
polynucleotide encoding at least one molecule of a Therapeutic
protein (or fragment or variant thereof); a nucleic acid molecule
comprising, or alternatively consisting of, a polynucleotide
encoding at least one molecule of albumin (or a fragment or variant
thereof) joined in frame to at least one polynucleotide encoding at
least one molecule of a Therapeutic protein (or fragment or variant
thereof) generated as described in Table 2 or in the Examples; or a
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide encoding at least one molecule of albumin (or a
fragment or variant thereof) joined in frame to at least one
polynucleotide encoding at least one molecule of a Therapeutic
protein (or fragment or variant thereof), further comprising, or
alternatively consisting of, for example, one or more of the
following elements: (1) a functional self-replicating vector
(including but not limited to, a shuttle vector, an expression
vector, an integration vector, and/or a replication system), (2) a
region for initiation of transcription (e.g., a promoter region,
such as for example, a regulatable or inducible promoter, a
constitutive promoter), (3) a region for termination of
transcription, (4) a leader sequence, and (5) a selectable
marker.
[0276] In a preferred embodiment, the invention provides an albumin
fusion protein comprising at least one molecule of a Therapeutic
protein disclosed in Table 1. In another preferred embodiment, the
invention provides an albumin fusion protein comprising at least a
fragment or variant of a Therapeutic protein disclosed in Table 1.
In a further embodiment, the invention provides an albumin fusion
protein comprising a mature polypeptide sequence of a Therapeutic
protein disclosed in Table 1. In another preferred embodiment, the
invention provides an albumin fusion protein comprising at least
one human serum albumin polypeptide sequence. In a further
preferred embodiment, the invention provides an albumin fusion
protein comprising at least a fragment or variant of human serum
albumin. In a still further embodiment, the invention provides an
albumin fusion protein comprising a mature human albumin
polypeptide sequence. In a preferred embodiment, the invention
provides an albumin fusion protein encoded by a polynucleotide or
albumin fusion construct described in Table 2.
[0277] In one embodiment, the invention provides an albumin fusion
protein comprising, or alternatively consisting of, a Therapeutic
protein (e.g., as described in Table 1) and a serum albumin
protein. In other embodiments, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
biologically active and/or therapeutically active fragment of a
Therapeutic protein and a serum albumin protein. In other
embodiments, the invention provides an albumin fusion protein
comprising, or alternatively consisting of, a biologically active
and/or therapeutically active variant of a Therapeutic protein and
a serum albumin protein. By "biologically active" and/or
"therapeutically active" fragment or variant of a Therapeutic
protein of the invention is meant a polypeptide that possesses one
or more known biological and/or therapeutic activities associated
with a therapeutic protein such as one or more of the Therapeutic
proteins described herein or otherwise known in the art (e.g., as
described in columns 2 or 4 of Table 1 for a particular reference
protein, and/or below under section heading "Biological
Activities"). Biological and/or therapeutic activity of a fusion
protein, or a Therapeutic protein, or a fragment or variant
thereof, may routinely be determined using assays described herein
(e.g., in Table, 1, column 3, and/or the Examples section) and/or
by using or routinely modifying assays/methods known in the art. In
preferred embodiments, the serum albumin protein component of the
albumin fusion protein is the mature portion of serum albumin.
[0278] In a further embodiment, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
Therapeutic protein, and a biologically active and/or
therapeutically active fragment of serum albumin. By "biologically
active" and/or "therapeutically active" fragment or variant of
human serum albumin is meant a polypeptide that possesses one or
more known biological and/or therapeutic activities associated with
human serum albumin such as maintaining osmolarity (Yeh et al.
Proc. Natl. Acad. Set. USA, 89:1904-1908 (1992)), slow in vivo
clearance from the circulation by the liver and, therefore, a long
half-life (Yeh et al., Proc. Natl. Acad. Sci. USA, 89:1904-1908
(1992); Waldmann, T. A., Albumin Structure, Function and Uses, pp.
255-273 (1977)), and a carrier. In a further embodiment, the
invention provides an albumin fusion protein comprising, or
alternatively consisting of, a Therapeutic protein and a
biologically active and/or therapeutically active variant of serum
albumin. in a preferred embodiment, the Therapeutic protein portion
of the albumin fusion protein is the mature portion of the
Therapeutic protein.
[0279] In a further embodiment, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
biologically active and/or therapeutically active fragment or
variant of a Therapeutic protein and a biologically active and/or
therapeutically active fragment or variant of serum albumin. In a
preferred embodiment, the invention provides an albumin fusion
protein comprising, or alternatively consisting of, the mature
portion of a Therapeutic protein and the mature portion of serum
albumin.
[0280] In a preferred embodiment, the albumin fusion protein
comprises HA as the N-terminal portion, and a Therapeutic protein
as the C-terminal portion. In alternative preferred embodiments, an
albumin fusion protein comprises HA as the C-terminal portion, and
a Therapeutic protein as the N-terminal portion may also be
used.
[0281] In another embodiment, the albumin fusion protein has a
Therapeutic protein fused to both the N-terminus and the C-terminus
of albumin. In one embodiment, the Therapeutic proteins fused at
the N- and C-termini are the same Therapeutic protein. In another
embodiment, the fragments or variants of Therapeutic proteins fused
at the N- and C-termini are from the same Therapeutic protein. In
an alternative embodiment, the Therapeutic proteins fused at the N-
and C-termini are different Therapeutic proteins. In a preferred
embodiment, where the Therapeutic proteins fused at the N- and
C-termini are different Therapeutic proteins, these Therapeutic
proteins are useful in treating or preventing the same or related
disease, disorder, or condition (e.g. as listed in the "Preferred
Indication Y" column of Table 1). In another preferred embodiment,
the Therapeutic proteins fused at the N- and C-termini are
different Therapeutic proteins which may be used to treat (e.g.,
ameliorate) or prevent diseases or disorders (e.g. as listed in the
"Preferred Indication Y" column of Table 1) which are known in the
art to commonly occur in patients simultaneously, concurrently, or
consecutively, and/or which commonly occur in patients in
association with one another.
[0282] For example, albumin fusion proteins of the invention
containing multiple Therapeutic protein portions fused at the N-
and C-termini of albumin may include, but are not limited to,
GLP1-HSA-GLP-1, GLP-HSA-Exendin 4, Exendin 4-HSA-Exendin 4, Exendin
4-HSA-GLP1, GLP1-HSA-Insulin, Exendin 4-HSA-Insulin,
Insulin-HSA-GLP1, Insulin-HSA-Exendin 4, GLP1-HSA-Resistin, Exendin
4-HSA-Resistin, Insulin-HSA-Resistin, Resistin-HSA-Resistin,
Resistin-HSA-GLP1, Resistin-HSA-Exendin 4, Resistin-HSA-Insulin,
GLP1-HSA-Leptin, Exendin 4-HSA-Leptin, Insulin-HSA-Leptin,
Resistin-HSA-Leptin, Leptin-HSA-Leptin, Leptin-HSA-GLP1,
Leptin-HSA-Exendin 4, Leptin-HSA-Insulin, Leptin-HSA-Resistin,
GLP1-HSA-IGF1, IGF1-HSA-IGF1, Exendin 4-HSA-IGF1, Insulin-HSA-IGF1,
Resistin-HSA-IGF1, Leptin-HSA-IGF1, IGF1-HSA-GLP1, IGF1-HSA-Exendin
4, IGF1-HSA-Insulin, IGF1-HSA-Resistin, IGF1-HSA-Leptin,
HCEIP80-HSA-HCEIP80, IGF1-HSA-HCEIP80, GLP3-HSA-HCEIP80, Exendin
4-HSA-IGF1-HCEIP80, Insulin-HSA-HCEIP80, Resistin-HSA-HCEIP80,
Leptin-HSA-HCEIP80, HCEIP80-HSA-IGF1, HCEIP80-HSA-GLP1,
HCEIP80-HSA-Exendin 4, HCEIP80-HSA-Insulin, HCEIP80-HSA-Resistin,
HCEIP80-HSA-Leptin, HCEIP80-HSA-HLDOU18, IGF1-HSA-HLDOU18,
GLP1-HSA-HLDOU18, Exendin 4-HSA-HLDOU18, Insulin-HSA-HLDOU18,
Resistin-HSA-HLDOU18, Leptin-HSA-HLDOU18, HLDOU18-HSA-HCEIP80,
HLDOU18-HSA-IGF1, HLDOU18-HSA-GLP1, HLDOU18-HSA-Exendin 4,
HLDOU18-HSA-Insulin, HLDOU18-HSA-Resistin, HLDOU18-HSA-Leptin, and
HLDOU18-HSA-HLDOU18, HCEIP80-HSA-RegIV, IGF1-HSA-RegIV,
GLP1-HSA-RegIV, Exendin 4-HSA-RegIV, Insulin-HSA-RegIV,
Resistin-HSA-RegIV, Leptin-HSA-RegIV, HLDOU18-HSA-RegIV,
RegIV-HSA-HCEIP80, RegIV-HSA-IGF1, RegIV-HSA-GLP1,
RegIV-HSA-Exendin 4, RegIV-HSA-Insulin, RegIV-HSA-Resistin,
RegIV-HSA-Leptin, RegIV-HSA-HLDOU18, RegIV-HSA-RegIV,
HCEIP80-HSA-HDRMI82, IGF1-HSA-HDRMI82, GLP1-HSA-HDRMI82, Exendin
4-HSA-HDRMI82, Insulin-HSA-HDRMI82, Resistin-HDRMI82,
Leptin-HSA-HDRMI82, HLDOU18-HSA-HDRMI82, RegIV-HSA-HDRMI82,
HDRMI82-HSA-HCEIP80, HDRMI82-HSA-IGF1, HDRMI82-HSA-GLP1,
HDRMI82-HSA-Exendin 4, HDRMI82-HSA-Insulin, HDRMI82-HSA-Resistin,
HDRMI82-HSA-Leptin, HDRMI82-HSA-HLDOU18, HDRMI82-HSA-RegIV,
HDRMI82-HSA-HDRMI82, HCEIP80-HSA-IFNa, IGF1-HSA-IFNa,
GLP1-HSA-IFNa, Exendin 4-HSA-IFNa, Insulin-HSA-IFNa,
Resistin-HSA-IFNa, Leptin-HSA-IFNa, HLDOU18-HSA-IFNa,
RegIV-HSA-IFNa, HDRMI82-HSA-IFNa, IFNs-HSA-HCEIP80, IFNa-HSA-IGF1,
IFNa-HSA-GLP1, IFNa-HSA-Exendin 4, IFNa-HSA-Insulin,
IFNa-HSA-Resistin, IFNa-HSA-Leptin, IFNa-HSA-HLDOU18,
IFNa-HSA-RegIV, IFNa-HSA-HDRMI82, IFNa-HSA-IFNa, IL22-HSA-insulin,
IL22-HSA-GLP-1, IL-22-HSA-Exendin-4, IL22-HSA-HLDOU18,
IL22-HSA-Resistin, IL22-HSA-Leptin, IL22-HSA-HCEIP80,
IL22-HSA-IGF1, IL22-HSA-IFNa, IL22-HSA-RegIV, IL22-HSA-HDRMI82,
GLP1-HSA-IL22, Exendin-4-HSA-IL22, HLDOU18-HSA-1192,
Resistin-HSA-IL22, Leptin-HSA-IL22, HCEIP80-HSA-IL22,
IFNa-HSA-IL22, RegIV-HSA-IL22, and HDRMI82-HSA-IL22.
[0283] Albumin fusion proteins of the invention encompass proteins
containing one, two, three, four, or more molecules of a given
Therapeutic protein X or variant thereof fused to the N- or
C-terminus of an albumin fusion protein of the invention, and/or to
the N- and/or C-terminus of albumin or variant thereof. Molecules
of a given Therapeutic protein X or variants thereof may be in any
number of orientations, including, but not limited to, a `head to
head` orientation (e.g., wherein the N-terminus of one molecule of
a Therapeutic protein X is fused to the N-terminus of another
molecule of the Therapeutic protein X), or a `head to tail`
orientation (e.g., wherein the C-terminus of one molecule of a
Therapeutic protein X is fused to the N-terminus of another
molecule of Therapeutic protein X).
[0284] In one embodiment, one, two, three, or more tandemly
oriented Therapeutic protein X polypeptides (or fragments in
variants thereof) are fused to the N- or C-terminus of an albumin
fusion protein of the invention, and/or to the N- and/or C-terminus
of albumin or variant thereof.
[0285] In a specific embodiment, one, two, three, four, five, or
more tandemly oriented molecules of GLP1 are fused to the N- or
C-terminus of albumin or variant thereof. For example, one, two,
three, four, five, or more tandemly oriented molecules of GLP1
(including, but not limited to, molecules of GLP1 comprising, or
alternatively consisting of, amino acids His-98 to Arg-127) are
fused to the N- or C-terminus of albumin or variant thereof.
Exemplary fusion proteins of the invention containing multiple
protein portions of GLP1, include, but are not limited to,
GLP1-GLP1-HSA, HSA-GLP1-GLP1, GLP1-GLP1-GLP1-HSA,
HSA-GLP1-GLP1-GLP1, GLP1-GLP1, GLP 1-GLP1-HSA,
HSA-GLP1-GLP1-GLP1-GLP1.
[0286] In another specific embodiment, one, two, three, four, five,
or more tandemly oriented molecules of GLP-1 are fused to the N- or
C-terminus of albumin or variant thereof. For example, one, two,
three, four, five, or more tandemly oriented molecules of GLP-1
(including, but not limited to, molecules of GLP-1 comprising, or
alternatively consisting of, amino acids 7 to 36, with residue 8
being mutated from an Alanine to a Glycine) (See for Example, the
mutants disclosed in U.S. Pat. No. 5,545,618, herein incorporated
by reference in its entirety) are fused to the N- or C-terminus of
albumin or variant thereof. Exemplary fusion proteins of the
invention containing multiple protein portions of GLP-1, include,
but are not limited to, GLP1-GLP1-HSA, HSA-GLP1-GLP1,
GLY1mutant-GLP1mutant-HSA, mutant-GLP1mutant, GLP1mutant-GLP1-HSA,
HSA-GLP1mutant-GLP1, GLP1-GLP1mutant-HSA, or HSA-GLP1-GLP1mutant.
Particularly preferred embodiments are GLP-1 tandem fusions such as
construct ID #3070 and the protein encoded by such construct.
[0287] Albumin fusion proteins of the invention further encompass
proteins containing one, two, three, four, or more molecules of a
given Therapeutic protein X or variant thereof fused to the N- or
C-terminus of an albumin fusion protein of the invention, and/or to
the N- and/or C terminus of albumin or variant thereof, wherein the
molecules are joined through peptide linkers. Examples include
those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby
incorporated by reference). Albumin fusion proteins comprising
multiple Therapeutic protein X polypeptides separated by peptide
linkers may be produced using conventional recombinant DNA
technology, Linkers are particularly important when fusing a small
peptide to the large INA molecule. The peptide itself can be a
linker by fusing tandem copies of the peptide (see for example
GLP-1) or other known linkers can be used. Constructs that
incorporate linkers are described in Table 2 or are apparent when
examining SEQ ID NO:Y.
[0288] Further, albumin fusion proteins of the invention may also
be produced by fusing a Therapeutic protein X or variants thereof
to the N-terminal and/or C-terminal of albumin or variants thereof
in such a way as to allow the formation of intramolecular and/or
intermolecular multimeric forms. In one embodiment of the
invention, albumin fusion proteins may be in monomeric or
multimeric forms (i.e., dimers, trimers, tetramers and higher
multimers). In a further embodiment of the invention, the
Therapeutic protein portion of an albumin fusion protein may be in
monomeric form or multimeric form (i.e., dimers, trimers, tetramers
and higher multimers). In a specific embodiment, the Therapeutic
protein portion of an albumin fusion protein is in multimeric form
(i.e., dimers, trimers, tetramers and higher multimers), and the
albumin protein portion is in monomeric form.
[0289] In addition to albumin fusion protein in which the albumin
portion is fused N-terminal and/or C-terminal of the Therapeutic
protein portion, albumin fusion proteins of the invention may also
be produced by inserting the Therapeutic protein or peptide of
interest (e.g., a Therapeutic protein X as disclosed in Table 1, or
an antibody that binds a Therapeutic protein or a fragment or
variant thereof) into an internal region of 1-TA. For instance,
within the protein sequence of the HA molecule a number of loops or
turns exist between the end and beginning of et-helices, which are
stabilized by disulphide bonds. The loops, as determined from the
crystal structure of HA (PDB identifiers 1AO6, 1BJ5, 1BKE, 1BM0,
1E7E to 1E7I and 1UOR) for the most part extend away from the body
of the molecule. These loops are useful for the insertion, or
internal fusion, of therapeutically active peptides, particularly
those requiring a secondary structure to be functional, or
Therapeutic proteins, to essentially generate an albumin molecule
with specific biological activity.
[0290] Loops in human albumin structure into which peptides or
polypeptides may be inserted to generate albumin fusion proteins of
the invention include: Val54-Asn61, Thr76-Asp89, Ala92-Glu100,
Gln170-Ala176, His 247-Glu252, Glu 266-Glu277, Glu 280-His288,
Ala362-Glu368, Lys439-Pro447, Val462-Lys475, Thr478-Pro486, and
Lys560-Thr566. In more preferred embodiments, peptides or
polypeptides are inserted into the Val54-Asn61, Gln170-Ala176,
and/or Lys560-Thr566 loops of mature human albumin (SEQ ID
NO:327).
[0291] Peptides to be inserted may be derived from either phage
display or synthetic peptide libraries screened for specific
biological activity or from the active portions of a molecule with
the desired function. Additionally, random peptide libraries may be
generated within particular loops or by insertions of randomized
peptides into particular loops of the HA molecule and in which all
possible combinations of amino acids are represented.
[0292] Such library(s) could be generated on HA or domain fragments
of HA by one of the following methods:
[0293] a. randomized mutation of amino acids within one or more
peptide loops of HA or HA domain fragments. Either one, snore or
all the residues within a loop could be mutated in this manner;
[0294] b. replacement of, or insertion into one or more loops of HA
or HA domain fragments (i.e., internal fusion) of a randomized
peptides) of length X.sub.n (where X is air amino acid and n is the
number of residues, c. N-, C- or N- and C-terminal peptide/protein
fusions in addition to (a) and/or (b).
[0295] The HA or HA domain fragment may also be made
multifunctional by grafting the peptides derived from different
screens of different loops against different targets into the same
HA or HA domain fragment.
[0296] In preferred embodiments, peptides inserted into a loop of
human serum albumin are peptide fragments or peptide variants of
the Therapeutic proteins disclosed in Table 1. More particularly,
the invention encompasses albumin fusion proteins which comprise
peptide fragments or peptide variants at least 7, at least 8, at
least 9, at least 10, at least 11, at least 12, at least 13, at
least 14, at least 15, at least 20, at least 25, at least 30, at
least 35, or at Least 40 amino acids in length inserted into a loop
of human serum albumin. The invention also encompasses albumin
fusion proteins which comprise peptide fragments or peptide
variants at least 7, at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
20, at least 25, at least 30, at least 35, or at least 40 amino
acids fused to the N-terminus of human serum albumin. The invention
also encompasses albumin fusion proteins which comprise peptide
fragments or peptide variants at least 7 at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 20, at least 25, at least 30, at least 35, or at
least 40 amino acids fused to the C-terminus of human serum
albumin. For example, short peptides described in Table 1 and 2
(e.g., Therapeutic Y) can be inserted into the albumin loops.
[0297] Generally, the albumin fusion proteins of the invention may
have one HA-derived region and one Therapeutic protein-derived
region. Multiple regions of each protein, however, may be used to
make an albumin fusion protein of the invention. Similarly, more
than one Therapeutic protein may be used to make an albumin fusion
protein of the invention. Far instance, a Therapeutic protein may
be fused to both the N- and C-terminal ends of the HA. In such a
configuration, the Therapeutic protein portions may be the same or
different Therapeutic protein molecules. The structure of
bifunctional albumin fusion proteins may be represented as: X-HA-Y
or Y-HA-X.
[0298] For example, an anti-BLyS.TM. scFv-HA-IFN.alpha.-2b fusion
may be prepared to modulate the immune response to IFN.alpha.-2b by
anti-BLyS.TM. scFv. An alternative is making a bi (or even multi)
functional dose of HA-fusions e.g. HA-IFN.alpha.-2b fusion mixed
with HA-anti-BLyS.TM. scFv fusion or other HA-fusions in various
ratio's depending on function, half-life etc.
[0299] Bi- or multi-functional albumin fusion proteins may also be
prepared to target the Therapeutic protein portion of a fusion to a
target organ or cell type via protein or peptide at the opposite
terminus of HA.
[0300] As an alternative to the fusion of known therapeutic
molecules, the peptides could be obtained by screening libraries
constructed as fusions to the N-, C- or N- and C-termini of HA, or
domain fragment of HA, of typically 6, 8, 12, 20 or 25 or X.sub.n
(where X is an amino acid (aa) and n equals the number of residues)
randomized amino acids, and in which all possible combinations of
amino acids were represented. A particular advantage of this
approach is that the peptides may be selected in situ on the HA
molecule and the properties of the peptide would therefore be as
selected for rather than, potentially, modified as might be the
case for a peptide derived by any other method then being attached
to HA.
[0301] Additionally, the albumin fusion proteins of the invention
may include a linker peptide between the fused portions to provide
greater physical separation between the moieties and thus maximize
the accessibility of the Therapeutic protein portion. For instance,
for binding to its cognate receptor. The linker peptide may consist
of amino acids such that it is flexible or more rigid.
[0302] The linker sequence may be cleavable by a protease or
chemically to yield the growth hormone related moiety. Preferably,
the protease is one which is produced naturally by the host, for
example the S. cerevisae protease kex2 or equivalent proteases.
[0303] Therefore, as described above, the albumin fusion proteins
of the invention may have the following formula R1-L-R2; R1; or
R1-L-R2-L-R1, wherein R1 is at least one Therapeutic protein,
peptide or polypeptide sequence, and not necessarily the same
Therapeutic protein, L is a linker and R2 is a serum albumin
sequence.
[0304] In preferred embodiments, Albumin fusion proteins of the
invention comprising a Therapeutic protein have extended shelf life
compared to the shelf life the same Therapeutic protein when not
fused to albumin. Shelf-life typically refers to the time period
over which the therapeutic activity of a Therapeutic protein in
solution or in some other storage formulation, is stable without
undue loss of therapeutic activity. Many of the Therapeutic
proteins are highly labile in their unfused state. As described
below, the typical shelf-life of these Therapeutic proteins is
markedly prolonged upon incorporation into the albumin fusion
protein of the invention.
[0305] Albumin fusion proteins of the invention with "prolonged" or
"extended" shelf-life exhibit greater therapeutic activity relative
to a standard that has been subjected to the same storage and
handling conditions. The standard may be the unfused full-length
Therapeutic protein. When the Therapeutic protein portion of the
albumin fusion protein is an analog, a variant, or is otherwise
altered or does not include the complete sequence for that protein,
the prolongation of therapeutic activity may alternatively be
compared to the unfitted equivalent of that analog, variant,
altered peptide or incomplete sequence. As an example, an albumin
fusion protein of the invention may retain greater than about 100%
of the therapeutic activity, or greater than about 105%, 110%,
120%, 130%, 150% or 200% of the therapeutic activity of a standard
when subjected to the same storage and handling conditions as the
standard when compared at a given time point.
[0306] Shelf-life may also be assessed in terms of therapeutic
activity remaining after storage, normalized to therapeutic
activity when storage began. Albumin fusion proteins of the
invention with prolonged or extended shelf-life as exhibited by
prolonged or extended therapeutic activity may retain greater than
about 50% of the therapeutic activity, about 60%, 70%, 80%, or 90%
or more of the therapeutic activity of the equivalent unfused
Therapeutic protein when subjected to the same conditions.
[0307] Expression of Fusion Proteins
[0308] A particular embodiment of the invention comprises a DNA
construct encoding a signal sequence effective for directing
secretion in yeast, particularly a yeast-derived signal sequence
(especially one which is homologous to the yeast host), and the
fused molecule of the first aspect of the invention, there being no
yeast-derived pro sequence between the signal and the mature
polypeptide.
[0309] As discussed herein, an albumin fusion protein comprising a
leader sequence may be secreted from a host cell and may be
processed into a mature form by host cell machinery. In one
embodiment, prior to protein processing, the albumin fusion protein
of the invention may comprise the wild type signal sequence of a
given Therapeutic protein portion. In a further embodiment, prior
to protein processing, the albumin fusion protein of the invention
may comprise the wild type signal sequence of HSA. In another
embodiment, prier to protein processing, the albumin fusion protein
of the invention may contain a chimeric signal sequence. In a
preferred embodiment, the chimeric signal sequence comprises, or
alternatively consists of, the full length HSA signal sequence or a
portion thereof. In a further embodiment, prior to protein
processing, the albumin fusion protein of the invention may contain
the wild type signal sequence of invertase, "INV". In an additional
embodiment, prior to protein processing, the albumin fusion protein
of the invention may contain the wild type signal sequence of
mating factor alpha, "MAF". In a still further embodiment, prior to
protein processing, the albumin fusion protein of the invention may
contain the wild type signal sequence of Myeloid Progenitor
Inhibitory Factor. "MPIF" (see GenBank Accession Number
AAB51134).
[0310] The Saccharomyces cerevisiae invertase signal is a preferred
example of a yeast-derived signal sequence.
[0311] Conjugates of the kind prepared by Poznanskv et al., (FEBS
Lett. 239:18 (1988)), in which separately-prepared polypeptides are
joined by chemical cross-linking, are not contemplated.
[0312] The present invention also includes a cell, preferably a
yeast cell transformed to express an albumin fusion protein of the
invention. In addition to the transformed host cells themselves,
the present invention also contemplates a culture of those cells,
preferably a monoclonal (clonally homogeneous) culture, or a
culture derived from a monoclonal culture, in a nutrient medium. If
the polypeptide is secreted, the medium will contain the
polypeptide, with the cells, or without the cells if they have been
filtered or centrifuged away. Many expression systems are known and
may be used, including bacteria (for example E. coli and Bacillus
subtilis), yeasts (for example Saccharomyces cerevisiae,
Muyverouryces lactis and Pichia pastoris, filamentous fungi (far
example Aspergillus), plant cells, animal cells and insect
cells.
[0313] Preferred yeast strains to be used in the production of
albumin fusion proteins are D88, DXY1 and BXP10. D88 [leu2-3,
leu2-122, can1, pra1, ubc4] is a derivative of parent strain
AH22his.sup.+ (also known as DB1; see, e.g., Sleep et al.
Biotechnology 8:42.46 (1990)). The strain contains a leu2 mutation
which allows for auxotrophic selection of 2 micron-based plasmids
that contain the LEU2 gene. D88 also exhibits a derepression of
PRB1 in glucose excess. The PRB1 promoter is normally controlled by
two checkpoints that monitor glucose levels and growth stage. The
promoter is activated in wild type yeast upon glucose depletion and
entry into stationary phase. Strain D88 exhibits the repression by
glucose but maintains the induction upon entry into stationary
phase. The PRAT gene encodes a yeast vacuolar protease, YscA
endoprotease A, that is localized in the ER. The UBC4 gene is in
the ubiquitination pathway and is involved in targeting short lived
and abnormal proteins for ubiquitin dependant degradation.
Isolation of this ubc4 mutation was found to increase the copy
number of an expression plasmid in the cell and cause an increased
level of expression of a desired protein expressed from the plasmid
(see, e.g., International Publication No. WO99/00504, hereby
incorporated in its entirety by reference herein).
[0314] DXY1, a derivative of D88, has the following genotype:
[leu2-3, leu2-122, can1, pra1, ubc4, ura3::yap3]. In addition to
the mutations isolated in 188, this strain also has a knockout of
the YAP3 protease. This protease causes cleavage of mostly di-basic
residues (RR, RK, KR, KK) but can also promote cleavage at single
basic residues in proteins. Isolation of this yap3 mutation
resulted in higher levels of full length HSA production (see. e.g.,
U.S. Pat. No. 5,965,386 and Kerry-Williams et al., Yeast 14:161-169
(1998), hereby incorporated in their entireties by reference
herein).
[0315] BXP10 has the following genotype: leu2-3, leu2-122, can1,
pra1, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, URA3. In addition
to the mutations isolated in DXY1, this strain also has a knockout
of the PMT1 gene and the HSP150 gene. The PMT1 gene is a member of
the evolutionarily conserved family of dolichyl-phosphate-D-mannose
protein O-mannosyltransferases (Pmts). The transmembrane topology
of PmtIp suggests that it is an integral membrane protein of the
endoplasmic reticulum with a role in O-linked glycosylation. This
mutation serves to reduce/eliminate O-linked glycosylation of HSA
fusions (see, e.g., International Publication No. WO00/44772,
hereby incorporated in its entirety by reference herein). Studies
revealed that the Hsp150 protein is inefficiently separated from
rHA by ion exchange chromatography. The ululation in the HSP150
gene removes a potential contaminant that has proven difficult to
remove by standard purification techniques. See, e.g., U.S. Pat.
No. 5,783,423, hereby incorporated in its entirety by reference
herein.
[0316] The desired protein is produced in conventional ways, for
example from a coding sequence inserted in the host chromosome or
on a free plasmid. The yeasts are transformed with a coding
sequence for the desired protein in any of the usual ways, for
example electroporation. Methods for transformation of yeast by
electroporation are disclosed in Becker & Guarente (1990)
Methods Enzymol. 194, 182.
[0317] Successfully transformed cells, i.e., cells that contain a
DNA construct of the present invention, can be identified by well
known techniques. For example, cells resulting from the
introduction of an expression construct can be grown to produce the
desired polypeptide. Cells can be harvested and lysed and their DNA
content examined for the presence of the DNA using a method such as
that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent
et at (1985) Biotech. 3, 208. Alternatively, the presence of the
protein in the supernatant can be detected using antibodies.
[0318] Fusion of albumin to the Therapeutic protein may be achieved
by genetic manipulation, such that the DNA coding for HA, or a
fragment thereof, is joined to the DNA coding for the Therapeutic
protein. In one embodiment, an exemplary reference nucleic acid
sequence from which a polynucleotide encoding a Therapeutic protein
portion of the corresponding albumin fusion protein comprises the
wild type sequence encoding a Therapeutic protein shown in Table 1.
In an alternative embodiment, an exemplary reference nucleic acid
sequence from which a polynucleotide encoding a Therapeutic protein
portion of the corresponding albumin fusion protein comprises a
variant or derivative of a wild type sequence encoding a
Therapeutic protein shown in Table 1, such as, for example, a
synthetic codon optimized variant of a wild type coding sequence
for a Therapeutic protein. In a further embodiment, oligonucleotide
primers may be used in overlapping PCR reactions known in the art
to generate mutations within a template DNA sequence, A suitable
host is then transformed or transferred with the fused nucleotide
sequences, so arranged on a suitable plasmid as to express a fusion
polypeptide. The expression may be effected in vitro from, for
example, prokaryotic or eukaryotic cells, or in vivo e.g., from a
transgenic organism. Expression vectors are known in the art, and
are available commercially or described herein. For example, as
described in the Examples, an "expression cassette" containing one
or more of: (1) a polynucleotide encoding a given albumin fusion
protein, (2) a leader sequence, (3) a promoter region, and (4) a
transcriptional terminator, may be assembled in a convenient
cloning vector and subsequently be moved into the appropriate
vector. In one embodiment, for expression in S. cerevisiae, an
expression cassette containing a nucleic acid molecule encoding an
albumin fusion protein is cloned into pSAC35. In another
embodiment, for expression in CHO cells, an expression cassette
comprising, or alternatively consisting of, a nucleic acid molecule
encoding an albumin fusion protein is cloned into pC4. In a further
embodiment, a polynucleotide containing a nucleic acid molecule
encoding the Therapeutic protein portion of an albumin fusion
protein is cloned into pC4:HSA. In a still further embodiment, for
expression in NS0 cells, an expression cassette containing a
nucleic acid molecule encoding an albumin fusion protein is cloned
into pEE12. The invention also encompasses embodiments making use
of other vectors and/or host systems that are known in the art and
that may be routinely applied to express the albumin fusion
proteins of the invention.
[0319] Useful yeast plasmid vectors include pRS403-406 and
pRS413-416 and are generally available from Stratagene Cloning
Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404,
pRS405 and pRS406 are Yeast Integrating plasmids (Ylps) and
incorporate the yeast selectable markers HIS3, 7RPI, LEU2 and URA3.
Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).
[0320] Preferred vectors for making albumin fusion proteins for
expression in yeast include pPPC0005, pScCHSA, pScNHSA, and pC4:HSA
which are described in detail in Example 2, FIG. 3 shows a map of
the pPPC0005 plasmid that can be used as the base vector into which
polynucleotides encoding Therapeutic proteins may be cloned to form
HA-fusions. It contains a PRP1 S. cerevisiae promoter (PRB1p), a
Fission leader sequence (FL), DNA encoding HA (rHA) and an ADP)) S.
cerevisiae terminator sequence. The sequence of the fusion leader
sequence consists of the first 19 amino acids of the signal peptide
of human serum albumin (SEQ ID NO:410) and the last five amino
acids of the mating factor alpha 1 promoter (SLDKR, see EP-A-387
319) which is hereby incorporated by reference in its entirety.
[0321] The plasmids, pPPC0005, pScCHSA, pSCNHSA, and pC4:HSA were
deposited on Apr. 11, 2001 at the American Type Culture Collection,
10801 University Boulevard, Manassas, Va. 20110-2209 and given
accession numbers ATCC PTA-3278, PTA-3276, PTA-3279, and PTA-3277,
respectively. Another vector useful for expressing an albumin
fusion protein in yeast the pSAC35 vector which is described in
Sleep et al., BioTechnology 8:42 (1990) which is hereby
incorporated by reference in its entirety. Another yeast promoter
that can be used to express the albumin fusion protein is the MET25
promoter. See, for example, Dominik Mumburg, Rolf Muller and Martin
Funk. Nucleic Acids Research, 1994, Vol. 22, No. 25, pp. 5767-5768.
The Met25 promoter is 383 bases long (bases 382 to 1) and the genes
expressed by this promoter are also known as Met/5, Met17, and
YLR303W. A preferred embodiment uses the sequence below, where, at
the 5' end of the sequence below, the Not 1 site used in the
cloning is underlined and at the 3' end, the ATG start codon is
underlined:
TABLE-US-00004 (SEQ ID NO: 831)
GCGGCCGCCGGATGCAAGGGTTCGAATCCCTTAGCTCTCATTATTTTTT
GCTTTTTCTCTTGAGGTCACATGATCGCAAAATGGCAAATGGCACGTGA
AGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATGACTAATTAAGT
TAGTCAAGGCGCCATCCTCATGAAAACTGTGTAACATAATAACCGAAGT
GTCGAAAAGGTGGCACCTTGTCCAATTGAACACGCTCGATGAAAAAAAT
AAGATATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGGAAGTTTTTC
CTTTTTCTTGCTCTCTTGTCTTTTCATCTACTATTTCCTTCGTGTAATA
CAGGGTCGTCAGATACATAGATACAATTCTATTACCCCCATCCATACAA TG
[0322] A variety of methods have been developed to operably link
DNA to vectors via complementary cohesive termini, For instance,
complementary homopolymer tracts can be added to the DNA segment to
be inserted to the vector DNA. The vector and DNA segment are then
joined by hydrogen bonding between the complementary homopolymeric
tails to form recombinant DNA molecules.
[0323] Synthetic linkers containing one or more restriction sites
provide an alternative method of joining the DNA segment to
vectors. The DNA segment, generated by endonuclease restriction
digestion, is treated with bacteriophage T4 DNA polymerase or E.
coli DNA polymerase I, enzymes that remove protruding,
gamma-single-stranded termini with their 3' 5'-exonucleolytic
activities, and fill in recessed 3'-ends with their polymerizing
activities.
[0324] The combination of these activities therefore generates
blunt-ended DNA segments. The blunt-ended segments are then
incubated with a large molar excess of linker molecules in the
presence of an enzyme that is able to catalyze the ligation of
blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
Thus, the products of the reaction are DNA segments carrying
polymeric linker sequences at their ends. These DNA segments are
then cleaved with the appropriate restriction enzyme and ligated to
an expression vector that has been cleaved with an enzyme that
produces termini compatible with those of the DNA segment.
[0325] Synthetic linkers containing a variety of restriction
endonuclease sites are commercially available from a number of
sources including International Biotechnologies Inc, New Haven,
Conn., USA.
[0326] A desirable way to modify the DNA in accordance with the
invention, if, for example, HA variants are to be prepared, is to
use the polymerase chain reaction as disclosed by Saiki et al.
(1988) Science 239, 487-491. In this method the DNA to be
enzymatically amplified is flanked by two specific oligonucleotide
primers which themselves become incorporated into the amplified
DNA. The specific primers may contain restriction endonuclease
recognition sites which can be used for cloning into expression
vectors using methods known in the art.
[0327] Exemplary genera of yeast contemplated to be useful in the
practice of the present invention as hosts for expressing the
albumin fusion proteins are Pichia (Hansenula), Saccharomyces,
Kluyveromyces, Candida, Torulopsis, Torulaspora,
Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces,
Merschunikowia, Rheidosporidium, Leucosporidium, Botryoascus,
Sporidiobolus, Endomycopsis, and the like, Preferred genera are
those selected from the group consisting of Saccharomyces,
Schizosaccharomyces, Kluyveromyces, Pichiu and Tontlaspote.
Examples of Saccharomyces spp. are S. cerevisiae. S. italicus and
S. rouxii.
[0328] Examples of Kluyveromyces spp. are K. fragilis, K. lactic
and K. marxianus. A suitable Torulaspora species is T. delbrueckii.
Examples of Pichia (Hansenula) spp. are P. angusta (formerly H.
polymorpha), P. anomala (formerly H. anamala) and P. pastoris.
Methods for the transformation of S. cerevisiae are taught
generally in EP 251 744, EP 258 067 and WO 90/01063, all of which
are incorporated herein by reference.
[0329] Preferred exemplary species of Saccharomyces include S.
cerevisiae, S. italicus, S. diastaticus, and Zygosaccharomyces
rouxii. Preferred exemplary species of Kluyveromyces include K.
fragilis and K. lactis. Preferred exemplary species of Hansenula
include H. polymorpha (now Pichia angusta), H. anomala (now Pichia
anomala), and Pichia capsulate. Additional preferred exemplary
species of Pichia include P. pastoris. Preferred exemplary species
of Aspergillus include A. niger and A. nidulans. Preferred
exemplary species of Yarrowia include Y. lipolytica. Many preferred
yeast species are available from the ATCC. For example, the
following preferred yeast species are available from the ATCC and
are useful in the expression of albumin fusion proteins:
Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 yap3
mutant (ATCC Accession No. 4022731); Saccharomyces cerevisiae
Hansen, teleomorph strain B14743 hsp150 mutant (ATCC Accession No.
4021266); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743
pmt1 mutant (ATCC Accession No. 4023792); Saccharomyces cerevisiae
Hansen, teleomorph (ATCC Accession Nos. 20626: 44773; 44774; and
62995); Saccharomyces diastaticus Andrews et Gilliland ex van der
Walt, teleomorph (ATCC Accession No. 62987); Kluyveromyces lactis
(Dombrowski) van der Walt, teleomorph (ATCC Accession No. 76492);
Pichia angusta (Teunisson at al.) Kurtzman, teleomorph deposited as
Hansenula polymorpha de Morais et Maia, teleomorph (ATCC Accession
No. 26012); Aspergillus niger van Tieghem, anamorph (ATCC Accession
No. 9029); Aspergillus niger van Tieghem, anamorph (ATCC Accession
No. 16404); Aspergillus nidulans (Eldam) Winter, anamorph (ATCC
Accession No. 48756); and Yarrowia lipolytica (Wickerham et al.)
van der Walt et von Arx, teleomorph (ATCC Accession No.
201847).
[0330] Suitable promoters for S. cerevisiae include those
associated with the PGKI gene, GAL1 or GAL10 genes, CYCI, PHO5,
TRPI, ADH1, ADH2, the genes for glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, triose phosphate isomerase, phosphoglucose
isomerase, glucokinase, alpha-mating factor pheromone, [a mating
factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDI
promoter, and hybrid promoters involving hybrids of parts of 5'
regulatory regions with parts of 5' regulatory regions of other
promoters or with upstream activation sites (e.g. the promoter of
EP-A-258 067).
[0331] Convenient regulatable promoters for use in
Schizosaccharomyces pombe are the thiamine-repressible promoter
front the rum gene as described by Maundrell (1990) J. Biol. Chem.
265, 10857-10864 and the glucose repressible jbpl gene promoter as
described by Hoffman & Winston (1990) Genetics 124,
8074316.
[0332] Methods of transforming Pichia for expression of foreign
genes are taught in, for example, Cregg et al. (1993), and various
Phillips patents (e.g. U.S. Pat. No. 4,857,467, incorporated herein
by reference), and Pichia expression kits are commercially
available from Invitrogen BV, Leek, Netherlands, and Invitrogen
Corp., San Diego, Calif. Suitable promoters include AOX1 and AOX2.
Gleeson et al. (1986) J. Gen. Microbiol. 132, 3459-3465 include
information on Hansenula vectors and transformation, suitable
promoters being MOX1 and FMD1; whilst EP 361 991, Fleer et al.
(1991) and other publications from Rhone-Poulenc Rorer teach how to
express foreign proteins in Kluyveromyces spp., a suitable promoter
being PGKI.
[0333] The transcription termination signal is preferably the 3'
flanking sequence of a eukaryotic gene which contains proper
signals for transcription termination and polyadenylation. Suitable
3' flanking sequences may, for example, be those of the gene
naturally linked to the expression control sequence used, i.e., may
correspond to the promoter. Alternatively, they may be different in
which case the termination signal of the S. cerevisiae ADH1 gene is
preferred.
[0334] The desired albumin fusion protein may be initially
expressed with a secretion leader sequence, which may be any leader
effective in the yeast chosen. Leaders useful in yeast include any
of the following: [0335] a) the MPIF-1 signal sequence (e.g., amino
acids 1-21 of GenBank Accession number AAB51134)
MKVSVAALSCLMLVTALGSQA (SEQ ID NO:825) [0336] b) the stanniocakin
signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:340) [0337] c) the
pre-pro region of the HSA signal sequence (e.g.,
MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:410) [0338] d) the pre region
of the HSA signal sequence (e.g., MKWVTFISLLFLFSSAYS, SEQ ID
NO:411) or variants thereof, such as, for example.
MKWVSFISLLPLFSSAYS, (SEQ ID NO:408) [0339] e) the invertase signal
sequence (e.g., MLLQAFLFLLAGFAAKISA, SEQ ID NO:393) [0340] f) the
yeast mating factor alpha signal sequence (e.g., [0341]
MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLF-
INTTIASIAAK EEGVSLEKR, SEQ ID NO:394 or [0342]
MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIAS-
IAAK EEGVSLDKR, SEQ ID NO:394) [0343] g) K. lactis killer toxin
leader sequence [0344] h) a hybrid signal sequence (e.g.,
MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID NO:395) [0345] i) an
HSA/MF.alpha.-1 hybrid signal sequence (also known as HSA/kex2)
(e.g., MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:396) [0346] j) a K.
lactis killer/MF.alpha.-1 fusion leader sequence (e.g.,
MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:409) [0347] k) the Immunoglobulin
Ig signal sequence (e.g., MGWSCIILFLVATATGVHS, SEQ ID NO:380)
[0348] l) the Fibulin B precursor signal sequence (e.g.,
MERAAPSRRVPLPLLLGGLALLAAGVDA, SEQ ID NO:381) [0349] m) the
clusterin precursor signal sequence (e.g., MMKTLLLFVGLLLTWESGQVLG,
SEQ ID NO:382) [0350] n) the insulin like growth factor-binding
protein 4 signal sequence (e.g., MLPLCLVAALLLAAGPGPSLG, SEQ ID
NO:383) [0351] o) variants of the pre-pro-region of the HSA signal
sequence such as, for example, MKWVSFISLLFLFSSAYSRGVFRR (SEQ ID NO
407), [0352] MKWVTFISLLFLFAGVLG (SEQ ID NO:384), [0353]
MKWVTFISLLFLFSGVLG (SEQ ID NO:385), [0354] MKWVTFISLLFLFGGVLG (SEQ
ID NO:386), [0355] Modified HSA leader HSA #64 [0356]
MKWVTFISLLFLFAGVSG (SEQ ID NO:826); [0357] Modified HSA leader NSA
#66 [0358] MKWVTFISLLFLFGGVSG (SEQ ID NO:827); [0359] Modified HSA
(A14) leader-- [0360] MKWVTFISLLFLFAGVSG (SEQ ID NO:387); [0361]
Modified HSA (S14) leader (also known as modified HSA #65)-- [0362]
MKWVTFISLLFLFSGVSG (SEQ ID NO:388), [0363] Modified HSA (G14)
leader-- [0364] MKWVTFISLLFLFGGVSG (SEQ ID NO:389), or
MKWVTFISILLFLFGGVLGDLHKS (SEQ ID NO:390) [0365] p) a consensus
signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:341) [0366] q)
acid phosphatase (PH05) leader (e.g., MFKSVVYSILAASLANA SEQ ID
NO:828) [0367] r) the pre-sequence of MFoz-1 [0368] s) the
pre-sequence of 0 glucanase (BGL2) [0369] t) killer toxin leader
[0370] u) the pre-sequence of killer toxin [0371] v) k. lactis
killer toxin prepro (29 amino acids; 16 amino acids of pre and 13
amino acids of pro) MNIFYIFLPLLSFVQGLEHTHRRGSLDKR (SEQ ID NO:829)
[0372] w) S. diastaticus glucoamylase II secretion leader sequence
[0373] x) S. carisbergensis .alpha.-galactosidase (MEL1) secretion
leader sequence Candida glucoanrylase leader sequence [0374] z) The
hybrid leaders disclosed in EP-A-387 319 (herein incorporated by
reference) [0375] aa) the gp67 signal sequence (in conjunction with
baculoviral expression systems) (e.g., amino acids 1-19 of GenBank
Accession Number AAA72759) or [0376] bb) the natural leader of the
therapeutic protein X; [0377] cc) S. cerevisiae invertase (SUC2)
leader, as disclosed in JP 62-096086 (granted as 911036516, herein
incorporate by reference), or [0378] dd)
Inulinase--MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:830). [0379] ee) A
modified TA57 propeptide leader variant
#1--MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR
(SEQ ID NO:821) [0380] ff) A modified TA57 propeptide leader
variant
#2--MKLKTVRSAVLSSLFASQVLGQPIDDTESQTSVNLMADDTEQTTSVNLMADDTESAFATQTNSCDVVCL-
ISMAEEGEPKR (SEQ ID NO:822)
[0381] Additional Methods of Recombinant and Synthetic Production
of Albumin Fusion Proteins
[0382] The present invention also relates to vectors containing a
polynucleotide encoding an albumin fusion protein of the present
invention, host cells, and the production of albumin fusion
proteins by synthetic and recombinant techniques. The vector may
be, for example, a phage, plasmid, viral, or retroviral vector.
Retroviral vectors may be replication competent or replication
defective. In the latter case, viral propagation generally will
occur only in complementing host cells.
[0383] The polynucleotides encoding albumin fusion proteins of the
invention may be joined to a vector containing a selectable marker
for propagation in a host. Generally, a plasmid vector is
introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0384] The polynucleotide insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp, phoA and tac promoters, the SV40 early and late
promoters and promoters of retroviral LTRs, to name a few. Other
suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initiation, termination, and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the transcripts
expressed by the constructs will preferably include a translation
initiating codon at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[0385] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418, glutamine synthase, or neomycin resistance for
eukaryotic cell culture, and tetracycline, kanamycin or ampicillin
resistance genes for culturing in E. coli and other bacteria.
Representative examples of appropriate hosts include, but are not
limited to, bacterial cells, such as E. coli, Streptomyces and
Salmonella typhimurium cells; fungal cells, such as yeast cells
(e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession
No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9
cells; animal cells such as CHO, COS, NSO. 293, and Bowes melanoma
cells; and plant cells. Appropriate culture mediums and conditions
for the above-described host cells are known in the art.
[0386] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors,
Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from
Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among
preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and
pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. Preferred expression vectors for use in
yeast systems include, but are not limited to pYES2, pYD1,
pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5,
pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from
Invitrogen, Carlbad, Calif.). Other suitable vectors will be
readily apparent to the skilled artisan, In one embodiment,
polynucleotides encoding an albumin fusion protein of the invention
may be fused to signal sequences which will direct the localization
of a protein of the invention to particular compartments of a
prokaryotic or eukaryotic cell and/or direct the secretion of a
protein of the invention from a prokaryotic or eukaryotic cell. For
example, in E. coli, one may wish to direct the expression of the
protein to the periplasmic space. Examples of signal sequences or
proteins (or fragments thereof) to which the albumin fusion
proteins of the invention may be fused in order to direct the
expression of the polypeptide to the periplasmic space of bacteria
include, but are not limited to, the pelB signal sequence, the
maltose binding protein (MBP) signal sequence, MBP, the ompA signal
sequence, the signal sequence of the periplasmic E. coli
heat-labile enterotoxin B-subunit, and the signal sequence of
alkaline phosphatase. Several vectors are commercially available
for the construction of fusion proteins which will direct the
localization of a protein, such as the pMAL series of vectors
(particularly the pMAL-p series) available from New England
Biolabs. In a specific embodiment, polynucleotides albumin fusion
proteins of the invention may be fused to the pelB pectate lyase
signal sequence to increase the efficiency of expression and
purification of such polypeptides in Gram-negative bacteria. See.
U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents of which are
herein incorporated by reference in their entireties.
[0387] Examples of signal peptides that may be fused to an albumin
fusion protein of the invention in order to direct its secretion in
mammalian cells include, but are not limited to: [0388] a) the
MPIF-1 signal sequence (e.g., amino acids 1-21 of GenBank Accession
number AAB51134) MKVSVAALSCLMLVTALGSQA (SEQ ID NO:825) [0389] b)
the stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:30)
[0390] c) the pre-pro region of the HSA signal sequence (e.g.,
MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:410) [0391] d) the pre region
of the HSA signal sequence (e.g., MKWVTFISLLFLFSSAYS, SEQ ID
NO:411) or variants thereof, such as, for example,
MKWVSFISLLFLFSSAYS, (SEQ ID NO:408) [0392] e) the invertase signal
sequence (e.g., MLLQAFLFLLAGFAAKISA, SEQ ID NO:393) [0393] f) the
yeast mating factor alpha signal sequence (e.g.,
MRFPSIFTAVLAFAASSALAAPVNTTFEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIAS-
IAAK EEGVSLEKR. SEQ ID NO:394 or
MRFPSIFTAVLAFAASSALAAPVNTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASI-
AAK EEGVSLDKR, SEQ ID NO:394) [0394] g) K. lactis killer toxin
leader sequence [0395] h) a hybrid signal sequence (e.g.,
MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID NO:395) [0396] i) an
HSA/MF.alpha.-1 hybrid signal sequence (also known as HSA/kex2)
(e.g., MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:396) [0397] j) a K.
lactis killed MF.alpha.-1 fusion leader sequence (e.g.
MNIFYFLFLILSFVQGSLDKR, SEQ ID NO:409) [0398] k) the Immunoglobulin
Ig signal sequence (e.g., MGWSCIILFLVATATGVHS, SEQ ID NO:380)
[0399] l) the Fibulin B precursor signal sequence (e.g.,
MERAAPSRRVPLPLLLLGGLALLAAAGVDA, SEQ ID NO:381) [0400] m) the
clusterin precursor signal sequence (e.g., MMKTLLLFVGLLLTWESGQVLG,
SEQ ID NO:382) [0401] n) the insulin-like growth factor-binding
protein 4 signal sequence (e.g., MLPLCLVAALLLAAGPGPSLG, SEQ ID
NO:383) [0402] o) variants of the pre-pro-region of the HSA signal
sequence such as, for example, MKWVSFISLLFLFSSAYSRGVFRR (SEQ ID
NO:407), [0403] MKWVITFISLLFLFAGVLG (SEQ ID NO:384), [0404]
MKWVTFISLLFLFSGVLG (SEQ ID NO:385), [0405] MKWVTFISLLFLFGGVLG (SEQ
ID NO:386), [0406] Modified HSA leader HSA #64 MKWVTFISLLFLFAGVSG
(SEQ ID NO:826); [0407] Modified HSA leader HSA #66
MKWVTFISULFLFGGVSG (SEQ ID NO:827); [0408] Modified HSA (A14)
leader--MKWVTFISLLFLFAGVSG (SEQ ID NO:387); [0409] Modified HSA
(S14) leader (also known as modified HSA #65)--MKWVTFISLLFLFSGVSG
(SEQ ID NO:388), [0410] Modified ISA (G14) leader-- [0411]
MKWVTFISLLLFGGVSG (SEQ ID NO:389), or MKWVTFISLLFLFGGVLGDLHKS (SEQ
ID NO:390) [0412] p) a consensus signal sequence
(MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:341) [0413] q) acid phosphatase
(PH05) leader (e.g., MFKSVVYSWLAASLANA SEQ ID NO:828) [0414] r) the
pre-sequence of MF.alpha.-1 [0415] s) the pre-sequence of 0
glucanase (HGL2) [0416] t) killer toxin leader [0417] u) the
presequence of killer toxin [0418] v) k. lactis killer toxin prepro
(29 amino acids; 16 amino acids of pre and 13 amino acids of pro)
MNIFYFLFLISFVQGLEHTHRRGSLDKR (SEQ ID NO:829)
[0419] w) S. diastaticus glucoamylase II secretion leader sequence
[0420] x) S. carlsbergenis .alpha.-galactosidase (MEL1) secretion
leader sequence Candida glucoamylase leader sequence [0421] z) The
hybrid leaders disclosed in EP-A-387 319 (herein incorporated by
reference) [0422] aa) the gp67 signal sequence (in conjunction with
baculoviral expression systems) (e.g., amino acids 1-19 of GenBank
Accession Number AAA72759) or [0423] bb) the natural leader of the
therapeutic protein X; [0424] cc) S. cerevisiae invertase (SUC2)
leader, as disclosed in JP 62-096086 (granted as 911036516, herein
incorporate by reference); or [0425] dd)
Inulinase--MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:830). [0426] ee) A
modified TA57 propeptide leader variant
#1--TVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR
(SEQ ID NO:821) [0427] ff) A modified TA57 propeptide leader
variant
#2--MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAEEGEPK-
R (SEQ ID NO:822)
[0428] Vectors which use glutamine synthase (GS) or DHFR as the
selectable markers can be amplified in the presence of the drugs
methionine sulphoximine or methotrexate, respectively. An advantage
of glutamine synthase based vectors are the availability of cell
lines (e.g., the murine myeloma cell line, NSO) which are glutamine
synthase negative. Glutamine synthase expression systems can also
function in glutamine synthase expressing cells (e.g., Chinese
Hamster Ovary (CHO) cells) by providing additional inhibitor to
prevent the functioning of the endogenous gene. A glutamine
synthase expression system and components thereof are detailed in
PCT publications: WO87/04462: WO86/05807; WO89/01036; WO89/10404;
and WO91/06657, which are hereby incorporated in their entireties
by reference herein. Additionally, glutamine synthase expression
vectors can be obtained from Lonza Biologics, Inc. (Portsmouth,
N.H.). Expression and production of monoclonal antibodies using a
GS expression system in murine myeloma cells is described in
Bebbington et al., Bio/technology 10:169 (1992) and in Biblia and
Robinson Biotechnol. Prog. 11:1 (1995) which are herein
incorporated by reference.
[0429] The present invention also relates to host cells containing
the above-described vector constructs described herein, and
additionally encompasses host cells containing nucleotide sequences
of the invention that are operably associated with one or more
heterologous control regions (e.g., promoter and/or enhancer) using
techniques known of in the art. The host cell can be a higher
eukaryotic cell, such as a mammalian cell (e.g., a human derived
cell), or a lower eukaryotic cell, such as a yeast cell, or the
host cell can be a prokaryotic cell, such as a bacterial cell. A
host strain may be chosen which modulates the expression of the
inserted gene sequences, or modifies and processes the gene product
in the specific fashion desired. Expression from certain promoters
can be elevated in the presence of certain inducers; thus
expression of the genetically engineered polypeptide may be
controlled. Furthermore, different host cells have characteristics
and specific mechanisms for the translational and
post-translational processing and modification (e.g.,
phosphorylation, cleavage) of proteins. Appropriate cell lines can
be chosen to ensure the desired modifications and processing of the
foreign protein expressed.
[0430] Introduction of the nucleic acids and nucleic acid
constructs of the invention into the host cell can be effected by
calcium phosphate transfection, DEAE-dextran mediated transfection,
cationic lipid-mediated transfection, electroporation,
transduction, infection, or other methods. Such methods are
described in many standard laboratory manuals, such as Davis et
al., Basic Methods In Molecular Biology (1986). It is specifically
contemplated that the polypeptides of the present invention may in
fact be expressed by a host cell lacking a recombinant vector.
[0431] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g., the coding
sequence corresponding to a Therapeutic protein may be replaced
with an albumin fusion protein corresponding to the Therapeutic
protein), and/or to include genetic material (e.g., heterologous
polynucleotide sequences such as for example, an albumin fusion
protein of the invention corresponding to the Therapeutic protein
may be included). The genetic material operably associated with the
endogenous polynucleotide may activate, alter, and/or amplify
endogenous polynucleotides.
[0432] In addition, techniques known in the art may be used to
operably associate heterologous polynucleotides (e.g.,
polynucleotides encoding an albumin protein, or a fragment or
variant thereof) and/or heterologous control regions (e.g.,
promoter and/or enhancer) with endogenous polynucleotide sequences
encoding a Therapeutic protein via homologous recombination (see,
e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International
Publication Number WO 96/29411; International Publication Number WO
94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935
(1989); and Zijlstra et al., Nature 342:435-438 (1989), the
disclosures of each of which are incorporated by reference in their
entireties).
[0433] Albumin fusion proteins of the invention can be recovered
and purified from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography, hydrophobic charge interaction chromatography and
lectin chromatography. Most preferably, high performance liquid
chromatography ("HPLC") is employed for purification.
[0434] In preferred embodiments the albumin fusion proteins of the
invention are purified using Anion Exchange Chromatography
including, but not limited to, chromatography on Q-sepharose, DEAE
sepharose, poros HQ, poros DEAE, Toyopearl Q, Toyopearl QAE,
Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Q and DRAB
columns.
[0435] In specific embodiments the albumin fusion proteins of the
invention are purified using Cation Exchange Chromatography
including, but not limited to, SP-sepharose, CM sepharose, pores
HS, poros CM, Toyopearl SP, Toyopearl CM, Resource/Source S and CM,
Fractogel S and CM columns and their equivalents and
comparables.
[0436] In specific embodiments the albumin fusion proteins of the
invention are purified using Hydrophobic Interaction Chromatography
including, but not limited to, Phenyl, Butyl, Methyl, Octyl,
Hexyl-sepharose, poros Phenyl, Butyl, Methyl, Octyl, Toyopearl
Phenyl, Butyl, Methyl, Octyl, Hexyl Resource/Source Phenyl, Butyl,
Methyl, Octyl, Hexyl, Fractogel Phenyl, Butyl, Methyl, Octyl, Hexyl
columns and their equivalents and comparables.
[0437] In specific embodiments the albumin fusion proteins of the
invention are purified using Size Exclusion Chromatography
including, but not limited to, sepharose S100, S200, S300, superdex
resin columns and their equivalents and comparables.
[0438] In specific embodiments the albumin fusion proteins of the
invention are purified using Affinity Chromatography including, but
not limited to, Mimetic Dye affinity, peptide affinity and antibody
affinity columns that are selective for either the HSA or the
"fusion target" molecules.
[0439] In preferred embodiments albumin fusion proteins of the
invention are purified using one or more Chromatography methods
listed above. In other preferred embodiments, albumin fusion
proteins of the invention are purified using one or more of the
following Chromatography columns, Q sepharose FF column, SP
Sepharose FF column, Q Sepharose High Performance Column, Blue
Sepharose FE column, Blue Column, Phenyl Sepharose FF column, DEAE
Sepharose FF, or Methyl Column.
[0440] Additionally, albumin fusion proteins of the invention may
be purified using the process described in PCT International
Publication WO 00/44772 which is herein incorporated by reference
in its entirety. One of skill in the art could easily modify the
process described therein for use in the purification of albumin
fusion proteins of the invention.
[0441] Albumin fusion proteins of the present invention may be
recovered from: products of chemical synthetic procedures; and
products produced by recombinant techniques from a prokaryotic or
eukaryotic host, including, for example, bacterial, yeast, higher
plant, insect, and mammalian cells. Depending upon the host
employed in a recombinant production procedure, the polypeptides of
the present invention may be glycosylated or may be
non-glycosylated. In addition, albumin fusion proteins of the
invention may also include an initial modified methionine residue,
in some eases as a result of host-mediated processes. Thus, it is
well known in the art that the N-terminal methionine encoded by the
translation initiation codon generally is removed with high
efficiency from any protein after translation in all eukaryotic
cells. While the N-terminal methionine on most proteins also is
efficiently removed in most prokaryotes, for some proteins, this
prokaryotic removal process is inefficient, depending on the nature
of the amino acid to which the N-terminal methionine is covalently
linked.
[0442] In one embodiment, the yeast Pichia pastoris is used to
express albumin fusion proteins of the invention in a eukaryotic
system. Pichia pastoris is a methylotrophic yeast which can
metabolize methanol as its sole carbon source. A main step in the
methanol metabolization pathway is the oxidation of methanol to
formaldehyde using O.sub.2. This reaction is catalyzed by the
enzyme alcohol oxidase. In order to metabolize methanol as its sole
carbon source, Pichia pastoris must generate high levels of alcohol
oxidase due, in part, to the relatively low affinity of alcohol
oxidase for O.sub.2. Consequently, in a growth medium depending on
methanol as a main emboli source, the promoter region of one of the
two alcohol oxidase genes (AOX1) is highly active. In the presence
of methanol, alcohol oxidase produced from the AOX1 gene comprises
up to approximately 30% of the total soluble protein in Pichia
pastoris. See Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21
(1985); Koutz, P. J, et al., Yeast 5:167-77 0989); Tschopp, J. F.,
et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous
coding sequence, such as, for example, a polynucleotide of the
present invention, under the transcriptional regulation of all or
part of the AOKI regulatory sequence is expressed at exceptionally
high levels in Pichia yeast grown in the presence of methanol.
[0443] In one example, the plasmid vector pPIC9K is used to express
DNA encoding an albumin fusion protein of the invention, as set
forth herein, in a Pichea yeast system essentially as described in
"Pichia Protocols: Methods in Molecular Biology," D. R. Higgins and
J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This
expression vector allows expression and secretion of a polypeptide
of the invention by virtue of the strong AOX1 promoter linked to
the Richly pastoris alkaline phosphatase (PHO) secretory signal
peptide leader) located upstream of a multiple cloning site.
[0444] Many other yeast vectors could be used in place of pPIC9K,
such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,
pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pPIC3.5K, and PAO815, as one
skilled in the art would readily appreciate, as long as the
proposed expression construct provides appropriately located
signals for transcription, translation, secretion (if desired), and
the like, including an in-frame AUG as required.
[0445] In another embodiment, high-level expression of a
heterologous coding sequence, such as, for example, a
polynucleotide encoding an albumin fusion protein of the present
invention, may be achieved by cloning the heterologous
polynucleotide of the invention into an expression vector such as,
for example, pGAPZ or pGAPZalpha, and growing the yeast culture in
the absence of methanol.
[0446] In a preferred embodiment, an albumin fusion protein of the
invention is processed by a host cell and secreted into the
surrounding culture medium. Processing of the nascent albumin
fusion protein that occurs in the secretory pathways of the host
used for expression may include, but is not limited to signal
peptide cleavage; formation of disulfide bonds; proper folding;
addition and processing of carbohydrates (such as for example, N-
and O-linked glycosylation); specific proteolytic cleavages; and
assembly into multimeric proteins. An albumin fusion protein of the
invention is preferably in the processed form. In a most preferred
embodiment, the "processed form of an albumin fusion protein"
refers to an albumin fusion protein product which has undergone
N-terminal signal peptide cleavage, herein also referred to as a
"mature albumin fusion protein".
[0447] In addition, albumin fusion proteins of the invention can be
chemically synthesized using techniques known in the art (e.g., see
Creighton, 1983, Proteins: Structures and Molecular Principles,
W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature,
310:105-111 (1984)). For example, a polypeptide corresponding to a
fragment of a polypeptide can be synthesized by use of a peptide
synthesizer. Furthermore, if desired, nonclassical amino acids or
chemical amino acid analogs can be introduced as a substitution or
addition into the polypeptide sequence. Non-classical amino acids
include, but are not limited to, to the D-isomers of the common
amino acids, 2,4-diaminobutyric acid, .alpha.-amino isobutyric
acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu,
6-amino hexanoic acid, Alb, 2-amino isobutyric acid, 3-amino
propionic acid, ornithine, norleucine, norvaline, hydroxyproline,
sarcosine, citrulline, homocitrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
b-alanine, fluoro-amino acids, designer amino acids such as
b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids,
and amino acid analogs in general. Furthermore, the amino acid can
be D (dextrorotary) or L (levorotary).
[0448] The invention encompasses albumin fusion proteins of the
present invention which are differentially modified during or after
translation, e.g., by glycosylation, acetylation, phosphorylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to an antibody molecule or other
cellular ligand, etc. Any of numerous chemical modifications may be
carried out by known techniques, including but not limited, to
specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin, papain, V8 protease, NaBH.sub.4; acetylation,
formylation, oxidation, reduction; metabolic synthesis in the
presence of tunicamycin; etc.
[0449] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The albumin fusion
proteins may also be modified with a detectable label, such as an
enzymatic, fluorescent, isotopic or affinity label to allow for
detection and isolation of the protein.
[0450] Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin; and examples
of suitable radioactive material include iodine (.sup.121I,
.sup.123I, .sup.125I, .sup.131I), carbon (.sup.14C), sulfur
(.sup.35S), tritium (.sup.3H), indium (.sup.111In, .sup.112In,
.sup.113mIn, .sup.115mIn), technetium (.sup.99Tc, .sup.99mTc),
thallium (.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga), palladium
(.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine
(.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149Pm,
.sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47Sc,
.sup.186Re, .sup.188Re, .sup.152Pr, .sup.105Rh, and .sup.97Ru.
[0451] In specific embodiments, albumin fusion proteins of the
present invention or fragments or variants thereof are attached to
macrocyclic dictators that associate with radiometal ions,
including but not limited to, .sup.177Lu, .sup.90Y, .sup.166Ho, and
.sup.153Sm, to polypeptides. In a preferred embodiment, the
radiometal ion associated with the macrocyclic chelators is
.sup.111In. In another preferred embodiment, the radiometal ion
associated with the macrocyclic chelator is .sup.90Y. In specific
embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid
(DOTA). In other specific embodiments, DOTA is attached to an
antibody of the invention or fragment thereof via linker molecule.
Examples of linker molecules useful for conjugating DOTA to a
polypeptide are commonly known in the art--see, for example,
DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et
al., Bioconjug. Chem. 10(4):1553-7 (1999); and Zimmerman et al.
Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated
by reference in their entirety.
[0452] As mentioned, the albumin fusion proteins of the invention
may be modified by either natural processes, such as
post-translational processing, or by chemical modification
techniques which are well known in the art. It will be appreciated
that the same type of modification may be present in the same or
varying degrees at several sites in a given polypeptide.
Polypeptides of the invention may be branched, for example, as a
result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic, branched, and branched cyclic polypeptides may
result from posttranslation natural processes or may be made by
synthetic methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a hems moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. (See, for instance, PROTEINS--STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and
Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION
OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990);
Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[0453] Albumin fusion proteins of the invention and antibodies that
hind a Therapeutic protein or fragments or variants thereof can be
fused to marker sequences, such as a peptide to facilitate
purification. In preferred embodiments, the marker amino acid
sequence is a hexa-histidine peptide, such as the tag provided in a
pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,
91311), among others, many of which are commercially available. As
described in Gentz et al., Proc, Natl. Acad. Sci. USA 86:821-824
(1989), for instance, hexa-histidine provides for convenient
purification of the fusion protein. Other peptide tags useful for
purification include, but are not limited to, the "HA" tag, which
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
[0454] Further, an albumin fusion protein of the invention may be
conjugated to a therapeutic moiety such as a cytotoxin, e.g., a
cytostatic or cytocidal agent, a therapeutic agent or a radioactive
metal ion, e.g., alpha-emitters such as for example, 213131. A
cytotoxin or cytotoxic agent includes any agent that is detrimental
to cells. Examples include paclitaxol, cytochalasin B, gramicidin
D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromornannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0455] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, alpha-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, TNF-alpha, TNF-beta, AIM I (See,
International Publication No. WO 97/33899), AIM I (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
at al. Int. Immunol. 6:1567-1574 (1994)), VEGI (See, International
Publication No. WO 99/23105), a thrombotic agent or an
anti-angiogenic agent, e.g., angiostatin or endostatin; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors. Techniques for conjugating such therapeutic
moiety to proteins (e.g., albumin fusion proteins) are well known
in the art.
[0456] Albumin fusion proteins may also be attached to solid
supports, which are particularly useful for immunoassays or
purification of polypeptides that are bound by, that bind to, or
associate with albumin fusion proteins of the invention. Such solid
supports include, but are not limited to, glass, cellulose,
polyacrylamide, nylon, polystyrene, polyvinyl chloride or
polypropylene.
[0457] Albumin fusion proteins, with or without a therapeutic
moiety conjugated to it, administered alone or in combination with
cytotoxic factor(s) and/or cytokine(s) can be used as a
therapeutic.
[0458] In embodiments where the albumin fusion protein of the
invention comprises only the VH domain of an antibody that binds a
Therapeutic protein, it may be necessary and/or desirable to
coexpress the fusion protein with the VL domain of the same
antibody that binds a Therapeutic protein, such that the VH-albumin
fusion protein and VL protein will associate (either covalently or
non-covalently) post-translationally.
[0459] In embodiments where the albumin fusion protein of the
invention comprises only the VL domain of an antibody that hinds a
Therapeutic protein, it may be necessary and/or desirable to
coexpress the fusion protein with the VH domain of the same
antibody that binds a Therapeutic protein, such that the VL-albumin
fusion protein and VH protein will associate (either covalently or
non-covalently) post-translation ally.
[0460] Some Therapeutic antibodies are bispecific antibodies,
meaning the antibody that hinds a Therapeutic protein is an
artificial hybrid antibody having two different heavy/light chain
pairs and two different binding sites. In order to create an
albumin fusion protein corresponding to that Therapeutic protein,
it is possible to create an albumin fusion protein which has an
scFv fragment fused to both the N- and C-terminus of the albumin
protein moiety. More particularly, the scFv fused to the N-terminus
of albumin would correspond to one of the heavy/light (VH/VL) pairs
of the original antibody that binds a Therapeutic protein and the
scFv fused to the C-terminus of albumin would correspond to the
other heavy/light (VH/VL) pair of the original antibody that binds
a Therapeutic protein.
[0461] Also provided by the invention are chemically modified
derivatives of the albumin fusion proteins of the invention which
may provide additional advantages such as increased solubility,
stability and circulating time of the polypeptide, or decreased
immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties
for derivitization may be selected from water soluble polymers such
as polyethylene glycol ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The albumin fusion proteins may be modified at random positions
within the molecule, or at predetermined positions within the
molecule and may include one, two, three or more attached chemical
moieties.
[0462] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a Therapeutic protein or analog). For example, the
polyethylene glycol may have an average molecular weight of about
200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,
5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000,
10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000,
18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000,
45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000,
85,000, 90,000, 95,000, or 100,000 kDa.
[0463] As noted above, the polyethylene glycol may have a branched
structure. Branched polyethylene glycols are described, for
example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl.
Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides
Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug.
Chem. 10:638-646 (1999), the disclosures of each of which are
incorporated herein by reference.
[0464] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, such as, for example, the method disclosed in EP 0 401 384
(coupling PEG to G-CSF), herein incorporated by reference; see also
Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting
pegylation of GM-CSF using tresyl chloride. For example,
polyethylene glycol may be covalently bound through amino acid
residues via reactive group, such as a free amino or carboxyl
group. Reactive groups are those to which an activated polyethylene
glycol molecule may be bound. The amino acid residues having a free
amino group may include lysine residues and the N-terminal amino
acid residues; those having a free carboxyl group may include
aspartic acid residues glutamic acid residues and the C-terminal
amino acid residue. Sulfhydryl groups may also be used as a
reactive group for attaching the polyethylene glycol molecules.
Preferred for therapeutic purposes is attachment at an amino group,
such as attachment at the N-terminus or lysine group.
[0465] As suggested above, polyethylene glycol may be attached to
proteins via linkage to any of a number of amino acid residues. For
example, polyethylene glycol can be linked to proteins via covalent
bonds to lysine, histidine, aspartic acid, glutamic acid, or
cysteine residues. One or more reaction chemistries may be employed
to attach polyethylene glycol to specific amino acid residues
(e.g., lysine, histidine, aspartic acid, glutamic acid, or
cysteine) of the protein or to more than one type of amino acid
residue (e.g., lysine, histidine, aspartic acid, glutamic acid,
cysteine and combinations thereof) of the protein.
[0466] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein
(polypeptide) molecules in the reaction mix, the type of pegylation
reaction to be performed, and the method of obtaining the selected
N-terminally pegylated protein. The method of obtaining the
N-terminally pegylated preparation (i.e., separating this moiety
from other monopegylated moieties if necessary) may be by
purification of the N-terminally pegylated material from a
population of pegylated protein molecules. Selective proteins
chemically modified at the N-terminus modification may be
accomplished by reductive alkylation which exploits differential
reactivity of different types of primary amino groups (lysine
versus the N-terminal) available for derivatization in a particular
protein. Under the appropriate reaction conditions, substantially
selective derivatization of the protein at the N-terminus with a
carbonyl group containing polymer is achieved.
[0467] As indicated above, pegylation of the albumin fusion
proteins of the invention may be accomplished by any number of
means. For example, polyethylene glycol may be attached to the
albumin fusion protein either directly or by an intervening linker.
Linkerless systems for attaching polyethylene glycol to proteins
are described in Delgado et al., Crit. Rev. Thera. Drug Carrier
Sys. 9:249-304 (1992); Francis et al Intern. J. of Hematol. 68:1-18
(1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO
95/06058; and WO 98/32466, the disclosures of each of which are
incorporated herein by reference.
[0468] One system for attaching polyethylene glycol directly to
amino acid residues of proteins without an intervening linker
employs tresylated MPEG, which is produced by the modification of
monmethoxy polyethylene glycol (MPEG) using tresylchloride
(ClSO.sub.2CH.sub.2CF.sub.3). Upon reaction of protein with
tresylated MPEG, polyethylene glycol is directly attached to amine
groups of the protein. Thus, the invention includes
protein-polyethylene glycol conjugates produced by reacting
proteins of the invention with a polyethylene glycol molecule
having a 2,2,2-trifluoreothane sulphonyl group.
[0469] Polyethylene glycol can also be attached to proteins using a
number of different intervening linkers. For example, U.S. Pat. No.
5,612,460, the entire disclosure of which is incorporated herein by
reference, discloses urethane linkers for connecting polyethylene
glycol to proteins. Protein-polyethylene glycol conjugates wherein
the polyethylene glycol is attached to the protein by a linker can
also be produced by reaction of proteins with compounds such as
MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcalbonate,
MPEG-p-nitrophenylcarbonate, and various MPEG-succinate
derivatives. A number of additional polyethylene glycol derivatives
and reaction chemistries for attaching polyethylene glycol to
proteins are described in International Publication No. WO
98/32466, the entire disclosure of which is incorporated herein by
reference. Pegylated protein products produced using the reaction
chemistries set out herein are included within the scope of the
invention.
[0470] The number of polyethylene glycol moieties attached to each
albumin fusion protein of the invention (i.e., the degree of
substitution) may also vary. For example, the pegylated proteins of
the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
Similarly, the average degree of substitution within ranges such as
1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, IL-13, 12-14,
13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol
moieties per protein molecule. Methods for determining the degree
of substitution are discussed, for example, in Delgado et al.,
Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
[0471] The polypeptides of the invention can be recovered and
purified nom chemical synthesis and recombinant cell cultures by
standard methods which include, but are not limited to, ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Well known techniques for refolding
protein may be employed to regenerate active conformation when the
polypeptide is denatured during isolation and/or purification.
[0472] The presence and quantity of albumin fusion proteins of the
invention may be determined using ELISA, a well known immunoassay
known in the art. In one ELISA protocol that would be useful for
detecting/quantifying albumin fusion proteins of the invention,
comprises the steps of coating an ELISA plate with an anti-human
serum albumin antibody, blocking the plate to prevent non-specific
binding, washing the ELISA plate, adding a solution containing the
albumin fusion protein of the invention (at one or more different
concentrations), adding a secondary anti-Therapeutic protein
specific antibody coupled to a detectable label (as described
herein or otherwise known in the art), and detecting the presence
of the secondary antibody. In an alternate version of this
protocol, the ELISA plate might be coated with the anti-Therapeutic
protein specific antibody and the labeled secondary reagent might
be the anti-human albumin specific antibody.
[0473] Uses of the Polynucleotides
[0474] Each of the polynucleotides identified herein can be used in
numerous ways as reagents. The following description should be
considered exemplary and utilizes known techniques.
[0475] The polynucleotides of the present invention are useful to
produce the albumin fusion proteins of the invention. As described
in more detail below, polynucleotides of the invention (encoding
albumin fusion proteins) may be used in recombinant DNA methods
useful in genetic engineering to make cells, cell lines; or tissues
that express the albumin fusion protein encoded by the
polynucleotides encoding albumin fusion proteins of the
invention.
[0476] Polynucleotides of the present invention are also useful in
gene therapy. One goal of gene therapy is to insert a normal gene
into an organism having a defective gene, in an effort to correct
the genetic defect. The polynucleotides disclosed in the present
invention offer a means of targeting such genetic defects in a
highly accurate manner. Another goal is to insert a new gene that
was not present in the host genome, thereby producing a new trait
in the host cell. Additional non-limiting examples of gene therapy
methods encompassed by the present invention are more thoroughly
described elsewhere herein (see, e.g., the sections labeled "Gene
Therapy", and Examples 47 and 48).
[0477] Uses of the Polypeptides
[0478] Each of the polypeptides identified herein can be used in
numerous ways. The following description should be considered
exemplary and utilizes known techniques.
[0479] Albumin fusion proteins of the invention are useful to
provide immunological probes for differential identification of the
tissue(s) (e.g., immunohistochemistry assays such as, for example,
ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem.
29:577-580 (1981)) or cell type(s) (e.g., immunocytochemistry
assays).
[0480] Albumin fusion proteins can be used to assay levels of
polypeptides in a biological sample using classical
immunohistological methods known to those of skill in the art
(e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985);
Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other
methods useful for detecting protein gene expression include
immunoassays, such as the enzyme linked immunosorbent assay (ELISA)
and the radioimmunoassay (RIA). Suitable assay labels are known in
the art and include enzyme labels, such as, glucose oxidase;
radioisotopes, such as incline (.sup.131I, .sup.125I, .sup.123I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115mIn, .sup.113mIn, .sup.112In,
.sup.111In), and technetium (.sup.99Tc, .sup.99mTc), thallium
(.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga), palladium
(.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine
(.sup.18F), .sup.133Sm, .sup.177Lu, .sup.159Gd, .sup.149Pm,
.sup.140La, .sup.166Ho, .sup.90Y, .sup.47Sc, .sup.186Re,
.sup.188Re, .sup.105Rh, .sup.97Ru; luminescent labels, such as
luminol; and fluorescent labels, such as fluorescein and rhodamine,
and biotin.
[0481] Albumin fusion proteins of the invention can also be
detected in vivo by imaging. Labels or markers for in vivo imaging
of protein include those detectable by X-radiography, nuclear
magnetic resonance (NMR) or electron spin relaxation (ESR). For
X-radiography, suitable labels include radioisotopes such as barium
or cesium, which emit detectable radiation but are not overtly
harmful to the subject. Suitable markers for NMR and ESR include
those with a detectable characteristic spin, such as deuterium,
which may be incorporated into the albumin fusion protein by
labeling of nutrients given to a cell line expressing the albumin
fusion protein of the invention.
[0482] An albumin fusion protein which has been labeled with an
appropriate detectable imaging moiety, such as a radioisotope (for
example, .sup.131I, .sup.112In, .sup.99mTc, (.sup.131I, .sup.125I,
.sup.123I, .sup.121I), carbon (.sup.14C), sulfur (.sup.35S),
tritium (.sup.3H), indium (.sup.115mIn, .sup.113mIn, .sup.112In,
.sup.111In), and technetium (.sup.99Tc, .sup.99mTc), thallium
(.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga), palladium
(.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine
(.sup.18F), .sup.133Sm, .sup.177Lu, .sup.159Gd, .sup.149Pm,
.sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47Sc,
.sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, .sup.97Ru, a
radio-opaque substance, or a material detectable by nuclear
magnetic resonance, is introduced (for example, parenterally,
subcutaneously or intraperitoneally) into the mammal to be examined
for immune system disorder. It will be understood in the art that
the size of the subject and the imaging system used will determine
the quantity of imaging moiety needed to produce diagnostic images.
In the case of a radioisotope moiety, for a human subject, the
quantity of radioactivity injected will normally range from about 5
to 20 millicuries of .sup.99mTc. The labeled albumin fusion protein
will then preferentially accumulate at locations in the body (e.g.,
organs, cells, extracellular spaces or matrices) where one or more
receptors, ligands or substrates (corresponding to that of the
Therapeutic protein used to make the albumin fusion protein of the
invention) are located. Alternatively, in the case where the
albumin fusion protein comprises at least a fragment or variant of
a Therapeutic antibody, the labeled albumin fusion protein will
then preferentially accumulate at the locations in the body (e.g.,
organs, cells, extracellular spaces or matrices) where the
polypeptides/epitopes corresponding to those bound by the
Therapeutic antibody (used to make the albumin fusion protein of
the invention) are located. In vivo tumor imaging is described in
S. W. Burchiel at al., "Immunopharmacokinetics of Radiolabeled
Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,
eds., Masson Publishing Inc. (1982)). The protocols described
therein could easily be modified by one of skill in the an for use
with the albumin fusion proteins of the invention.
[0483] In one embodiment, the invention provides a method for the
specific delivery of albumin fusion proteins of the invention to
cells by administering albumin fusion proteins of the invention
(e.g., polypeptides encoded by polynucleotides encoding albumin
fusion proteins of the invention and/or antibodies) that are
associated with heterologous polypeptides or nucleic acids. In one
example, the invention provides a method for delivering a
Therapeutic protein into the targeted cell. In another example, the
invention provides a method for delivering a single stranded
nucleic acid (e.g., antisense or ribozymes) or double stranded
nucleic acid (e.g., DNA that can integrate into the cell's genome
or replicate episomally and that can be transcribed) into the
targeted cell.
[0484] In another embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering albumin fusion proteins of the invention in
association with toxins or cytotoxic prodrugs.
[0485] By "toxin" is meant one or more compounds that bind and
activate endogenous cytotoxic effector systems, radioisotopes,
holotoxins, modified toxins, catalytic subunits of toxins, or any
molecules or enzymes not normally present in or on the surface of a
cell that tinder defined conditions cause the cell's death. Toxins
that may be used according to the methods of the invention include,
but are not limited to, radioisotopes known in the art, compounds
such as, for example, antibodies (or complement fixing containing
portions thereof) that bind an inherent or induced endogenous
cytotoxic effector system, thymidine kinase, endonuclease, RNAse,
alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria
toxin, saporin, momordin, gelonin, pokeweed antiviral protein,
alpha-sarcin and cholera toxin. "Toxin" also includes a cytostatic
or cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, .sup.213Bi, or other
radioisotopes such as, for example, .sup.103Pd, .sup.133Xe,
.sup.131I, .sup.5Ge, .sup.57Co, .sup.65Zn, .sup.85Sr, .sup.32P,
.sup.35S, .sup.90Y, .sup.153Sm, .sup.153Gd, .sup.169Yb, .sup.51Cr,
.sup.54Mn, .sup.72Se, .sup.113Sn, .sup.90Yttrium, .sup.117Tin,
.sup.186Rhenium, .sup.166Holmium, and .sup.188Rhenium; luminescent
labels, such as luminol; and fluorescent labels, such as
fluorescein and rhodamine, and biotin. In a specific embodiment,
the invention provides a method for the specific destruction of
cells (e.g., the destruction of tumor cells) by administering
polypeptides of the invention or antibodies of the invention in
association with the radioisotope .sup.90Y. In another specific
embodiment, the invention provides a method for the specific
destruction of cells (e.g., the destruction of tumor cells) by
administering polypeptides of the invention or antibodies of the
invention in association with the radioisotope .sup.131In . In a
further specific embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering polypeptides of the invention or antibodies
of the invention in association with the radioisotope
.sup.131I.
[0486] Techniques known in the art may be applied to label
polypeptides of the invention. Such techniques include, but are not
limited to, the use of bifunctional conjugating agents (see e.g.,
U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361;
5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;
4,994,560; and 5,808,003; die contents of each of which are hereby
incorporated by reference in its entirety).
[0487] The albumin fusion proteins of the present invention are
useful for diagnosis, treatment, prevention and/or prognosis of
various disorders in mammals, preferably humans. Such disorders
include, but are not limited to, those described in column 4 of
Table corresponding to a Therapeutic protein of interest, and/or as
described under the section heading "Biological Activities,"
below.
[0488] Thus, the invention provides a diagnostic method of a
disorder, which involves (a) assaying the expression level of a
certain polypeptide in cells or body fluid of an individual using
an albumin fusion protein of the invention; and (b) comparing the
assayed polypeptide expression level with a standard polypeptide
expression level, whereby an increase or decrease in the assayed
polypeptide expression level compared to the standard expression
level is indicative of a disorder. With respect to cancer, the
presence of a relatively high amount of transcript in biopsied
tissue from an individual may indicate a predisposition for the
development of the disease, or may provide a means for detecting
the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[0489] Moreover, albumin fusion proteins of the present invention
can be used to treat or prevent diseases or conditions such as, for
example, neural disorders, immune system disorders, muscular
disorders, reproductive disorders, gastrointestinal disorders,
pulmonary disorders, cardiovascular disorders, renal, disorders,
proliferative disorders, and/or cancerous diseases and conditions.
For example, patients can be administered a polypeptide of the
present invention in an effort to replace absent or decreased
levels of the polypeptide (e.g., insulin), to supplement absent or
decreased levels of a different polypeptide (e.g., hemoglobin S for
hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the
activity of a polypeptide (e.g., an oncogene or tumor supressor),
to activate the activity of a polypeptide (e.g., by binding to a
receptor), to reduce the activity of a membrane bound receptor by
competing with it for free ligand (e.g., soluble TNF receptors used
in reducing inflammation), or to bring about a desired response
(e.g., blood vessel growth inhibition, enhancement of the immune
response to proliferative cells or tissues).
[0490] In particular, albumin fusion proteins comprising of at
least a fragment or variant of a Therapeutic antibody can also be
used to treat disease (as described supra, and elsewhere herein).
For example, administration of an albumin fusion protein comprising
of at least a fragment or variant of a Therapeutic antibody can
bind, and/or neutralize the polypeptide to which the Therapeutic
antibody used to make the albumin fusion protein specifically
binds, and/or reduce overproduction of the polypeptide to which the
Therapeutic antibody used to make the albumin fusion protein
specifically binds. Similarly, administration of an albumin fusion
protein comprising of at least a fragment or variant of a
Therapeutic antibody can activate the polypeptide to which the
Therapeutic antibody used to make the albumin fusion protein
specifically binds, by binding to the polypeptide bound to a
membrane (receptor).
[0491] At the very least, the albumin fusion proteins of the
invention of the present invention can be used as molecular weight
markers on SDS-PACE gels or on molecular sieve gel filtration
columns using methods well known to those of skill in the alt.
Albumin fusion proteins of the invention can also be used to raise
antibodies, which in turn may be used to measure protein expression
of the Therapeutic protein, albumin protein, and/or the albumin
fusion protein of the invention from a recombinant cell, as a way
of assessing transformation of the host cell, or in a biological
sample. Moreover, the albumin fusion proteins of the present
invention can be used to test the biological activities described
herein.
[0492] Diagnostic Assays
[0493] The compounds of the present invention are useful for
diagnosis, treatment, prevention and/or prognosis of various
disorders in mammals, preferably humans. Such disorders include,
but are not limited to, those described for each Therapeutic
protein in the corresponding row of Table 1 and herein under the
section headings "Immune Activity," "Blood Related Disorders,"
"Hyperproliferative Disorders," "Renal Disorders," "Cardiovascular
Disorders," "Respiratory Disorders," "Anti-Angiogenesis Activity,"
"Diseases at the Cellular Level," "Wound Healing and Epithelial
Cell Proliferation," "Neural Activity and Neurological Diseases,"
"Endocrine Disorders," "Reproductive System Disorders," "Infectious
Disease," "Regeneration," and/or "Gastrointestinal Disorders,"
infra.
[0494] For a number of disorders, substantially altered (increased
or decreased) levels of gene expression can be detected in tissues,
cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial
fluid or spinal fluid) taken from an individual having such a
disorder, relative to a "standard" gene expression level, that is,
the expression level in tissues or bodily fluids from an individual
not having the disorder. Thus, the invention provides a diagnostic
method useful during diagnosis of a disorder, which involves
measuring the expression level of the gene encoding a polypeptide
in tissues, cells or body fluid from an individual and comparing
the measured gene expression level with a standard gene expression
level, whereby an increase or decrease in the Gene expression
level(s) compared to the standard is indicative of a disorder.
These diagnostic assays may be performed in vita or in vitro, such
as, for example, on blood samples, biopsy tissue or autopsy
tissue.
[0495] The present invention is also useful as a prognostic
indicator, whereby patients exhibiting enhanced or depressed gene
expression will experience a worse clinical outcome.
[0496] By "assaying the expression level of the gene encoding a
polypeptide" is intended qualitatively or quantitatively measuring
or estimating the level of a particular polypeptide (e.g. a
polypeptide corresponding to a Therapeutic protein disclosed in
Table 1) or the level of the mRNA encoding the polypeptide of the
invention in a first biological sample either directly (e.g., by
determining or estimating absolute protein level or mRNA level) or
relatively (e.g., by comparing to the polypeptide level or mRNA
level in a second biological sample). Preferably, the polypeptide
expression level or in RNA level in the first biological sample is
measured or estimated and compared to a standard polypeptide level
or mRNA level, the standard being taken from a second biological
sample obtained from an individual not having the disorder or being
determined by averaging levels from a population of individuals not
having the disorder. As will be appreciated in the art, once a
standard polypeptide level or mRNA level is known, it can be used
repeatedly as a standard for comparison.
[0497] By "biological sample" is intended any biological sample
obtained from an individual, cell line, tissue culture, or other
source containing polypeptides of the invention (including portions
thereof) or mRNA. As indicated, biological samples include body
fluids such as sera, plasma, urine, synovial fluid and spinal
fluid) and tissue sources found to express the full length or
fragments thereof of a polypeptide or mRNA. Methods for obtaining
tissue biopsies and body fluids from mammals are well known in the
art. Where the biological sample is to include mRNA, a tissue
biopsy is the preferred source.
[0498] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described in
Chomezynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels
of mRNA encoding the polypeptides of the invention are then assayed
using any appropriate method. These include Northern blot analysis,
S1 nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0499] The present invention also relates to diagnostic assays such
as quantitative and diagnostic assays for detecting levels of
polypeptides that bind to, are bound by, or associate with albumin
fusion proteins of the invention, in a biological sample (e.g.,
cells and tissues), including determination of normal and abnormal
levels of polypeptides. Thus, for instance, a diagnostic assay in
accordance with the invention for detecting abnormal expression of
polypeptides that bind to: are bound by, or associate with albumin
fusion proteins compared to normal control tissue samples may be
used to detect the presence of tumors. Assay techniques that can be
used to determine levels of a polypeptide that bind to are bound
by, or associate with albumin fusion proteins of the present
invention in a sample derived from a host are well-known to those
of skill in the art. Such assay methods include radioimmunoassays,
competitive-binding assays, Western Blot analysis and ELISA assays.
Assaying polypeptide levels in a biological sample can occur using
any art-known method.
[0500] Assaying polypeptide levels in a biological sample can occur
using a variety of techniques. For example, polypeptide expression
in tissues can be studied with classical immunohistological methods
(Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,
et al., J. Cell. Biol. 105:3087-3096 (1987)). Other methods useful
for detecting polypeptide gene expression include immunoassays,
such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme Labels, such as, glucose oxidase, and
radioisotopes, such as iodine (.sup.125I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99mTc), and fluorescent labels,
such as fluorescent and rhodamine, and biotin.
[0501] The tissue or cell type to be analyzed will generally
include those which are known, or suspected, to express the gene of
interest (such as, for example, cancer). The protein isolation
methods employed herein may, for example, be such as those
described in Harlow and Lane (Harlow, E. and Lane, D., 1988,
"Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.), which is incorporated herein by
reference in its entirety. The isolated cells can be derived from
cell culture or from a patient. The analysis of cells taken from
culture may be a necessary step in the assessment of cells that
could be used as part of a cell-based gene therapy technique or,
alternatively, to test the effect of compounds on the expression of
the gene.
[0502] For example, albumin fusion proteins may be used to
quantitatively or qualitatively detect the presence of polypeptides
that bind to, are bound by, or associate with albumin fusion
proteins of the present invention. This can be accomplished, for
example, by immunofluorescence techniques employing a fluorescently
labeled albumin fusion protein coupled with light microscopic, flow
cytometric, or fluorimetric detection.
[0503] In a preferred embodiment, albumin fusion proteins
comprising at least a fragment or variant of an antibody that
specifically binds at least a Therapeutic protein disclosed herein
(e.g., the Therapeutic proteins disclosed in Table 1) or otherwise
known in the art may be used to quantitatively or qualitatively
detect the presence of gene products or conserved variants or
peptide fragments thereof. This can be accomplished, for example,
by immunofluorescence techniques employing a fluorescently labeled
antibody coupled with light microscopic, flow cytometric, or
fluorimetric detection.
[0504] The albumin fusion proteins of the present invention may,
additionally, be employed histologically, as in immunofluorescence,
immunoelectron microscopy or non-immunological assays, for in situ
detection of polypeptides that bind to are bound by, or associate
with an albumin fusion protein of the present invention. In situ
detection may be accomplished by removing a histological specimen
from a patient, and applying thereto a labeled antibody or
polypeptide of the present invention. The albumin fusion proteins
are preferably applied by overlaying the labeled albumin fusion
proteins onto a biological sample. Through the use of such a
procedure, it is possible to determine not only the presence of the
polypeptides that bind to, are bound by, or associate with albumin
fusion proteins, but also its distribution in the examined tissue.
Using the present invention, those of ordinary skill will readily
perceive that any of a wide variety of histological methods (such
as staining procedures) can be modified in order to achieve such in
situ detection.
[0505] Immunoassays and non-immunoassays that detect polypeptides
that bind to, are bound by, or associate with albumin fusion
proteins will typically comprise incubating a sample, such as a
biological fluid, a tissue extract, freshly harvested cells, or
lysates of cells which have been incubated in cell culture, in the
presence of a delectably labeled antibody capable of binding gene
products or conserved variants or peptide fragments thereof, and
detecting the bound antibody by any of a number of techniques
well-known in the art.
[0506] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the delectably labeled albumin fusion protein of the invention. The
solid phase support may then be washed with the buffer a second
time to remove unbound antibody or polypeptide. Optionally the
antibody is subsequently labeled. The amount of bound label on
solid support may then be detected by conventional means.
[0507] By "solid phase support or carrier" is intended any support
capable of binding a polypeptide (e.g., an albumin fusion protein,
or polypeptide ilea binds, is bound by, or associates with an
albumin fusion protein of the invention.) Well-known supports or
carriers include glass, polystyrene, polypropylene, polyethylene,
dextran, nylon, amylases, natural and modified celluloses,
polyacrylamides, gabbros, and magnetite. The nature of the carrier
can be either soluble to some extent or insoluble for the purposes
of the present invention. The support material may have virtually
any possible structural configuration so long as the coupled
molecule is capable of binding to a polypeptide. Thus, the support
configuration may be spherical, as in a bead, or cylindrical, as in
the inside surface of a test tube, or the external surface of a
rod. Alternatively, the surface may be fiat such as a sheet, test
strip, etc. Preferred supports include polystyrene beads. Those
skilled in the art will know many other suitable carriers for
binding antibody or antigen, or will be able to ascertain the same
by use of routine experimentation.
[0508] The binding activity of a given lot of albumin fusion
protein may be determined according to well known methods. Those
skilled in the art will be able to determine operative and optimal
assay conditions for each determination by employing routine
experimentation.
[0509] In addition to assaying polypeptide levels in a biological
sample obtained from an individual, polypeptide can also be
detected in viva by imaging. For example, in one embodiment of the
invention, albumin fusion proteins of the invention are used to
image diseased or neoplastic cells.
[0510] Labels or markers for in vivo imaging of albumin fusion
proteins of the invention include those detectable by
X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography,
suitable labels include radioisotopes such as barium or cesium,
which emit detectable radiation but are not overtly harmful to the
subject. Suitable markers for NMR and ESR include those with a
detectable characteristic spin, such as deuterium, which may be
incorporated into the albumin fusion protein by labeling of
nutrients of a cell line (or bacterial or yeast strain)
engineered.
[0511] Additionally, albumin fusion proteins of the invention whose
presence can be detected, can be administered. For example, albumin
fusion proteins of the invention labeled with a radio-opaque or
other appropriate compound can be administered and visualized in
vivo, as discussed, above for labeled antibodies. Further, such
polypeptides can be utilized for in vitro diagnostic
procedures.
[0512] A polypeptide-specific antibody or antibody fragment which
has been labeled with an appropriate detectable imaging moiety,
such as a radioisotope (for example, .sup.131I, .sup.212In,
.sup.99mTc), a radio-opaque substance, or a material detectable by
nuclear magnetic resonance, is introduced (for example,
parenterally, subcutaneously or intraperitoneally) into the mammal
to be examined for a disorder. It will be understood in the art
that the size of the subject and the imaging system used will
determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a
human subject, the quantity of radioactivity injected will normally
range from about 5 to 20 millicuries of .sup.99mTc. The labeled
albumin fusion protein will then preferentially accumulate at the
locations in the body which contain a polypeptide or other
substance that binds to, is bound by or associates with art albumin
fusion protein of the present invention. In vivo tumor imaging is
described in S. W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor
Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and
B. A. Rhodes, eds., Masson Publishing Inc, (1982)).
[0513] One of the ways in which an albumin fusion protein of the
present invention can be detectably labeled is by linking the same
to a reporter enzyme and using the linked product in an enzyme
immunoassay (ETA) (Volley, A., "The Enzyme Linked Immunosorbent
Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological
Associates Quarterly Publication, Walkersville, Md.); Voller et
al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth.
Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme
Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. et al.,
(eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter
enzyme which is bound to the antibody will react with an
appropriate substrate, preferably a chromogenic substrate, in such
a manner as to produce a chemical moiety which can be detected, for
example, by spectrophotometric, fluorimetric or by visual means.
Reporter enzymes which can be used to detectably label the antibody
include, but are not limited to, malate dehydrogenase,
staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose
phosphate isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase
and acetylcholinesterase. Additionally, the detection can be
accomplished by colorimetric methods which employ a chromogenic
substrate for the reporter enzyme. Detection may also be
accomplished by visual comparison of the extent of enzymatic
reaction of a substrate in comparison with similarly prepared
standards.
[0514] Albumin fusion proteins may also be radiolabeled and used in
any of a variety of other immunoassays. For example, by
radioactively labeling the albumin fusion proteins, it is possible
to the use the albumin fusion proteins in a radioimmunoassay (RIA)
(see, for example, Weintraub, B., Principles of Radioimmunoassays,
Seventh Training Course on Radioligand Assay Techniques, The
Endocrine Society, March, 1986, which is incorporated by reference
herein). The radioactive isotope can be detected by means
including, but not limited to, a gamma counter, a scintillation
counter, in autoradiography.
[0515] Additionally, chelator molecules, are known in the art and
can be used to label the Albumin fusion proteins. Chelator
molecules may be attached Albumin fusion proteins of the invention
to facilitate labeling said protein with metal ions including
radionuclides or fluorescent labels. For example, see Subramanian,
R. and Meares, C. F., "Bifunctional Chelating Agents for
Radiometal-labeled monoclonal Antibodies," in Cancer Imaging with
Radiolabeled Antibodies (D. M. Goldenberg, Ed.) Kluwer Academic
Publications, Boston; Saji, H., "Targeted delivery of radiolabeled
imaging and therapeutic agents: bifunctional radiopharmaceuticals."
Crit. Rev. Ther. Drug Carrier Syst. 16:209-244 (1999); Srivastava
S. C. and Mease R. C., "Progress in research on ligands, nuclides
and techniques for labeling monoclonal antibodies." Int. J. Rad.
Appl. Instrum. B 18:589 603 (1991); and Liu, S. and Edwards, D. S.,
"Bifunctional chelators for therapeutic lanthanide
radiopharmaceuticals." Bioconjug, Chem. 12:7-34 (2001). Any
chelator which can be covalently bound to said Albumin fusion
proteins may be used according to the present invention. The
chelator may further comprise a linker moiety that connects the
chelating moiety to the Albumin fusion protein.
[0516] In one embodiment, the Albumin fusion protein of the
invention are attached to an acyclic chelator such as diethylene
triamine-N,N,N',N'',N''-pentaacetic acid (DPTA), analogues of DPTA,
and derivatives of DPTA. As non-limiting examples, the chelator may
be 2-(p-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic
acid (1B4M-DPTA, also known as MX-DTPA),
2-methyl-6-(rho-nitrobenzyl)-1,4,7-triazaheptane-N,N,N',N'',N''-pentaacet-
ic acid (nitro-1B4M-DTPA or nitro-MX-DTPA);
2-(p-isothiocyanatobenzyl)-cyclohexyldiethylenetriaminepentaacetic
acid (CHX-DTPA), or
N-[2-amino-3-(rho-nitrophenyl)propyl]-trans-cyclohexane-1,2-diamine-N,N',-
N''-pentaacetic acid (nitro-CHX-A-DTPA). In another embodiment, the
Albumin fusion protein of the invention are attached to an acyclic
terpyridine chelator such as
6,6''-bis[[N,N,N'',N''-tetra(carboxymethyl)amino]methyl]-4'-(3-amino-4-tr-
iethoxyphenyl)-2,2':6',2''-terpyridine (TMT-amine).
[0517] In specific embodiments, the macrocyclic chelator which is
attached to the Albumin fusion protein of the invention is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA). In other specific embodiments, the DOTA is attached to the
Albumin fusion protein of the invention via a linker molecule.
Examples of linker molecules useful for conjugating DOTA to a
polypeptide are commonly known in the art--see, for example,
DeNardo et al., Clin. Cancer Res. 4(10):2483-90, 1998; Peterson et
al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al.,
Natl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporated
by reference in their entirety. In addition, U.S. Pat. Nos.
5,652,361 and 5,756,065, which disclose chelating agents that may
be conjugated to antibodies, and methods for making and using them,
are hereby incorporated by reference in their entireties. Though
U.S. Pat. Nos. 5,652,361 and 5,756,065 focus on conjugating
chelating agents to antibodies, one skilled in the art could
readily adapt the method disclosed therein in order to conjugate
chelating agents to other polypeptides.
[0518] Bifunctional chelators based on macrocyclic ligands in which
conjugation is via an activated arm, or functional group, attached
to the carbon backbone of the ligand can be employed as described
by M. Moi et al. J. Amer. Chem. Soc. 49:2639 (1989)
(2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic
acid); S. V. Deshpande et al., J. Nucl. Med. 31:473 (1990); G.
Ruser et al., Bioconj. Chem., 1:345 (1990); C. J. Email et al., J.
C. S. Chem. Comm. 23:1739 (1990); and C. J. Anderson et al., J.
Nucl. Med. 36:850 (1995).
[0519] In one embodiment, a macrocyclic chelator, such as
polyazamacrocyclic chamois, optionally containing uric or more
carboxy, amino, hydroxamate, phosphonate, or phosphate groups, are
attached to the Albumin fusion protein of the invention. In another
embodiment, the chelator is a chelator selected from the group
consisting of DOTA, analogues of DOTA, and derivatives of DOTA.
[0520] In one embodiment, suitable chelator molecules that may be
attached to the Albumin fusion protein of the invention include
DOXA (1-oxa-4,7,10-triazacyclododecanetriacetic acid), NOTA
(1,4,7-triazacyclononanetriacetic acid), TETA
(1,4,8,11-tetraazacyclotetradecanetetraacetic acid), and THT
(4'-(3-amino-4-methoxy-phenyl)-6,6''-bis(N',N'-dicarboxymethyl-N-methylhy-
drazino)-2,2':6',2''-terpyridine), and analogs and derivatives
thereof. See, e.g., Ohmono et al., J. Med. Cheer. 35: 157-162
(1992); Kong et al., J. Nucl. Med. 25: 326-332 (1984); Jurisson et
al., Chem. Rev. 93:1137-1156 (1993); and U.S. Pat. No. 5,367,080.
Other suitable chelators include chelating agents disclosed in U.S.
Pat. Nos. 4,647,447; 4,687,659; 4,885,363; EP-A-71564; WO89/00557;
and EP-A-232751.
[0521] In another embodiment, suitable macrocyclic carboxylic acid
chelators which can be used in the present invention include
1,4,7,10 tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA); 1,4,8,12-tetraazacyclopentadecane-N,N',N'',N'''-tetraacetic
acid (15N4); 1,4,7-triazacyclononane-N,N',N''-triacetic acid (9N3);
1,5,9-triazacyclododecane-N,N',N''-triacetic acid (12N3); and
6-bromoacetamido-benzyl-1,4,8,11-tetrazacyclotetradecane-N,N',N'',N'''-te-
traacetic acid (BAT).
[0522] A preferred chelator that can be attached to the Albumin
Fusion protein of the invention is
.alpha.-(5-isothiocyanate-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecane-
-1,4,7,10-tetraacetic acid, which is also known as MeO-DOTA-NCS. A
salt or ester of
.alpha.-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacycl-
odoclecane-1,4,7,0-tetraacetic acid may also be used.
[0523] Albumin fusion proteins of the invention to which chelators
such as those described are covalently attached may be labeled (via
the coordination site of the chelator) with radionuclides that are
suitable for therapeutic, diagnostic, or both therapeutic and
diagnostic purposes. Examples of appropriate metals include Ag, At,
Au, Bi, Cu, Ca, Ho, In, Lu, Pb, Pd, Pm, Pr, Rb, Re, Rh, Sc, Sr, Tc,
Tl, Y, and Yb. Examples of the radionuclide used for diagnostic
purposes are Fe, Gd, .sup.111In, .sup.67Ga, or .sup.68Ga. In
another embodiment, the radionuclide used for diagnostic purposes
is or .sup.111In, or .sup.67Ga. Examples of the radionuclide used
for therapeutic purposes are .sup.166Ho, .sup.165Dy, .sup.90Y,
.sup.115mIn, .sup.52Fe, or .sup.72Ga. In one embodiment, the
radionuclide used for diagnostic purposes is .sup.166Ho or
.sup.90Y. Examples of the radionuclides used for both therapeutic
and diagnostic purposes include .sup.153Sm, .sup.177Lu, .sup.159Gd,
.sup.175Yb, or .sup.47Sc. In one embodiment, the radionuclide is
.sup.153Sm, .sup.177Lu, .sup.175Yb, or .sup.159Gd.
[0524] Preferred metal radionuclides include .sup.90Y, .sup.99mTc,
.sup.111In, .sup.47Sc, .sup.67Ga, .sup.51Cr, .sup.177mSn,
.sup.67Cu, .sup.167Tm, .sup.97Ru, .sup.188Re, .sup.177Lu,
.sup.199Au, .sup.47Sc, .sup.67Ga, .sup.51Cr, .sup.177mSn,
.sup.67Cu, .sup.167Tm, .sup.95Ru, .sup.188Re, .sup.173Lu,
.sup.199Au, .sup.203Pb, and .sup.141Ce.
[0525] In a particular embodiment, Albumin fusion proteins of the
invention to which chelators are covalently attached may be labeled
with a metal ion selected from the group consisting of .sup.90Y,
.sup.111In, .sup.177Lu, .sup.166Ho, .sup.215Bi, and .sup.225Ac.
[0526] Moreover, .gamma.-remitting radionuclides, such as
.sup.99mTc, .sup.111In, .sup.67Ga, and .sup.169Yb have been
approved or under investigation for diagnostic imaging, while
.beta.-emitters, such as .sup.67Cu, .sup.111Ag, .sup.186Re, and
Y.sup.90Y are useful for the applications in tumor therapy. Also
other useful radionuclides include .gamma.-emitters, such as
.sup.99mTc, .sup.111In, .sup.67 Ga, and .sup.169Yb, and
.beta.-emitters, such as .sup.67Cu, .sup.111Ag, .sup.186Re,
.sup.186Re and .sup.90Y, as well as other radionuclides of interest
such as .sup.211At, .sup.212Bi, .sup.177Lu, .sup.86Rb, .sup.106Rh,
.sup.153Sm, .sup.198Au, .sup.149Pm, .sup.85Sr, .sup.142Pr,
.sup.214Pb, .sup.103Pd, .sup.166Ho, .sup.103Tl, and .sup.44Sc.
Albumin fusion proteins of the invention to which chelators are
covalently attached may be labeled with the radionuclides described
above.
[0527] In another embodiment, Albumin fusion proteins of the
invention to which chelators are covalently attached may be labeled
with paramagnetic metal ions including ions of transition and
lanthanide metal, such as metals having atomic numbers of 21-29,
42, 43, 44, or 57-71, in particular ions of Cr, V, Mn, Fe, Co, Ni,
Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Th, Dy, Ho, Er, Tm, Yb, and Lu.
The paramagnetic metals used in compositions for magnetic resonance
imaging include the elements having atomic numbers of 22 to 29, 42,
44 and 58-70.
[0528] In another embodiment, Albumin fusion proteins of the
invention to which chelators are covalently attached may be labeled
with fluorescent metal ions including lanthanides, in particular
La, Ce, Pr, Nd, Pm, Sm, Eu (e.g., .sup.152Eu), Gd, Tb, Dy, Ho, Er,
Tm, Yb, and Lu.
[0529] In another embodiment, Albumin fusion proteins of the
invention to which chelators are covalently attached may be labeled
with heavy metal-containing reporters may include atoms of Mo, Bi,
Si, and W.
[0530] It is also possible to label the albumin fusion proteins
with a fluorescent compound. When the fluorescently labeled
antibody is exposed to light of the proper wave length, its
presence can then be detected due to fluorescence. Among the most
commonly used fluorescent labeling compounds are fluorescein
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, ophthaldehyde and fluorescamine.
[0531] The albumin fusion protein can also be detectably labeled
using fluorescence emitting metals such as .sup.152Eu, or others of
the lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0532] The albumin fusion proteins can also can be detectably
labeled by coupling it to a chemiluminescent compound. The presence
of the chemiluminescent-tagged albumin fusion protein is then
determined by detecting the presence of luminescence that arises
during the course of a chemical reaction. Examples of particularly
useful chemiluminescent labeling compounds are luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate
ester.
[0533] Likewise, a bioluminescent compound may be used to label
albumin fusion proteins of the present invention. Bioluminescence
is a type of chemiluminescence found in biological systems in,
which a catalytic protein increases the efficiency of the
chemiluminescent reaction. The presence of a bioluminescent protein
is determined by detecting the presence of luminescence. Important
bioluminescent compounds for purposes of labeling are luciferin,
luciferase and aequorin.
[0534] Transgenic Organisms
[0535] Transgenic organisms that express the albumin fusion
proteins of the invention are also included in the invention.
Transgenic organisms are genetically modified organisms into which
recombinant, exogenous or cloned genetic material has been
transferred. Such genetic material is often referred to as a
transgene. The nucleic acid sequence of the transgene may include
one or more transcriptional regulatory sequences and other nucleic
acid sequences such as introns, that may be necessary for optimal
expression and secretion of the encoded protein. The transgene may
be designed to direct the expression of the encoded protein in a
manner that facilitates its recovery from the organism or from a
product produced by the organism, e.g. from the milk, blood, urine,
eggs, hair or seeds of the organism. The transgene may consist of
nucleic acid sequences derived front the genome of the same species
or of a different species than the species of the target animal.
The transgene may be integrated either at a locus of a genome where
that particular nucleic acid sequence is not otherwise normally
found of at the normal locus for the transgene.
[0536] The term "germ cell line transgenic organism" refers to a
transgenic organism in which the genetic alteration or genetic
information was introduced into a germ line cell, thereby
conferring the ability of the transgenic organism to transfer the
genetic information to offspring. If such offspring in fact possess
some or all of that alteration or genetic information, then they
too are transgenic organisms. The alteration or genetic information
may be foreign to the species of organism to which the recipient
belongs, foreign only to the particular individual recipient, or
may be genetic information already possessed by the recipient. In
the last case, the altered or introduced gene may be expressed
differently than the native gene.
[0537] A transgenic organism may be a transgenic animal or a
transgenic plant. Transgenic animals can be produced by a variety
of different methods including transfection, electroporation,
microinjection, gene targeting in embryonic stem cells and
recombinant viral and retroviral infection (see, e.g., U.S. Pat.
No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins et al. (1993)
Hypertension 22(4):630-633; Brenin et al. (1997) Surg. Oncol.
6(2)99-110; Tuan (ed.), Recombinant Gene Expression Protocols,
Methods in Molecular Biology No. 62, Humana Press (1997)). The
method of introduction of nucleic acid fragments into recombination
competent mammalian cells can be by any method which favors
co-transformation of multiple nucleic acid molecules. Detailed
procedures for producing transgenic animals are readily available
to one skilled in the art, including the disclosures in U.S. Pat.
No. 5,489,743 and U.S. Pat. No. 5,602,307.
[0538] A number of recombinant or transgenic mice have been
produced, including those which express an activated oncogene
sequence (U.S. Pat. No. 4,736,866); express simian SV40 T-antigen
(U.S. Pat. No. 5,728,915); lack the expression of interferon
regulatory factor 1 (IRE-1) (U.S. Pat. No. 5,731,490); exhibit
dopaminergic dysfunction (U.S. Pat. No. 5,723,719); express at
least one human gene which participates in blood pressure control
(U.S. Pat. No. 5,731,489); display greater similarity to the
conditions existing in naturally occurring Alzheimer's disease
(U.S. Pat. No. 5,720,936); have a reduced capacity to mediate
cellular adhesion (U.S. Pat. No. 5,602,307); possess a bovine
growth hormone gene (Clutter et al. (1996) Genetics
143(4):1753-1760); or, are capable of generating a fully human
antibody response (McCarthy (1997) The Lancet 349(9049):405).
[0539] While mice and rats remain the animals of choice for most
transgenic experimentation, in some instances it is preferable or
even necessary to use alternative animal species. Transgenic
procedures have been successfully utilized in a variety of
non-murine animals, including sheep, goats, pigs, dogs, cats,
monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see,
e.g., Kim et al. (1997) Mol. Reprod. Dev. 46(4):515-526; Houdebine
(1995) Reprod. Nutr. Dev. 35(6):609-617; Petters (1994) Reprod.
Perth. Dev. 6(5):643-645; Schnieke et al, (1997) Science
278(5346):2130-2133; and Amoah (1997) J. Animal Science
75(2):578-585).
[0540] To direct the secretion of the transgene-encoded protein of
the invention into the milk of transgenic mammals, it may be put
under the control of a promoter that is preferentially activated in
mammary epithelial cells. Promoters that control the genes encoding
milk proteins are preferred, for example the promoter for casein,
beta lactoglobulin, whey acid protein, Of lactalbumin (see, e.g.,
DiTullio (1992) BioTechnology 10:74-77; Clark et al. (1989)
BioTechnology 7; 487-492; Gorton et al. (1987) BioTechnology
5:1183-1187; and Soldier et al. (1992) FEBS Letts. 297:13). The
transgenic mammals of choice would produce large volumes of milk
and have long lactating periods, for example goats, cows, camels or
sheep.
[0541] An albumin fusion protein of the invention can also be
expressed in a transgenic plant, e.g. a plant in which the DNA
transgene is inserted into the nuclear or plastidic genome, Plant
transformation procedures used to introduce foreign nucleic acids
into plant cells or protoplasts are known in the art. See, in
general, Methods in Enzymology Vol. 153 ("Recombinant DNA Part D")
1987, Wu and Grossman Eds., Academic Press and European Patent
Application EP 693554. Methods for generation of genetically
engineered plants are further described in U.S. Pat. No. 5,283,184,
U.S. Pat. No. 5,482,852, and European Patent Application EP 693
554, all of which are hereby incorporated by reference.
[0542] Pharmaceutical or Therapeutic Compositions
[0543] The albumin fusion proteins of the invention or formulations
thereof may be administered by any conventional method including
parenteral (e.g. subcutaneous or intramuscular) injection or
intravenous infusion. The treatment may consist of a single dose or
a plurality of doses over a period of time.
[0544] While it is possible for an albumin fusion protein of the
invention to be administered alone, it is preferable to present it
as a pharmaceutical formulation, together with one or more
acceptable carriers. The carrier(s) must be "acceptable" in the
sense of being compatible with the albumin fusion protein and not
deleterious to the recipients thereof. Typically, the carriers will
be water or saline which will be sterile and pyrogen free. Albumin
fusion proteins of the invention are particularly well suited to
formulation in aqueous carriers such as sterile pyrogen free water,
saline or other isotonic solutions because of their extended
shelf-life in solution. For instance, pharmaceutical compositions
of the invention may be formulated well in advance in aqueous form,
for instance, weeks or months or longer time periods before being
dispensed.
[0545] In instances where aerosol administration is appropriate,
the albumin fusion proteins of the invention can be formulated as
aerosols using standard procedures. The term "aerosol" includes any
gas-borne suspended phase of an albumin fusion protein of the
instant invention which is capable of being inhaled into the
bronchioles or nasal passages. Specifically, aerosol includes a
gas-borne suspension of droplets of an albumin fusion protein of
the instant invention, as may be produced in a metered dose inhaler
or nebulizer, or in a mist sprayer. Aerosol also includes a dry
powder composition of a compound of the instant invention suspended
in air or other cattier gas, which may be delivered by insufflation
from an inhaler device, for example. See Ganderton & Jones,
Drug Delivery to the Respiratory Tract. Ellis Horwood (1987); Gonda
(1990) Critical Reviews in Therapeutic Drug Carrier Systems 6;
273-313; and Raeburn et al., (1992) Pharmacol. Toxicol. Methods
27:143-159.
[0546] The formulations of the invention are also typically
non-immunogenic, in part, because of the use of the components of
the albumin fusion protein being derived from the proper species.
For instance, for human use, both the Therapeutic protein and
albumin portions of the albumin fusion protein will typically be
human. In some cases, wherein either component is non
human-derived, that component may be humanized by substitution of
key amino acids so that specific epitopes appear to the human
immune system to be human in nature rather than foreign.
[0547] The formulations may conveniently be presented in unit
dosage form and may be prepared by any of the methods well known in
the art of pharmacy. Such methods include the step of bringing into
association the albumin fusion protein with the carrier that
constitutes one or more accessory ingredients. In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0548] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation appropriate for the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example sealed
ampules, vials or syringes, and may be stored in a freeze-dried
(lyophilised) condition requiring only the addition of the sterile
liquid carrier, for example water for injections, immediately prior
to use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders. Dosage formulations may contain the
Therapeutic protein portion at a lower molar concentration or lower
dosage compared to the non fused standard formulation for the
Therapeutic protein given the extended serum half-life exhibited by
many of the albumin fusion proteins of the invention.
[0549] As an example, when an albumin fusion protein of the
invention comprises growth hormone as one or more of the
Therapeutic protein regions, the dosage form can be calculated on
the basis of the potency of the albumin fusion protein relative to
the potency of hGH, while taking into account the prolonged serum
half-life and shelf-life of the albumin fusion proteins compared to
that of native hGH. Growth hormone is typically administered at 0.3
to 30.0 ID/kg/week, for example 0.9 to 12.0 IU/kg/week, given in
three or seven divided doses for a year or more. In an albumin
fusion protein consisting of full length HA fused to full length an
equivalent dose in terms of units would represent a greater weight
of agent but the dosage frequency can be reduced, for example to
twice a week, once a week or less.
[0550] Formulations or compositions of the invention may be
packaged together with, or included in a kit with, instructions or
a package insert referring to the extended shelf-life of the
albumin fusion protein component. For instance, such instructions
or package inserts may address recommended storage conditions, such
as time, temperature and light, taking into account the extended or
prolonged shelf-life of the albumin fusion proteins of the
invention. Such instructions or package inserts may also address
the particular advantages of the albumin fusion proteins of the
inventions, such as the ease of storage for formulations that may
require use in the field, outside of controlled hospital, clinic or
office conditions. As described above, formulations of the
invention may be in aqueous form and may be stored under less than
ideal circumstances without significant loss of therapeutic
activity.
[0551] Albumin fusion proteins of the invention can also be
included in nutraceuticals. For instance, certain albumin fusion
proteins of the invention may be administered in natural products,
including milk or milk product obtained from a transgenic mammal
which expresses albumin fusion protein. Such compositions can also
include plant or plant products obtained from a transgenic plant
which expresses the albumin fusion protein. The albumin fusion
protein can also be provided in powder or tablet form, with or
without other known additives, carriers, fillers and diluents.
Nutraceuticals are described in Scott Hegenhart, Food Product
Design, December 1993.
[0552] The invention also provides methods of treatment and/or
prevention of diseases or disorders (such as, for example, any one
or more of the diseases or disorders disclosed herein) by
administration to a subject of an effective amount of an albumin
fusion protein of the invention or a polynucleotide encoding an
albumin fusion protein of the invention ("albumin fusion
polynucleotide") in a pharmaceutically acceptable carrier.
[0553] The albumin fusion protein and/or polynucleotide will be
formulated and dosed in a fashion consistent with good medical
practice, taking into account the clinical condition of the
individual patient (especially the side effects of treatment with
the albumin fusion protein and/or polynucleotide alone), the site
of delivery, the method of administration, the scheduling of
administration, and other factors known to practitioners. The
"effective amount" for purposes herein is thus determined by such
considerations.
[0554] As a general proposition, the total pharmaceutically
effective amount of the albumin fusion protein administered
parenterally per dose will be in the range of about 1 ug/kg/day to
10 mg/kg/day of patient body weight, although, as noted above, this
will be subject to therapeutic discretion. More preferably, this
dose is at least 0.01 mg/kg/day, and most preferably for humans
between about 0.01 and 1 mg/kg/day for the hormone. If given
continuously, the albumin fusion protein is typically administered
at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either
by 1-4 injections per day or by continuous subcutaneous infusions,
for example, using a mini-pump. An intravenous bag solution may
also be employed. The length of treatment needed to observe changes
and the interval following treatment for responses to occur appears
to vary depending on the desired effect.
[0555] Albumin fusion proteins and/or polynucleotides can be are
administered orally, rectally, parenterally, intracistemally,
intravaginally, intraperitoneally, topically (as by powders,
ointments, gels, drops or transdermal patch), bucally, or as an
oral or nasal spray. "Pharmaceutically acceptable carrier" refers
to a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any. The term
"parenteral" as used herein refers to modes of administration which
include intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous and intraarticular injection and infusion.
[0556] Albumin fusion proteins and/or polynucleotides of the
invention are also suitably administered by sustained-release
systems. Examples of sustained-release albumin fusion proteins
and/or polynucleotides are administered orally, rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, gels, drops or transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically
acceptable carrier" refers to a non-toxic solid, semisolid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any type. The term "parenteral" as used herein refers
to modes of administration which include intravenous,
intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular injection and infusion. Additional examples of
sustained-release albumin fusion proteins and/or polynucleotides
include suitable polymeric materials (such as, for example,
semi-permeable polymer matrices in the form of shaped articles,
e.g., films, or mirocapsules), suitable hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, and sparingly soluble derivatives (such as, for example, a
sparingly soluble salt).
[0557] Sustained-release matrices include polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556
(1983)), poly(2-hydroxyethyl methacrylate) (Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech.
12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0558] Sustained-release albumin fusion proteins and/of
polynucleotides also include liposomally entrapped albumin fusion
proteins and/or polynucleotides of the invention (see generally,
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. 317-327 and 353-365 (1989)).
Liposomes containing the albumin fusion protein and/or
polynucleotide are prepared by methods known per se: DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci, (USA) 82:3688-3642 (1985);
Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP
52,322; EP 36,676; EP 88.046; EP 143,949; EP 142,641; Japanese Pat.
Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP
102,324. Ordinarily, the liposomes are of the small (about 200-800
Angstroms) unilamellar type in which the lipid content is greater
than about 30 mol. percent cholesterol, the selected proportion
being adjusted for the optimal Therapeutic.
[0559] In yet an additional embodiment, the albumin fusion proteins
and/or polynucleotides of the invention are delivered by way of a
pump (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)).
[0560] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0561] For parenteral administration, in one embodiment, the
albumin fusion protein and/or polynucleotide is formulated
generally by mixing it at the desired degree of purity, in a unit
dosage injectable form (solution, suspension, or emulsion), with a
pharmaceutically acceptable carrier, i.e., one that is non-toxic to
recipients at the dosages and concentrations employed and is
compatible with other ingredients of the formulation. For example,
the formulation preferably does not include oxidizing agents and
other compounds that are known to be deleterious to the
Therapeutic.
[0562] Generally, the formulations are prepared by contacting the
albumin fusion protein and/or polynucleotide uniformly and
intimately with liquid carriers or finely divided solid carriers or
both. Then, if necessary, the product is shaped into the desired
formulation. Preferably the carrier is a parenteral carrier, more
preferably a solution that is isotonic with the blood of the
recipient. Examples of such carrier vehicles include water, saline,
Ringer's solution, and dextrose solution. Non-aqueous vehicles such
as fixed oils and ethyl oleate are also useful herein, as well as
liposomes.
[0563] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose, or its derivatives, glucose, manose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0564] The albumin fusion protein is typically formulated in such
vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml,
preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be
understood that the use of certain of the foregoing excipients,
carriers, or stabilizers will result in the formation of
polypeptide salts.
[0565] Any pharmaceutical used for therapeutic administration can
be sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes). Albumin
fusion proteins and/or polynucleotides generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0566] Albumin fusion proteins and/or polynucleotides ordinarily
will be stored in unit or multi-dose containers, for example,
sealed ampoules or vials, as an aqueous solution or as a
lyophilized formulation for reconstitution. As an example of a
lyophilized formulation, 10-ml vials are filled with 5 ml of
sterile-filtered 1% (w/v) aqueous albumin fusion protein and/or
polynucleotide solution, and the resulting mixture is lyophilized.
The infusion solution is prepared by reconstituting the lyophilized
albumin fusion protein and/or polynucleotide using bacteriostatic
Water-for-Injection.
[0567] In a specific and preferred embodiment, the Albumin fusion
protein formulations comprises 0.01 M sodium phosphate, 0.15 mM
sodium chloride, 0.16 micromole sodium octanoate/milligram of
fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2. In
another specific and preferred embodiment, the Albumin fusion
protein formulations consists 0.01 M sodium phosphate, 0.15 mM
sodium chloride, 0.16 micromole sodium octanoate/milligram of
fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2.
The pH and buffer are chosen to match physiological conditions and
the salt is added as a tonicifier. Sodium octanoate has been chosen
due to its reported ability to increase the thermal stability of
the protein in solution. Finally, polysorbate has been added as a
generic surfactant, which lowers the surface tension of the
solution and lowers non-specific adsorption of the albumin fusion
protein to the container closure system.
[0568] The invention also provides a pharmaceutical pack or kit
comprising one or more containers tilled with one or more of the
ingredients of the albumin fusion proteins and/or polynucleotides
of the invention. Associated with such container(s) can be a notice
in the form prescribed by as governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration. In addition, the albumin fusion
proteins and/or polynucleotides may be employed in conjunction with
other therapeutic compounds.
[0569] The albumin fusion proteins and/or polynucleotides of the
invention may be administered alone or in combination with
adjuvants. Adjuvants that may be administered with the albumin
fusion proteins and/or polynucleotides of the invention include,
but are not limited to, alum, alum plus deoxycholate (ImmunoAg),
MTP-PE (Biocine Corp.), QS2I (Genentech, Inc.), BCG (e.g.,
THERACYS.RTM.), MPL and nonviable preparations of Corynebacterium
parvum. In a specific embodiment, albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with alum. In another specific embodiment, albumin fusion proteins
and/or polynucleotides of the invention are administered in
combination with QS-21. Further adjuvants that may be administered
with the albumin fusion proteins and/or polynucleotides of the
invention include, but are not limited to, Monophosphoryl lipid
immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum
salts, MF-59, and Virosomal adjuvant technology. Vaccines that may
be administered with the albumin fusion proteins and/or
polynucleotides of the invention include, but are not limited to,
vaccines directed toward protection against MMR (measles, mumps,
rubella), polio, varicella, tetanus/diptheria, hepatitis A,
hepatitis B, Haemophilus influenzae B, whooping cough, pneumonia,
influenza, Lyme's Disease, rotavirus, cholera, yellow fever,
Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and
pertussis. Combinations may be administered either concomitantly,
e.g., as an admixture, separately but simultaneously or
concurrently; or sequentially. This includes presentations in which
the combined agents are administered together as a therapeutic
mixture, and also procedures in which the combined agents are
administered separately but simultaneously, e.g., as through
separate intravenous lines into the same individual. Administration
"in combination" further includes the separate administration of
one of the compounds or agents given first, followed by the
second.
[0570] Therapeutic Compositions Alone or in Combination
[0571] The albumin fusion proteins and/or polynucleotides of the
invention may be administered alone or in combination with other
therapeutic agents. Albumin fusion protein and/or polynucleotide
agents that may be administered in combination with the albumin
fusion proteins and/or polynucleotides of the invention, include
but are not limited to, GLP-1, insulin (including an insulin
fragment or variant), an insulin secretagogue, IGF-1, an IGF-1
secretagogue, an insulin sensitizer (e.g., a Thiazolidinedione, or
Resistin antagonist (e.g., anti-resistin antibody)), a beta cell
growth factor, an alpha glucosidase inhibitor, a sulfonylureas,
biguanide, chemotherapeutic agents, antibiotics, steroidal and
non-steroidal anti-inflammatories, conventional immunotherapeutic
agents, and/or therapeutic treatments described below. Combinations
may be administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially.
This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and also procedures
in which the combined agents are administered separately but
simultaneously, e.g., as through separate intravenous lines into
the same individual. Administration "in combination" further
includes the separate administration of one of the compounds or
agents given first, followed by the second. In preferred
embodiments, this administration immediately prior to, during, or
immediately after nutrient consumption (e.g., a meal).
[0572] In preferred embodiments, a composition of the invention
(i.e., albumin fusion proteins and/or polynucleotides of the
invention) is administered in combination with Glucagon-Like
Peptide 1 or fragments or variants thereof (including fusion
proteins containing GLP-1 peptides, fragments and/or variants).
Glucagon-Like Peptide 1 that may be administered in combination
with a composition of the invention include, but are not limited
to, AC-2993 (Exendin-4), insulinotropin (GLP-1-(7-37)), and NNC
90-1170.
[0573] In particular embodiments, the use of a composition of the
invention in combination with Glucagon-Like Peptide 1 is
contemplated for the treatment (e.g., amelioration) and/or
prevention of diabetes mellitus, i.e., IDDM and/or NIDDM and/or a
condition associated with diabetes.
[0574] In other preferred embodiments, a composition of the
invention (i.e. albumin fusion proteins and/or polynucleotides of
the invention) is administered in combination one or more with
beta-cell growth factors. Beta-cell growth factors (Stewart et al.,
Journal of Clinical Endocrinology & Metabolism 86(3):984-988
(2001)) that may be administered in combination with a composition
of the invention include, but are not limited to, betacellulin,
exendin-4, glucagons-like peptide-1, hepatocyte growth factors,
insulin-like growth factor-I, insulin-like growth factor-II, islet
neogenesis-associated protein, placental lactogen, PTH-related
protein, and cytokeratin 20 (Anastasi et al., Eur 3 Endocrinol
141(6):644-52 (1999)). In another preferred embodiment, a
composition of the invention is administered in combination with
RegIV (The RegIV gene and protein have also been identified by the
names "Colon Specific Gene" and "Colon Specific Protein",
respectively. See e.g., U.S. Pat. No. 5,861,494, U.S. Pat. No.
6,080,722, and PUT Publication No. WO96/39541).
[0575] In particular embodiments, the use of a composition of the
invention in combination with one or more beta-cell growth factors
is contemplated for the treatment (e.g., amelioration) or
prevention of diabetes mellitus, i.e., IDDM and/or NIDDM and/or a
condition associated with diabetes.
[0576] In other preferred embodiments, a composition of the
invention is administered in combination with one or more
alpha-glucosidase inhibitors. Alpha-Glucosidase inhibitors that may
be administered in combination with a composition of the invention
include, but are not limited to, miglitol (Glyset), acarbose
(Precose), voglibose (Basen; Glustat).
[0577] In particular embodiments, the use of a composition of the
invention in combination with one or more alpha-glucosidase
inhibitors is contemplated for the treatment (e.g., amelioration)
or prevention of diabetes mellitus, i.e., IDDM and/or NIDDM and/or
a condition associated with diabetes.
[0578] In other preferred embodiments, a composition of the
invention (i.e., albumin fusion proteins and/or polynucleotides of
the invention) is administered in combination with one or more
insulin and related agents. Insulin and related agents that may be
administered in combination with a composition of the invention
include, but are not limited to, Insulin Mixtures (Humulin 50/50,
Humulin 7000, Novolin 70/30), intermediate acting insulin (Humulin
L, Humulin N, Iletin II Lente, Iletin II NPH, Novolin L, Novolin
N), long acting insulin (Humulin U, Latus), rapid acting insulin
(Humalog, Insulin lispro, Insulin Aspart), short acting insulin
(Humulin R, Iletin II Regular, Novolin R, Novolin BR), AFRx Insulin
Inhaler, Basulin (Insulin Flamel), Inhaled Insulin, Insulin detemir
(long-acting insulin, N,N-304), Macrulin (oral insulin), Mecasermin
(Somazon), Oral Insulin, Oralin (Oralgen, RapidMist), and
Transfersulin (insulin, Transfersome).
[0579] In particular embodiments, the use of a composition of the
invention in combination with one or more insulin and related
agents is contemplated for the treatment (e.g., amelioration)
and/or prevention of diabetes mellitus, i.e., IDDM and/or NIDDM
and/or a condition associated with diabetes. In a highly preferred
embodiment a composition of the invention is administered in
combination with insulin and/or related molecules (e.g., insulin
fragments and variants, and insulin secretagogues).
[0580] In other preferred embodiments, it composition of the
invention ise administered in combination with one or more hormone
inhibitors. Hormone inhibitors that may be administered in
combination with a composition of the invention include, but are
not limited to, BAY-27-9955 and pegvisomant (Somavert,
Trovert).
[0581] In particular embodiments, the use of a composition of the
invention in combination with one or more hormone inhibitors is
contemplated for the treatment (e.g., amelioration) or prevention
of conditions associated with diabetes mellitus, for example,
diabetic retinopathy.
[0582] In particular embodiments, the use of a composition of the
invention in combination with the mature (secreted) portion, the
cysteine rich region(s), the precursor polypeptide, the propeptide
polypeptide, or any fragment thereof, of one or more of the
polypeptides selected from the group: TGF-.beta.1, TGF-.beta.2,
TGF-.beta.3, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7,
BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15,
BMP-15, GDF-1, GDF-3, GDF-8, GDF-9, and MIS.
[0583] In other preferred embodiments, a composition of the
invention is administered in combination with one or more
sulfonylureas. Sulfonylureas that may be administered in
combination with a composition of the invention include, but are
not limited to, glimepiride (Amaryl), glyburide (DiaBeta, Glynase
PresTab, Micronase), chlorpropamide (Diabinese), acetohexamide
(Dymelor), glipizide (Glueutrol, Glucotrol XL), tolbutamide
(Orinase), tolazamide (Tolinase), gliclazide (Adianor), and
glipentide (Staticum).
[0584] In particular embodiments, the use of a composition of the
invention in combination with one or more sulfonylureas is
contemplated for the treatment (e.g., amelioration) and/or
prevention of diabetes mellitus, i.e., IDDM and/or NIDDM and/or a
condition associated with diabetes.
[0585] In other preferred embodiment, composition of the invention
is administered in combination with one or more biguanides.
Biguanides that may be administered in combination with a
composition of the invention include, but are not limited to,
metformin (Glucophage) and a combination of metformin with
glibenclamide (Glucovance, Glucophage+Glylautide).
[0586] In particular embodiments, the use of a composition of the
invention in combination with biguanides is contemplated for the
treatment (e.g., amelioration) and/or prevention of diabetes
mellitus, i.e. IDDM and/or NIDDM and/or a condition associated with
diabetes.
[0587] In other preferred embodiments, a composition of the
invention (i.e., albumin fusion proteins and/or polynucleotides of
the invention) is administered in combination with one or more
Thiazolidinediones (TZDs). Thiazolidinediones that may be
administered in combination with a composition of the invention
include, but are not limited to, rosiglitazone maleate (Avandia),
pioglitazone hydrochloride (Actos), isaglitazone (MCC-555,
RWJ241947), and troglitazone (Rezulin, Romozin, Prelay, Noscal.
[0588] In particular embodiments, the use of a composition of the
invention in combination with one or more Thiazolidinediones is
contemplated for the treatment (e.g., amelioration) and/or
prevention of diabetes mellitus, i.e., IDDM and/or NIDDM and/or a
condition associated with diabetes.
[0589] In other preferred embodiments, a composition of the
invention is administered in combination with Resistin antagonists
and/or anti-Resistin antibodies.
[0590] In other preferred embodiments, a composition of the
invention is administered in combination with one or more other
insulin sensitizers. Other Insulin Sensitizers that may be
administered in combination with a Therapeutics of the invention
include, but are not limited to, Bexarotene (Targretin), Chiro
inositol (INS-1), Chromium picolinate (Chromax Plus; Chromax),
Vanadium (KP-102, LP-100), and PPAR-gamma Activators which include,
but are not limited to, GI-262570 (GW-2570), GW-409544 (GW-544),
and KRP-297.
[0591] In other preferred embodiments, a composition of the
invention is administered in combination with Resistin antagonists
and/or anti-Resistin antibodies.
[0592] In particular embodiments, the use of a composition of the
invention in combination with one or more insulin sensitizers is
contemplated for the treatment (e.g., amelioration) and/or
prevention of diabetes mellitus, i.e., IDDM and/or NIDDM and/or a
condition associated with diabetes.
[0593] In other preferred embodiments, a composition of the
invention is administered in combination with one or more
non-sulfonylureas including Meglitinides. Non-Sulfonylureas that
may be administered in combination with a composition of the
invention include, but are not limited to, repaglinide (Prandin,
Aculin), rateglinide (Starlix), BTS 67582, Mitiglinide (KAD-1229),
and ProBeta.
[0594] In particular embodiments, the use of a composition of the
invention in combination with one or more non-sulfonylureas is
contemplated for the treatment (e.g., amelioration) and/or
prevention of diabetes mellitus, i.e., IDDM and/or NIDDM and/or a
condition associated with diabetes.
[0595] In preferred embodiments, a composition of the invention is
administered in combination with one or more immunomodulators.
Immunomodulators that may be administered in combination with a
composition of the invention include, but are not limited to,
AI-401, CDP-571 (anti-TNF monoclonal antibody), CG-1088, Diamyd
(diabetes vaccine), ICM3 (anti-ICAM-3 monoclonal antibody),
linomide (Roquinimex), NBI-6024 (altered peptide ligand), TM-27, VX
740 (HMR-3480), caspase 8 protease inhibitors, thalidomide,
hOKT3gamma1 (Ala-ala) (anti-CD3-monoclonal antibody), Oral
Interferon-Alpha, oral lactobacillus, and LymphoStat-B.TM..
[0596] In particular embodiments, the use of a composition of the
invention in combination with one or more immunomodulators is
contemplated for the treatment (e.g., amelioration) and/or
prevention of autoimmune diabetes, i.e. IDDM Insulin-Dependent
Diabetes Mellitus.
[0597] In still other preferred embodiments, a composition of the
invention is administered in combination with one or more of the
following: bromocriptine (Ergoset), etomoxir, iloprost (Endoprost),
acetylcholine, ascorbic acid (Vitamin C), and antagonists of
resistin (Steppan et al., Nature 409(6818):307-12 2001)) and is
contemplated for the treatment (e.g., amelioration) and/or
prevention of diabetes mellitus, i.e., IDDM and/or NIDDM and/or a
condition associated with diabetes.
[0598] In one embodiment, a composition of the invention (i.e.,
albumin fusion proteins and/or polynucleotides of the invention) is
administered in combination with an anticoagulant. Anticoagulants
that may be administered with a compositions of the invention
include, but are not limited to, heparin, low molecular weight
heparin, warfarin sodium (e.g., COUMADIN.RTM.), dicumarol,
4-hydroxycoumarin, anisindione MIRADON.TM.), acenocoumarol (e.g.,
nicoumalone, SINTHROME.TM.), indan-1,3-dione, phenprocoumon (e.g.,
MARCUMAR.TM.), ethyl biscoumacetate (e.g., TROMEXAN.TM.), and
aspirin. In a specific embodiment, a composition of the invention
is administered in combination with heparin and/or warfarin. In
another specific embodiment, a composition of the invention is
administered in combination with warfarin. In another specific
embodiment, a composition of the invention is administered in
combination with warfarin and aspirin. In another specific
embodiment, a composition of the invention is administered in
combination with heparin. In another specific embodiment, a
composition of the invention is administered in combination with
heparin and aspirin.
[0599] In another embodiment, a composition of the invention is
administered in combination with one or more thrombolytic drugs.
Thrombolytic drugs that may be administered with a composition of
the invention include, but are not limited to, plasminogen,
lys-plasminogen, alpha2-antiplasmin, streptokinase KABIKINASE.TM.),
antiresplace (e.g., EMINASE.TM.), tissue plasminogen activator
(t-PA, altevase, ACTIVASE.TM.), urokinase (e.g., ABBOKINASE.TM.),
sauruplase, (Prourokinase, single chain urokinase), and
aminocaproic acid (e.g., AMICAR.TM.). In a specific embodiment, a
composition of the invention is administered in combination with
tissue plasminogen activator and aspirin.
[0600] In another embodiment, a composition of the invention is
administered in combination with one or more antiplatelet drugs,
Antiplatelet drugs that may be administered with a composition of
the invention include, but are not limited to, aspirin,
dipyridamole (e.g., PERSANTINE.TM.), and ticlopidine (e.g.,
TICLID.TM.).
[0601] In specific embodiments, the use of one or more
anti-coagulants, thrombolytic and/or antiplatelet drugs in
combination with a composition of the invention is contemplated for
the prevention, diagnosis, and/or treatment of thrombosis, arterial
thrombosis, venous thrombosis, thromboembolism, pulmonary embolism,
atherosclerosis, myocardial infarction, transient ischemic attack,
unstable angina. In specific embodiments, the use of one or more
anticoagulants, thrombolytic drugs and/or antiplatelet drugs in
combination with a composition of the invention is contemplated for
the prevention of occlusion of saphenous grafts, for reducing the
risk of periprocedural thrombosis as might accompany angioplasty
procedures, for reducing the risk of stroke in patients with atrial
fibrillation including nonrheumatic atrial fibrillation, for
reducing the risk of embolism associated with mechanical heart
valves and or initial valves disease, Other uses for a composition
of the invention, alone or in combination with one or more
antiplatelet, anticoagulant, and/or thrombolytic drugs, include,
but are not limited to, the prevention of occlusions in
extracorporeal devices (e.g., intravascular cannulas, vascular
access shunts in hemodialysis patients, hemodialysis machines, and
cardiopulmonary bypass machines).
[0602] In certain embodiments, a composition of the invention
(i.e., albumin fusion proteins and/or polynucleotides of the
invention) is administered in combination with one or more
antiretroviral agents, nucleoside/nucleotide reverse transcriptase
inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors
(NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be
administered in combination with a composition of the invention,
include, but are not limited to, RETROVIR.TM. (zidovudine/AZT),
VIDEX.TM. (didanosine/ddI), HIVID.TM. (zalcitabine/ddC), ZERIT.TM.
(stavudine/d4T), EPIVAR.TM. (lamivudine/3TC), and COMBIVIR.TM.
(zidovudine/lamivudine). NNRTIs that may be administered in
combination with the a composition of the invention, includes, but
are not limited to, VIRAMUNE.TM. (nevirapine), RESCRIPTOR.TM.
(delavirdine), and SUSTIVA.TM. (efavirenz). Protease inhibitors
that may be administered in combination with a composition of the
invention, include, but are not limited to, CRIXIVAN.TM.
(indinavir), NORVIR.TM. (ritonavir), INVIRASE.TM. (saquinavir), and
VIRACEPT.TM. (nelfinavir). In a specific embodiment, one or more
antiretroviral agents, nucleoside reverse transcriptase inhibitors,
non-nucleoside reverse transcriptase inhibitors, and/or protease
inhibitors may be used in any combination with a composition of the
invention to treat AIDS and/or to prevent or treat WV
infection.
[0603] Additional NRTIs that may be administered in combination
with a composition of the invention, include LODENOSINE.TM. (F-ddA;
an acid-stable adenosine NRTI; Triangle/Abbott; COVIRACIL.TM.
(emtricitabine/FTC; structurally related to lamivudine (3TC) but
with 3- to 10-fold greater activity in vitro; Triangle/Abbott);
dOTC (BCH-10652, also structurally related to lamivudine but
retains activity against a substantial proportion of
lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused
approval for anti-HIV therapy by FDA; Gilead Sciences);
PREVEON.RTM. (Adefovir Dipivoxil, the active prodrug of adefovir;
its active form is PMEA-pp); TENOFOVIR.TM. (bis-POC PMPA, a PMPA
prodrug; Gilead); DAPD/DXG (active metabolite of DAPD;
Triangle/Abbott); D-D4FC (related to 3TC, with activity against
AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGEN.TM.
(abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3'
azido-2',3'-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl
(SATE)-bearing prodrug forms of .beta.-L-FD4C and .beta.-L-FddC (WO
98/17281).
[0604] Additional NRTIs that may be administered in combination
with a composition of the invention, include COACTINON.TM.
(Emivirine/MKC-442, potent NNRTI of the HEPT class;
Triangle/Abbott); CAPRAVIRINE.TM. (AG-1549/S-1153, a next
generation NNRTI with activity against viruses containing the K103N
mutation; Agouron); PNU-142721 (has 20- to 50-fold greater activity
than its predecessor delavirdine and is active against K103N
mutants; Pharmacia & Upjohn); DPC-961 and DPC-963
(second-generation derivatives of cfavirenz, designed to be active
against viruses with the K103N mutation; DuPont); GW-420867X (has
25-fold greater activity than HBY097 and is active against K103N
mutants; Glaxo Wellcome); CALANOLIDE A (naturally occurring agent
from the latex tree; active against viruses containing either or
both the Y181C and K103N mutations); and Propolis (WO
99/49830).
[0605] Additional protease inhibitors that may be administered in
combination with a composition of the invention, include
LOPINAVIR.TM. (ABT378/r; Abbott Laboratories); BMS-232632 (an
azapeptide; Bristol-Myres Squibb); TIPRANAVIR.TM. (PNU-140690, a
non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (a
nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide;
Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck);
DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a
peptidomimetic with in vitro activity against protease
inhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphate
prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755
(Ciba); and AGENERASE.TM. (amprenavir; Glaxo Wellcome Inc.).
[0606] Additional antiretroviral agents that may be administered in
combination with a composition of the invention, include one or
more fusion inhibitors/gp41 hinders. Fusion inhibitors/gp41 binders
include T-20 (a peptide from residues 643-678 of the HIV gp41
transmembrane protein ectodomain which binds to gp41 in its resting
state and prevents transformation to the fusogenic state; Trimeris)
and T-1249 (a second-generation fusion inhibitor; Trimeris).
[0607] Additional antiretroviral agents that may be administered in
combination with a composition of the invention, include fusion
inhibitors/chemokine receptor antagonists. Fusion
inhibitors/chemokine receptor antagonists include CXCR4 antagonists
such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C
(a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and
the T22 analogs T134 and T140; CCR5 antagonists such as RANTES
(9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4
antagonists such as NSC 651016 (a distamycin analog). Also included
are CCR213, CCR3, and CCR6 antagonists. Chemokine receptor agonists
such as RANTES, SDF-1, MIP-1.alpha., MIP-1.beta., etc., may also
inhibit fusion.
[0608] Additional antiretroviral agents that may be administered in
combination with it composition of the invention, include integrase
inhibitors. Integrase inhibitors include dicaffeoylquinie (DFQA)
acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid);
quinalizarin (QLC) and related anthraquinones; ZINTEVIR.TM. (AR
177, an oligonucleotide that probably acts at cell surface rather
than being a true integrate inhibitor; Arondex); and naphthols such
as those disclosed in WO 98/50347.
[0609] Additional antiretroviral agents that may be administered in
combination with a composition of the invention, include
hydroxyurea-like compounds such as BCX-34 (a purine nucleoside
phosphorylase inhibitor; Biocryst); ribonucleotide reductase
inhibitors such as DIDOX.TM. (Molecules for Health); inosine
monophosphate dehydrogenase (IMPDH) inhibitors such as VX-497
(Vertex); and mycopholic acids such as CellCept (mycophenolate
moferil; Roche).
[0610] Additional antiretroviral agents that may be administered in
combination with a composition of the invention, include inhibitors
of viral integrase, inhibitors of viral genome nuclear
translocation such as arylene bis(methylketone) compounds;
inhibitors of HIV entry such as AOP-RANTES, NNY-RANTES, RANTES-IgG
fusion protein, soluble complexes of RANTES and glycosaminoglycans
(GAG), and AMD-3100; nucleocapsid zinc finger inhibitors such as
dithiane compounds; targets of HIV Tat and Rev; and
pharmacoenhancers such as ABT-378.
[0611] Other antiretroviral therapies and adjunct therapies that
may be administered in combination with a composition of the
invention, include one or more cytokines and/or lymphokines such
as, MW-1.alpha., MIP-1.beta., SDP-1.alpha., IL-2, PROLEUKIN.TM.
(aldesleukin/L2-7001; Chiron), IL-4, IL-12, and IL-13; interferons
such as IFN-.alpha.2a; antagonists of TNFs, NF.kappa.B, GM-CSF,
M-CSF, and IL-10; agents that modulate immune activation such as
cyclosporin and prednisone; vaccines such as Remune.TM. (HIV
Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments,
bivalent (B/E) recombinant envelope glycoprotein, rgp120CM235, MN
rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL
protein, branched synthetic peptide derived from discontinuous
gp120 C3/C4 domain, fusion-competent immunogens, and Gag, Pol, Nef,
and Tat vaccines; gene-based therapies such as genetic suppressor
elements (GSEs; WO 98/54366), and intrakines (genetically modified
CC chemokines targeted to the ER to block surface expression of
newly synthesized CCR5 (Yang et ah, PNAS 94:11567-72 (1997); Chen
et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the
anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8,
PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and
RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b,
48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies,
anti-TNF-.alpha. antibodies, and monoclonal antibody 33A; aryl
hydrocarbon (Ali) receptor agonists and antagonists such as TCDD,
3,3',4,4',5-pentachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and
.alpha.-naphthoflavone (WO 98/30213); and antioxidants such as
.gamma.-L-glutamyl-L-cysteine ethyl ester (.gamma.-GCE; WO
99/56764).
[0612] In a further embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered in combination
with an antiviral agent, Antiviral agents that may be administered
with the albumin fusion proteins and/or polynucleotides of the
invention include, but are not limited to, acyclovir, ribavirin,
amantadine, remantidine, maxamine, or thymalfasin. Specifically,
interferon albumin fusion protein cats be administered in
combination with any of these agents. Moreover, interferon alpha
albumin fusion protein can also be administered with any of these
agents, and preferably, interferon alpha 2a or 2b albumin fusion
protein can be administered with any of these agents. Furthermore,
interferon beta albumin fusion protein can also be administered
with any of these agents. Additionally, any of the IFN hybrids
albumin fusion proteins can be administered in combination with any
of these agents.
[0613] In a most preferred embodiment, interferon albumin fusion
protein is administered in combination with ribavirin. In a further
preferred embodiment, interferon alpha albumin fusion protein is
administered in combination with ribavirin. In a further preferred
embodiment, interferon alpha 2a albumin fusion protein is
administered in combination with ribavirin. In a further preferred
embodiment, interferon alpha 2b albumin fusion protein is
administered in combination with ribavirin. In a further preferred
embodiment, interferon beta albumin fusion protein is administered
in combination with ribavirin. In a further preferred embodiment,
hybrid interferon albumin fusion protein is administered in
combination with ribavirin.
[0614] In other embodiments, a composition of the invention may be
administered in combination with anti-opportunistic infection
agents. Anti-opportunistic agents that may be administered in
combination with a composition of the invention, include, but are
not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM.,
PENTAMIDINE.TM., ATOVAQUONE.TM., ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., ETHAMBUTOL.TM., RIFABUTIN.TM.,
CLARITHROMYCIN.TM., AZITHROMYCIN.TM., GANCICLOVIR.TM.,
FOSCARNET.TM., CIDOFOVIR.TM., FLUCONAZOLE.TM., ITRACONAZOLE.TM.,
KETOCONAZOLE.TM., ACYCLOVIR.TM., FAMCICOLVIR.TM.,
PYRIMETHAMINE.TM., LEUCOVORIN.TM., NEUPOGEN.TM. (filgrastim/G-CSF),
and LEUKINE.TM. (sargramostim/GM-CSF). In a specific embodiment,
composition of the invention are used in any combination with
TRIMETHOPRIM-SULFAIVIETHOXAZOLE.TM., DAPSONE.TM., PENTAMIDINE.TM.,
and/or ATOVAQUONE.TM. to prophylactically treat or prevent an
opportunistic Pneumocystis carinii pneumonia infection. In another
specific embodiment, a composition of the invention is used in any
combination with ISONIAZID.TM., RIFAMPIN.TM., PYRAZINAMIDE.TM.,
and/or ETHAMBUTOL.TM. to prophylactically treat or prevent an
opportunistic Mycobacterium avium complex infection. In another
specific embodiment, a composition of the invention is used in any
combination with RIFABUTIN.TM., CLARITHROMYCIN.TM., and/or
AZITHROMYCIN.TM. to prophylactically treat or prevent an
opportunistic Mycobacterium tuberculosis infection. In another
specific embodiment, a composition of the invention is used in any
combination with GANCICLOVIR.TM., FOSCARNET.TM., and/or
CIDOFOVIR.TM. to prophylactically treat or prevent an opportunistic
cytomegalovirus infection. In another specific embodiment, a
Composition of the invention is used in any combination with
FLUCONAZOLE.TM., ITRACONAZOLE.TM., and/or KETOCONAZOLE.TM. to
prophylactically treat or prevent an opportunistic fungal
infection. In another specific embodiment, a Composition of the
invention is used in any combination with ACYCLOVIR.TM. and/or
FAMCICOLVIR.TM. to prophylactically treat or prevent an
opportunistic herpes simplex virus type I and/or type II infection.
In another specific embodiment, a Composition of the invention is
used in any combination with PYRIMETHAMINE.TM. and/or
LEUCOVORIN.TM. to prophylactically treat or prevent an
opportunistic Toxoplamsa gondii infection. In another specific
embodiment, a Composition of the invention is used in any
combination with LEUCOVORIN.TM. and/or NEUPOGEN.TM. to
prophylactically treat or prevent an opportunistic bacterial
infection.
[0615] In a further embodiment, a composition of the invention is
administered in combination with an antibiotic agent. Antibiotic
agents that may be administered with a composition of the invention
include, but are not limited to, amoxicillin, beta-lactamases,
aminoglycosides, beta-lactam (glycopeptide), beta-lactamases,
Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin,
erythromycin, fluoroquinolones, macrolides, metronidazole,
penicillins, quinolones, rapamycin, rifampin, streptomycin,
sulfonamide, tetracyclines, trimethoprim,
trimethoprim-sulfamethoxazole, and vancomycin.
[0616] In other embodiments, a composition of the invention is
administered in combination with one or more immunostimulants.
Immunostimulants that may be administered in combination with a
composition of the invention include, but are not limited to,
levamisole (e.g., ERGAMISOL.TM.), isoprinosine (e.g.,
INOSIPLEX.TM.), interferons (e.g., interferon alpha), and
interleukins (e.g., IL-2).
[0617] In other embodiments, a Composition of the invention (i.e.,
albumin fusion proteins and/or polynucleotides of the invention) is
administered in combination with one or more immunosuppressive
agents. Immunosuppressive agents that may be administered in
combination with a composition of the invention include, but are
not limited to, steroids, cyclosporine, cyclosporine analogs,
cyclophosphamide methylprednisone, prednisone, azathioprine,
FK-506, 15-deoxyspergualin, and other immunosuppressive agents that
act by suppressing the function of responding T cells. Other
immunosuppressive agents that may be administered in combination
with a composition of the invention include, but are not limited
to, prednisolone, methotrexate, thalidomide, methoxsalen,
rapamycin, leflunomide, mizoribine (BREDININ.TM.), brequinar,
deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT.RTM. 3
(muromonab-CD3), SANDIMMUNE.TM., NEORAL.TM., SANGDYA.TM.
(cyclosporine), PROGRAF.RTM. (FK506, tacrolimus), CELLCEPT.RTM.
(mycophenolate motefil, of which the active metabolite is
mycophenolic acid). IMURAN.TM. (azathioprine),
glucocorticosteroids, adrenocortical steroids such as DELTASONE.TM.
(prednisone) and HYDELTRASOL.TM. (prednisolone), FOLEX.TM. and
MEXATE.TM. (methotrexate), OXSORALEN-ULTRA.TM. (methoxsalen) and
RAPAMUNE.TM. (sirolimus). In a specific embodiment,
immunosuppressants may be used to prevent rejection of organs or
bone marrow transplantation.
[0618] In an additional embodiment, albumin fusion proteins and/or
polynucleotides of the invention are administered alone or in
combination with one or more intravenous immune globulin
preparations. Intravenous immune globulin preparations that may be
administered with the albumin fusion proteins and/or
polynucleotides of the invention include, but not limited to,
GAMMAR.TM., IVEEGAM.TM., SANDOGLOBULIN.TM., GAMMAGARD S/D.TM.,
ATGAM.TM. (antithymocyte glubulin), and GAMIMUNE.TM.. In a specific
embodiment, albumin fusion proteins and/or polynucleotides of the
invention are administered in combination with intravenous immune
globulin preparations in transplantation therapy (e.g., bone marrow
transplant).
[0619] In another embodiment, the albumin fusion proteins and/or
polynucleotides of the invention are administered alone or as part
of a combination therapy, either in also to patients or in vitro to
cells, for the treatment of cancer. In a specific embodiment, the
albumin fusion proteins, particularly IL-2-albumin fusions, are
administered repeatedly during passive immunotherapy for cancer,
such as adoptive cell transfer therapy for metastatic melanoma as
described in Dudley et al. (Science Express, 19 Sep. 2002 at
www.scienceexpress.org, hereby incorporated by reference in its
entirety).
[0620] In certain embodiments, a Compositions of the invention
(i.e. albumin fusion proteins and/or polynucleotides of the
invention) is administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that may be
administered with a composition of the invention include, but are
not limited to, corticosteroids (e.g., betamethasone, budesonide,
cortisone, dexamethasone, hydrocortisone, methylprednisolone,
prednisolone, prednisone, and triamcinolone), nonsteroidal
anti-inflammatory drugs (e.g., diclofenac, diflunisal, etodolac,
fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin,
ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone,
naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac,
tenoxicam, tiaprofenic acid, and tolmetin), as well as
antihistamines, aminoarylcarboxylic acid derivatives, arylacetic
acid derivatives, arylbutyric acid derivatives, arylcarboxylic
acids, arylpropionic acid derivatives, pyrazoles, pyrazolones,
salicylic acid derivatives, thiazinecarboxamides,
e-acetamidocaproic acid, S-adenosylmethionine,
3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,
bucoloine, difenpiramide, ditazol, emorfazone, guaiazulene,
nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
[0621] In an additional embodiment, a composition of the invention
is administered alone or in combination with art anti-angiogenic
anent Anti-angiogenic agents that may be administered with a
composition of the invention include, but are not limited to,
Angiostatin (Entremed, Rockville, Md.), Troponin-1 (Boston Life
Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and
derivatives thereof, paclitaxel (Taxol), Suramin, Tissue Inhibitor
of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,
VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator
inhibitor-2, and various forms of the lighter "d group" transition
metals.
[0622] Lighter "d group" transition metals include, for example,
vanadium, molybdenum, tungsten, titanium, niobium, and tantalum
species. Such transition metal species may form transition metal
complexes. Suitable complexes of the above-mentioned transition
metal species include oxo transition metal complexes.
[0623] Representative examples of vanadium complexes include oxo
vanadium complexes such as vanadate and vanadyl complexes. Suitable
vanadate complexes include metavanadate and orthovanadate complexes
such as, for example, ammonium metavanadate, sodium metavanadate,
and sodium orthovanadate. Suitable vanadyl complexes include, for
example, vanadyl acetylacetonate and vanadyl sulfate including
vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
[0624] Representative examples of tungsten and molybdenum complexes
also include oxo complexes. Suitable oxo tungsten complexes include
tungstate and tungsten oxide complexes. Suitable tungstate
complexes include ammonium tungstate, calcium tungstate, sodium
tungstate dihydrate, and tungstic acid. Suitable tungsten oxides
include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and
molybdenyl complexes, Suitable molybdate complexes include ammonium
molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium molybdate and its hydrates. Suitable molybdenum oxides
include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic
acid. Suitable molybdenyl complexes include, for example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes include hydroxo derivatives derived from, for example,
glycerol, tartaric acid, and sugars.
[0625] A wide variety of other anti-angiogenic (actors may also be
utilized within the context of the present invention.
Representative examples include, but are not limited to, platelet
factor 4; protamine sulphate; sulphated chitin derivatives
(prepared from queen crab shells), (Murata et al., Cancer Res.
51:22-2 6 (1991)); Sulphated Polysaccharide Peptidoglycan Complex
(SP-PG) (the function of this compound may be enhanced by the
presence of steroids such as estrogen, and tamoxifen citrate);
Staurosporine; modulators of matrix metabolism, including for
example, praline analogs, cishydroxyproline,
d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl,
aminopropionitrile fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate;
Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3
(Pavloff et al., J. Bio. Chem. 267:17321-17326 (1992)); Chymostatin
(Tomkinson et al., Biochem J. 286:475-480 (1992)); Cyclodextrin
Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et
al, Nature 348:555-557, (1990)); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446 (1987));
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol.
Chem. 262(4):1659-1664 (1987)); Bisantrene (National Cancer
Institute); Lobenzarit disodium
(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA";
(Takeuchi et al., Agents Actions 36:312-316, (1992)); and
metalloproteinase inhibitors such as BB94.
[0626] Additional anti-angiogenic factors that may also be utilized
within the context of the present invention include Thalidomide,
(Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (Brent et
al., J. Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta
3 antagonist (Storgard et al., J. Clin. Invest. 103:47-54 (1999));
carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer
Institute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE,
Boston, Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth
Meeting, Pa.); TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.);
ZD-0101 AstraZeneca (London, UK); APRA (CT2584); Benefin,
Byrostatin-1 (SC339555); CGP-41251 (PKC 412); CM101; Dexrazoxane
(ICRF187); DMXAA; Endostatin; Flavopridion Genestein; GTE; lmmTher;
Iressa (ZD1839); Octreotide (Somatostatin); Panretin;
Penacillamine; Photopoint; Prinomastat (AG-3340) Purlytin;
Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene;
Tetrathiomolybdate; Xeloda (Capecitabine); and 5-Fluorouracil.
[0627] Anti-angiogenic agents that may be administed in combination
with a compound of the invention may work through a variety of
mechanisms including, but not limited to, inhibiting proteolysis of
the extracellular matrix, blocking the function of endothelial
cell-extracellular matrix adhesion molecules, by antagonizing the
function of angiogenesis inducers such as growth factors, and
inhibiting integrin receptors expressed on proliferating
endothelial cells. Examples of antiangiogenic inhibitors that
interfere with extracellular matrix proteolysis and which may be
administered in combination with a composition of the invention
include, but are not limited to, AG-3340 (Agouron, La Jolla,
Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291
(Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East
Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and
Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenic
inhibitors that act by blocking the function of endothelial
cell-extracellular matrix adhesion molecules and which may be
administered in combination with a composition of the invention
include, but are not limited to, EMD 121974 (Merck KegaA Darmstadt,
Germany) and Vitaxin (Ixsys, La Jolla, Calif. CA/Medimmune,
Gaithersburg, Md.). Examples of anti-angiogenic agents that act by
directly antagonizing or inhibiting angiogenesis inducers and which
may be administered in combination with a composition of the
invention include, but are not limited to, Angiozyme (Ribozyme,
Boulder, Colo.), Anti-VEGF antibody (Genentech, S. San Francisco,
Calif.), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101
(Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn,
Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic
agents act to indirectly inhibit angiogenesis. Examples of indirect
inhibitors of angiogenesis which may be administered in combination
with a compositor of the invention include, but are not limited to,
IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche,
Nutley, N.J.), and Pentosan polysulfate (Georgetown University,
Washington, D.C.).
[0628] In particular embodiments, the use of a composition of the
invention in combination with one or more anti-angiogenic agents is
contemplated for the treatment (e.g., amelioration) and/or
prevention of an autoimmune disease, such as for example, an
autoimmune disease described herein.
[0629] In a particular embodiment, the use of a composition of the
invention in combination with one or more anti-angiogenic agents is
contemplated for the treatment (amelioration) and/or prevention of
arthritis. In a more particular embodiment, the use of a
composition of the invention in combination with anti-angiogenic
agents is contemplated for the treatment (amelioration) and/or
prevention of rheumatoid arthritis.
[0630] In another embodiment, the polynucleotides encoding a
polypeptide of the present invention are administered in
combination with an angiogenic protein, or polynucleotides encoding
an angiogenic protein. Examples of angiogenic proteins that may be
administered with the compositions of the invention include, but
are not limited to, acidic and basic fibroblast growth factors,
VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta,
platelet-derived endothelial cell growth factor, platelet-derived
growth factor, tumor necrosis factor alpha, hepatocyte growth
factor, insulin-like growth factor, colony stimulating factor,
macrophage colony stimulating factor, granulocyte/macrophage colony
stimulating factor, and nitric oxide synthase.
[0631] In additional embodiments, a composition of the invention is
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with a composition
of the invention include, but are not limited to alkylating agents
such as nitrogen mustards (for example, Mechlorethamine,
cyclophosphamide, Cyclophosphamide Ifosfamide, Melphalan
(L-sarcolysin), and Chlorambucil), ethylenimines and
methylmelamines (for example, Hexamethylmelamine and Thiotepa),
alkyl sulfonates (for example, Busulfan), nitrosoureas (for
example. Carmustine (BCNU), Lomustine (CCNU), Semustine
(methyl-CCNU), and Streptozocin (streptozotocin)), triazenes (for
example, Dacarbazine (DTIC; dimethyltriazenimidazolecarboxamide)),
folic acid analogs (for example, Methotrexate (amethopterin)),
pyrimidine analogs (for example, Fluorouacil (5-fluorouracil;
5-FU), Floxuridine (fluorodeoxyuridine; FudR), and Cytarabine
(cytosine arabinoside)), purine analogs and related inhibitors (for
example, Mercaptopurine (6-mercaptopurine; 6-MP), Thioguanine
(6-thioguanine; TG), and Pentostatin (2'-deoxycoformycin)), vinca
alkaloids (for example, Vinblastine (VLB, vinblastine sulfate)) and
Vincristine (vincristine sulfate)), epipodophyllotoxins (for
example, Etoposide and Teniposide), antibiotics (for example,
Dactinomycin (actinomycin D), Daunorubicin (daunomycin;
rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), and
Mitomycin (mitomycin C), enzymes (for example, L-Asparaginase),
biological response modifiers (for example, Interferon-alpha and
interferon-alpha-2b), platinum coordination compounds (for example,
Cisplatin (cis-DPP) and Carboplatin), anthracenedione
(Mitoxantrone), substituted cress (for example, Hydroxyurea),
methylhydrazine derivatives (for example, Procarbazine
(N-methylhydrazine; MIH), adrenocorticosteroids (for example,
Prednisone), progestins (for example, Hydroxyprogesterone caproate,
Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol
acetate), estrogens (for example, Diethylstilbestrol (DES),
Diethylstilbestrol diphosphate, Estradiol, and Ethinyl estradiol),
antiestrogens (for example, Tamoxifen), androgens (Testosterone
proprionate, and Fluoxymesterone), antiandrogens (for example,
Flutamide), gonadotropin-releasing hormone analogs (for example,
Leuprolide), other hormones and hormone analogs (for example,
methyltestosterone, estramustine, estramustine phosphate sodium,
chlorotrianisene, and testolactone), and others (for example,
dicarbazine, glutamic acid, and mitotane).
[0632] In one embodiment, a composition of the invention is
administered in combination with one or more of the following
drugs: infliximab (also known as Remicade.TM. Centocor, Inc.),
Trocade (Roche, RO-32-3555), Leflunomide (also known as Arava.TM.
from Hoechst Marion Roussel), Kineret.TM. (an IL-1 Receptor
antagonist also known as Anakinra from Amgen, Inc.)
[0633] In a specific embodiment, a composition of the invention is
administered in combination with CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) or combination of one or
more of the components of CHOP. In one embodiment, a compositions
of the invention is administered in combination with anti-CD20
antibodies, human monoclonal anti-CD20 antibodies. In another
embodiment, a composition of the invention is administered in
combination with anti-CD20 antibodies and CHOP, or anti-CD20
antibodies and any combination of one or more of the components of
CHOP, particularly cyclophosphamide and/or prednisone. In a
specific embodiment, a composition of the invention is administered
in combination with Rituximab. In a further embodiment, a
composition of the invention is administered with Rituximab and
CHOP, or Rituximab and any combination of one or more of the
components of CHOP, particularly cyclophosphamide and/or
prednisone. In a specific embodiment, a composition of the
invention is too administered in combination with tositumomab. In a
further embodiment, a composition of the invention is administered
with tositumomab and CHOP, or tositumomab and any combination of
one or more of the components of CHOP, particularly
cyclophosphamide and/or prednisone. The anti-CD20 antibodies may
optionally be associated with radioisotopes, toxins or cytotoxic
prodrugs.
[0634] In another specific embodiment, a composition of the
invention is administered in combination Zevalin.TM.. In a further
embodiment, a composition of the invention is administered with
Zevalin.TM. and CHOP, or Zevalin.TM. and any combination of one or
more of the components of CHOP, particularly cyclophosphamide
and/or prednisone. Zevalin.TM. may be associated with one or more
radisotopes. Particularly preferred isotopes are .sup.90Y and
.sup.111In.
[0635] In an additional embodiment, a composition of the invention
is administered in combination with one or more cytokines.
Cytokines that may be administered with a composition of the
invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6,
IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and
TNF-alpha. In another embodiment, a Composition of the invention
may be administered with an interleukin, including, but not limited
to, IL-1alpha, L-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, and IL-21.
[0636] In one embodiment, a composition of the invention is
administered in combination with one or more members of the TNF
family. TNF, TNF-related or TNF-like molecules that may be
administered with a composition of the invention include, but are
not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha
(LT-alpha, also known as TNF-beta), LT-beta (found in complex
heterotrimer LT-alpha2-beta). OPGL, FasL, CD27L, CD30L, CD40L,
4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO
96/14328), AIM-I (International Publication No. WO 97/33899),
endokine-alpha (International Publication No. WO 98/07880), GPO,
and neutrokine-alpha (International Publication No. WO 98/18921,
OX40, and nerve growth factor (NGF), and soluble forms of Fas,
CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO
96/34095), DR3 (International Publication No. WO 97/33904), DR4
(International Publication No. WO 98/32856), TR5 (International
Publication No. WO 98/30693), TRANK, TR9 (International Publication
No. WO 98/56892), TR10 (International Publication No. WO 98/54202),
312C2 (International Publication No. WO 98/06842), and TR12, and
soluble forms CD154, CD70, and CD153.
[0637] In an additional embodiment, a composition of the invention
is administered in combination with one or more angiogenic
proteins. Angiogenic proteins that may be administered with a
composition of the invention include, but are not limited to,
Glioma Derived Growth Factor (GDGF), as disclosed in European
Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A),
as disclosed in European Patent Number EP-682110; Platelet Derived
Growth Factor-B (PDGF-B), as disclosed in European Patent Number
EP-282317; Placental Growth Factor (PlGR), as disclosed in
International Publication Number WO 92/06194; Placental Growth
Factor-2 (PlGF-2), as disclosed in Hauser et al., Growth Factors,
4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as
disclosed in International Publication Number WO 90/13649; Vascular
Endothelial Growth Factor-A (VEGF-A), as disclosed in European
Patent Number EP-506477; Vascular Endothelial Growth Factor-2
(VEGF-2), as disclosed in International Publication Number WO
96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular
Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in
International Publication Number WO 96/26736; Vascular Endothelial
Growth Factor-D (VEG-D), as disclosed in International Publication
Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D),
as disclosed in International Publication Number WO 98/07832; and
Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in
German Patent Number DE19639601. The above mentioned references are
herein incorporated by reference in their entireties.
[0638] In an additional embodiment, a composition of the invention
is administered in combination with one or more Fibroblast Growth
Factors. Fibroblast Growth Factors that may be administered with a
composition of the invention include, but are not limited to,
FGF-1, FGF-2, FGF-3, FGF-4. FGF-5, FGF-6, FGF-7, FGF-8, FGF-9,
FOE-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
[0639] In an additional embodiment, a composition of the invention
is administered in combination with one or more hematopoietic
growth factors. Hematopoietic growth factors that may be
administered with a composition of the invention include, but are
not limited to, granulocyte macrophage colony stimulating factor
(GM-CSF) (sargramostim, LEUKINE.TM., PROKINE.TM.), granulocyte
colony stimulating factor (G-CSF) (filgrastim, NEUPOGEN.TM.),
macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin
(epoetin alfa, EPOGEN.TM., PROCRIT.TM.), stem cell factor (SCF,
c-kit ligand, steel factor), megakaryocyte colony stimulating
factor, PIXY321 (a GMCS/IL-3 fusion protein), interleukins,
especially any one or more of IL-1 through IL-12, interferon-gamma,
or thrombopoietin.
[0640] In certain embodiments, a composition of the invention is
administered in combination with one or more adrenergic blockers,
such as, for example, acebutolol, atenolol, betaxolol, bisoprolol,
carteolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol,
pindolol, propranolol, sotalol, and timolol.
[0641] In another embodiment, a composition of the invention is
administered in combination with an antiarrhythmic drug (e.g.,
adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin,
diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine,
moricizine, phenyloin, procainamide, N-acetyl procainamide,
propafenone, propranolol, quinidine, sotalol, tocainide, and
verapamil).
[0642] In another embodiment, a composition of the invention is
administered in combination with one or more diuretic agents, such
as carbonic anhydrase-inhibiting agents acetazolamide,
dichlorphenamide, and methazolamide), osmotic diuretics (e.g.,
glycerin, isosorbide, mannitol, and urea), diuretics that inhibit
Na.sup.+--K.sup.+-2Cl.sup.- symport (e.g., furosemide, bumetanide,
azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and
torsemide), thiazide and thiazide-like diuretics (e.g.,
bendroflumethiazide, benzthiazide, chlorothiazide,
hydrochlorothiazide, hydroflumethiazide, methyclothiazide,
polythiazide, trichormethiazide, chlorthalidone, indapamide,
metolazone, and quinethazone), potassium sparing diuretics (e.g.,
amiloride and triamterene), and mineralocorticoid receptor
antagonists (e.g., spironolactone, canrenone, and potassium
canrenoate).
[0643] In one embodiment, a composition of the invention is
administered in combination with one or more treatments for
endocrine and/or hormone imbalance disorders. Treatments for
endocrine and/or hormone imbalance disorders include, but are not
limited to, .sup.127I, radioactive isotopes of iodine such as
.sup.131I and .sup.173I; recombinant growth hormone, such as
HUMATROPE.TM. (recombinant somatropin); growth hormone analogs such
as PROTROPIN.TM. (somatrem); dopamine agonists such as PARLODEL.TM.
(bromocriptine); somatostatin analogs such as SANDOSTATIN.TM.
(octreotide); gonadotropin preparations such as PREGNYL.TM.,
A.P.L..TM. and PROFAST.TM. (chorionic gonadotropin (CG)),
PERGONAL.TM. (menotropins), and METRODIN.TM. (urofollitropin
(uFSH)); synthetic human gonadotropin releasing hormone
preparations such as FACTREL.TM. and LUTREPULSE.TM. (gonadorclin
hydrochloride); synthetic gonadotropin agonies such as LUPRON.TM.
(leuprolide acetate), SUPPRELIN.TM. (histrelin acetate),
SYNAREL.TM. (nafarelin acetate), and ZOLADEX.TM. (goserelin
acetate); synthetic preparations of thyrotropin-releasing hormone
such as RELEFACT TRH.TM. and THYPINONE.TM. (protirelin);
recombinant human TSH such as THYROGEN.TM.; synthetic preparations
of the sodium salts of the natural isomers of thyroid hormones such
as L-T.sub.4.TM., SYNTHROID.TM. and LEVOTHROID.TM. (levothyroxine
sodium), L-T.sub.3.TM., CYTOMEL.TM. and TRIOSTAT.TM. (liothyroine
sodium), and THYROLAR.TM. (liotrix); antithyroid compounds such as
6-n-propylthiouracil (propylthiouracil),
1-methyl-2-mercaptoimidazole and TAPAZOLE.TM. (methimazole),
NEO-MERCAZOLE.TM. (carbimazole); beta-adrenergic receptor
antagonists such as propranolol and esmolol; Ca.sup.2+ channel
blockers; dexamethasone and iodinated radiological contrast agents
such as TELEPAQUE.TM. (iopanoic acid) and ORAGRAFIN.TM. (sodium
ipodate).
[0644] Additional treatments for endocrine and/or hormone imbalance
disorders include, but are not limited to, estrogens or congugated
estrogens such as ESTRACE.TM. (estradiol), ESTINYL.TM. (ethinyl
estradiol), PREMARIN.TM., ESTRATAB.TM., ORTHO-EST.TM., OGEN.TM. and
estropipate (estrone), ESTROVIS.TM. (quinestrol), ESTRADERM.TM.
(estradiol), DELESTROGEN.TM. and VALERGEN.TM. (estradiol valerate),
DEPO-ESTRADIOL CYPIONATE.TM. and ESTROJECT LA.TM. (estradiol
cypionate); antiestrogens such as NOLVADEX.TM. (tamoxifen),
SEROPHENE.TM. and CLOMID.TM. (clomiphene); progestins such as
DURALUTIN.TM. (hydroxyprogesterone caproate), MPA.TM. and
DEPO-PROVERA.TM. (medroxyprogesterone acetate), PROVERA.TM. and
CYCRIN.TM. (MPA), MEGACE.TM. (megestrol acetate), NORLUTIN.RTM.
(norethindrone), and NORLUTATE.TM. and AYGESTIN.TM. (norethindrone
acetate); progesterone implants such as NORPLANT SYSTEM.TM.
(subdermal implants of norgestrel); antiprugestins such as RU
486.TM. (mifepristone); hormonal contraceptives such as ENOVID.TM.
(norethynodrel plus mestranol), PROGESTASERT.TM. (intrauterine
device that releases progesterone), LOESTRIN.TM., BREVICON.TM.,
MODICON.TM., GENORA.TM., NELONA.TM., NORINYL.TM., OVACON-35.TM. and
OVACON-50.TM. (ethinyl estradiol/norethindrone), LEVLEN.TM.,
NORDETTE.TM., TRI-LEVLEN.TM. and TRIPHASIL-21.TM. (ethinyl
estradiol/levonorgestrel) LO/OVRAL.TM. and OVRAL.TM. (ethinyl
estradiol/norgestrel), DEMULEN.TM. (ethinyl estradiol/ethynodiol
diacetate), NORINYL.TM., ORTHO-NOVUM.TM., NORETHIN.TM., GENORA.TM.,
and NELOVA.TM. (norethindrone/mestranol), DESOGEN.TM. and
ORTHO-CEPT.TM. (ethinyl estradiol/desogestrel), ORTHO-CYCLEN.TM.
and ORTHO-TRICYCLEN.TM. (ethinyl estradiol/norgestimate),
MICRONOR.TM. and NOR-QD.TM. (norethindrone), and OVRETTE.TM.
(norgestrel).
[0645] Additional treatments for endocrine and/or hormone imbalance
disorders include, but are not limited to, testosterone esters such
as methenolone acetate and testosterone undecanoate; parenteral and
oral androgens such as TESTOJECT-50.TM. (testosterone), TESTEX.TM.
(testosterone propionate), DELATESTRYL.TM. (testosterone
enanthate), DEPO-TESTOSTERONE.TM. (testosterone cypionate),
DANOCRINE.TM. (danazol). HALOTESTIN.TM. (fluoxymesterone), ORETON
METHYL.TM., TESTRED.TM. and VIRILON.TM. (methyltestosterone), and
OXANDRIN.TM. (oxandrolone); testosterone transdermal systems such
as TESTODERM.TM.; androgen receptor antagonist and
5-alpha-reductase inhibitors such as ANDROCUR.TM. (cyproterone
acetate), EULEXIN.TM. (flutamide), and PROSCAR.TM. (finasteride);
adrenocorticotropic hormone preparations such as CORTROSYN.TM.
(cosyntropin); adrenocortical steroids and their synthetic analogs
such as ACLOVATE.TM. (alclometasone dipropionate), CYCLOCORT.TM.
(amcinonide), BECLOVENT.TM. and VANCERIL.TM. (beclomethasone
dipropionate), CELESTONE.TM. (betamethasone), BENISONE.TM. and
UTICORT.TM. (betamethasone benzoate), DIPROSONE.TM. (betamethasone
dipropionate), CELESTONE PHOSPHATE.TM. (betamethasone sodium
phosphate), CELESTONE SOLUSPAN.TM. (betamethasone sodium phosphate
and acetate), BETA-VAL.TM. and VALISONE.TM. (betamethasone
valerate), TEMOVATE.TM. (clobetasol propionate), CLODERM.TM.
(clocortolone pivalate), CORTEF.TM. and HYDROCORTONE.TM. (cortisol
(hydrocortisone)), HYDROCORTONE ACETATE.TM. (cortisol
(hydrocortisone) acetate), LOCOID.TM. (cortisol (hydrocortisone)
butyrate), HYDROCORTONE PHOSPHATE.TM. (cortisol (hydrocortisone)
sodium phosphate), A-HYDROCORT.TM. and SOLU CORTEF.TM. (cortisol
(hydrocortisone) sodium succinate), WESTCORT.TM. (cortisol
(hydrocortisone) valerate). CORTISONE ACETATE.TM. (cortisone
acetate), DESOWEN.TM. and TRIDESILON.TM. (desonide), TOPICORT.TM.
(desoximetasone), DECADRON.TM. (dexamethasone), DECADRON LA.TM.
(dexamethasone acetate), DECADRON PHOSPHATE.TM. and HEXADROL
PHOSPHATE.TM. (dexamethasone sodium phosphate), FLORONE.TM. and
MAXIFLOR.TM. (diflorasone diacetate), FLORINEF ACETATE.TM.
(fludrocortisone acetate), AEROBID.TM. and NASALIDE.TM.
(flunisolide), FLUONID.TM. and SYNALAR.TM. (fluocinolone
acetonide), LIDEX.TM. (fluocinonide), FLUOR-OP.TM. and FML.TM.
(fluorometholone), CORDRAN.TM. (flurandrenolide). HALOG.TM.
(halcinonide), HMS LIZUIFILM.TM. (medrysone), MEDROL.TM.
(methylprednisolone), DEPO-MEDROL.TM. and MEDROL ACETATE.TM.
(methylprednisone acetate), A-METHAPRED.TM. and SOLUMEDROL.TM.
(methylprednisolone sodium succinate), ELOCON.TM. (mometasone
furoate), HALDRONE.TM. (paramethasone acetate), DELTA-CORTEF.TM.
(prednisolone), ECONOPRED.TM. (prednisolone acetate),
HYDELTRASOL.TM. (prednisolone sodium phosphate), HYDELTRA-T.B.A.TM.
(prednisolone tebutate), DELTASONE.TM. (prednisone). ARISTOCORT.TM.
and KENACORT.TM. (triamcinolone), KENALOG.TM. (triamcinolone
acetonide), ARISTOCORT.TM. and KENACORT DIACETATE.TM.
(triamcinolone diacetate), and ARISTOSPAN.TM. (triamcinolone
hexacetonide); inhibitors of biosynthesis and action of
adrenocortical steroids such as CYTADREN.TM. (aminoglutethimide),
NIZORAL.TM. (ketoconazole), MODRASTANE.TM. (trilostane), and
METOPIRONE.TM. (metyrapone); bovine, porcine or human insulin or
mixtures thereof; insulin analogs; recombinant human insulin such
as HUMULIN.TM. and NOVOLIN.TM.; oral hypoglycemic agents such as
ORAMIE.TM. and ORINASE.TM. (tolbutamide), DIABINESE.TM.
(chlorpropamide), TOLAMIDE.TM. and TOLINASE.TM. (tolazamide),
DYMELOR.TM. (acetohexamide), glibenclamide, MICRONASE.TM.,
DIBETA.TM. and GLYNASE.TM. (glyburide), GLUCOTROL.TM. (glipizide),
and DIAMICRON.TM. (gliclazide), GLUCOPHAGE.TM. (metformin),
ciglitazone, pioglitazone, and alpha-glucosidase inhibitors, bovine
or porcine glucagon; somatostatins such as SANDOSTATIN.TM.
(octreotide); and diazoxides such as PROGLYCEM.TM. (diazoxide).
[0646] Additional treatments for endocrine and/or hormone imbalance
disorders include, but are not limited to, bovine, porcine or human
insulin or mixtures thereof; insulin analogs; recombinant human
insulin such as HUMULIN.TM. and NOVOLIN.TM.; oral hypoglycemic
agents such as ORAMIDE.TM. and ORINASE.TM. (tolbutamide),
DIABINESE.TM. (chlorpropamide), TOLAMIDE.TM. and TOLINASE.TM.
(tolazamide), DYMELOR.TM. (acetohexamide), glibenclamide,
MICRONASE.TM., META.TM. and GLYNASE.TM. (glyburide), GLUCOTROL.TM.
(glipizide), and DIAMICRON.TM. (gliclazide), GLUCOPHAGE.TM.
(metformin), PRECOSE.TM. (acarbose), AMARYL.TM. (glimepiride), and
ciglitazone; thiazolidinediones (TZDs) such as rosiglitazone,
AVANDIA.TM. (rosiglitazone maleate) ACTOS.TM. (piogliatazone), and
troglitazone; alpha-glucosidase inhibitors; bovine or porcine
glucagon; somatostatins such as SANDOSTATIN.TM. (octreotide); and
diazoxides such as PROGLYCEM.TM. (diazoxide).
[0647] In still other embodiments, a composition of the invention
is administered in combination with one or more of the following: a
biguanide antidiabetic agent, a glitazone antidiabetic agent, and a
sulfonylurea antidiabetic agent.
[0648] In one embodiment, a composition of the invention is
administered in combination with one or more treatments for uterine
motility disorders. Treatments for uterine motility disorders
include, but are not limited to, estrogen drugs such as conjugated
estrogens (e.g., PREMARIN.RTM. and ESTRATAB.RTM.), estradiols
(e.g., CLIMARA.RTM. and ALORA.RTM.), estropipate, and
chlorotrianisene; progestin drugs (e.g., AMEN.RTM.
(medroxyprogesterone), MICRONOR.RTM. (norethidrone acetate),
PROMETRIUM.RTM. progesterone, and megestrol acetate); and
estrogen/progesterone combination therapies such as, for example,
conjugated estrogens/medroxyprogesterone (e.g., PREMPRO.TM. and
PREMPHASE.RTM.) and norethindrone acetate/ethinyl estsradiol
FEMHRT.TM.).
[0649] In an additional embodiment, a composition of the invention
is administered in combination with one or more drugs effective in
treating iron deficiency and hypochromic anemias, including but not
limited to, ferrous sulfate (iron sulfate, FEOSOL.TM.), ferrous
fumarase (e.g., FEOSTAT.TM.), ferrous gluconate (e.g., FERGON.TM.),
polysaccharide-iron complex (e.g., NIFEREX.TM.), iron dextran
injection (e.g., INFED.TM.), cupric sulfate, pyroxidine,
riboflavin, Vitamin B.sub.12, cyancobalamin injection (e.g.,
REDISOL.TM., RUBRAMIN PC.TM.), hydroxocobalamin, folic acid (e.g.,
FOLVITE.TM.), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum
factor) or WELLCOVORIN (Calcium salt of leucovorin), transferrin or
ferritin.
[0650] In certain embodiments, a composition of the invention is
administered in combination with one or more agents used to treat
psychiatric disorders, Psychiatric drugs that may be administered
with a composition of the invention include, but are not limited
to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene,
clozapine, fluphenazine, haloperidol, loxapine, mesoridazine,
molindone, olanzapine, perphenazine, pimozide, quetiapine,
risperidone, thioridazine, thiothixene, trifluoperazine, and
triflupromazine), antimanic agents (e.g., carbamazepine, divalproex
sodium, lithium carbonate, and lithium citrate), antidepressants
(e.g., amitriptyline, amoxapine, bupropion, citalopram,
clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine,
imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone,
nortriptyline, paroxetine, phenelzine, protriptyline, sertraline,
tranylcypromine, trazodone, trimipramine, and venlafaxine),
antianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide,
clorazepate, diazepam, halazepam, lorazepam, oxazepam, and
prazepam), and stimulants (e.g., d-amphetamine, methylphenidate,
and pemoline).
[0651] In other embodiments, a composition of the invention is
administered in combination with one or more agents used to treat
neurological disorders, Neurological agents that may be
administered with a composition of the invention include, but are
not limited to, antiepileptic agents (e.g., carbamazepine,
clonazepam, ethosuximide, phenobarbital, phenyloin, primidone,
valproic acid, divalproex sodium, felbamate, gabapentin,
lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate,
zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian
agents (e.g., levodopa/carbidopa, selegiline, amantidine,
bromocriptine, pergolide, ropinirole, pramipexole, benztropine;
biperiden; ethapropazine; procyclidine; trihexyphenidyl,
tolcapone), and ALS therapeutics (e.g., riluzole).
[0652] In another embodiment, a composition of the invention is
administered in combination with one or more vasodilating agents
and/or calcium channel blocking agents. Vasodilating agents that
may be administered with a composition of the invention include,
but are not limited to, Angiotensin Converting Enzyme (ACE)
inhibitors (e.g., papaverine, isoxsuprine, benazepril, captopril,
cilazapril, enalapril, enalaprilat, fosinopril, lisinopril,
moexipril, perindopril, quinapril, ramipril, spirapril
trandolapril, and nylidrin), and nitrates (e.g., isosorbide
dinitrate, isosorbide mononitrate, and nitroglycerin). Examples of
calcium channel blocking agents that may be administered in
combination with a composition of the invention include, but are
not limited to amlodipine, bepridil, diltiazem, felodipine,
flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and
verapamil.
[0653] In certain embodiments, a composition of the invention is
administered in combination with one or more treatments for
gastrointestinal disorders. Treatments for gastrointestinal
disorders that may be administered with a composition of the
invention include, but are trot limited to, H.sub.2 histamine
receptor antagonists (e.g., TAGAMET.TM. (cimetidine), ZANTAC.TM.
(ranitidine), PEPCID.TM. (famotidine), and AXID.TM. (nizatidine));
inhibitors of H.sup.+, K.sup.+ ATPase (e.g., PREVACID.TM.
(lansoprazole) and PRILOSEC.TM. (omeprazole)); Bismuth compounds
(e.g., PEPTO-BISMOL.TM. (bismuth subsalicylate) and DE-NOL.TM.
(bismuth subcitrate)); various antacids; sucralfate; prostaglandin
analogs (e.g., CYTOTEC.TM. (misoprostol)); muscarinic cholinergic
antagonists; laxatives (e.g., surfactant laxatives, stimulant
laxatives, saline and osmotic laxatives); antidiarrheal agents
(e.g., LOMOTIL.TM. (diphenoxylate), MOTOFEN.TM. (diphenoxin), and
IMODIUM.TM. (loperamide hydrochloride)), synthetic analogs of
somatostatin such as SANDOSTATIN.TM. (octreotide), antiemetic
agents (e.g., ZOFRAN.TM. (ondansetron), KYTRIL.TM. (granisetron
hydrochloride), tropisetron, dolasetron, metoclopramide,
chlorpromazine, perphenazine, prochlorperazine, promethazine,
thiethylperazine, triflupromazine, domperidone, haloperidol,
droperidol, trimethobenzamide, dexamethasone, methylprednisolone,
dronabinol, and nabilone); D2 antagonists (e.g., metoclopramide,
trimethobenzamide and chlorpromazine) bile salts; chenodeoxycholic
acid; ursodeoxycholic acid; and pancreatic enzyme preparations such
as pancreatin and pancrelipase.
[0654] In additional embodiments, a composition of the invention is
administered in combination with one or more other therapeutic or
prophylactic regimens, such as, for example, radiation therapy.
[0655] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions comprising albumin
fusion proteins of the invention. Optionally associated with such
container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0656] Gene Therapy
[0657] Constructs encoding albumin fusion proteins of the invention
can be used as a part of a gene therapy protocol to deliver
therapeutically effective doses of the albumin fusion protein. A
preferred approach for in vivo introduction of nucleic acid into a
cell is by use of a viral vector containing nucleic acid, encoding
an albumin fusion protein of the invention. Infection of cells with
a viral vector has the advantage that a large proportion of the
targeted cells can receive the nucleic acid. Additionally,
molecules encoded within the viral vector, e.g., by a cDNA
contained in the viral vector, are expressed efficiently in cells
which have taken up viral vector nucleic acid.
[0658] Retrovirus vectors and adeno-associated virus vectors can be
used as a recombinant gene delivery system for the transfer of
exogenous nucleic acid molecules encoding albumin fusion proteins
in vivo. These vectors provide efficient delivery of nucleic acids
into cells, and the transferred nucleic acids are stably integrated
into the chromosomal DNA of the host. The development of
specialized cell lines (termed "packaging cells") which produce
only replication-defective retroviruses has increased the utility
of retroviruses for gene therapy, and defective retroviruses we
characterized for use in gene transfer for gene therapy purposes
(for a review see Miller, A. D. (1990) Blood 76:27 1). A
replication defective retrovirus can be packaged into virions which
can be used to infect a target cell through the use of a helper
virus by standard techniques. Protocols for producing recombinant
retroviruses and for infecting cells in vitro or in vivo with such
viruses can be found in Current Protocols in Molecular Biology,
Ausubel, F. M. et al., (eds.) Greene Publishing Associates, (1989),
Sections 9.10-9.14 and other standard laboratory manuals.
[0659] Another viral gene delivery system useful in the present
invention uses adenovirus-derived vectors. The genome of an
adenovirus can be manipulated such that it encodes and expresses a
gene product of interest but is inactivated in terms of its ability
to replicate in a normal lytic viral life cycle. See, for example,
Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al.,
Science 252:431-434 (1991); and Rosenfeld et al., Cell 65:143-155
(1992). Suitable adenoviral vectors derived from the adenovirus
strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2,
Ad3, Ad7 etc.) are known to those skilled in the art. Recombinant
adenoviruses can be advantageous in certain circumstances in that
they are not capable of infecting nondividing cells and can be used
to infect a wide variety of cell types, including epithelial cells
(Rosenfeld et al., (1992) cited supra). Furthermore, the virus
particle is relatively stable and amenable to purification and
concentration, and as above, can be modified so as to affect the
spectrum of infectivity. Additionally, introduced adenoviral DNA
(and foreign DNA contained therein) is not integrated into the
genome of a host cell but remains episomal, thereby avoiding
potential problems that can occur as a result of insertional
mutagenesis in situations where introduced DNA becomes integrated
into the host genome (e.g., retroviral DNA). Moreover, the carrying
capacity of the adenoviral genome for foreign DNA is large (up to 8
kilobases) relative to other gene delivery vectors (Beriorer et
al., cited supra; Haj-Ahmand et al., J. Virol. 57:267 (1986)).
[0660] In another embodiment, non-viral gene delivery systems of
the present invention rely on endocytic pathways for the uptake of
the subject nucleotide molecule by the targeted cell. Exemplary
gene delivery systems of this type include liposomal derived
systems, poly-lysine conjugates, and artificial viral envelopes. In
a representative embodiment, a nucleic acid molecule encoding an
albumin fusion protein of the invention can be entrapped in
liposomes bearing positive charges on their surface (e.g.,
lipofectins) and (optionally) which are tagged with antibodies
against cell surface antigens of the target tissue (Mizuno et al.
(1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309;
Japanese patent application 1047381; and European patent
publication ERA-43075).
[0661] Gene delivery systems for a gene encoding an albumin fusion
protein of the invention can be introduced into a patient by any of
a number of methods. For instance, a pharmaceutical preparation of
the gene delivery system can be introduced systemically, e.g. by
intravenous injection, and specific transduction of the protein in
the target cells occurs predominantly from specificity of
transfection provided by the gene delivery vehicle, cell-type or
tissue-type expression due to the transcriptiopal regulatory
sequences controlling expression of the receptor gene, or a
combination thereof. In other embodiments, initial delivery of the
recombinant gene is more limited with introduction into the animal
being quite localized. Pot example, the gene delivery vehicle can
be introduced by catheter (see U.S. Pat. No. 5,328,470) or by
Stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3 054-3 05
7). The pharmaceutical preparation of the gene therapy construct
can consist essentially of the gene delivery system in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Where the albumin fusion
protein can be produced intact from recombinant cells, e.g.
retroviral vectors, the pharmaceutical preparation can comprise one
or more cells which produce the albumin fusion protein.
[0662] Additional Gene Therapy Methods
[0663] Also encompassed by the invention are gene therapy methods
for treating or preventing disorders, diseases and conditions. The
gene therapy methods relate to the introduction of nucleic acid
(DNA, RNA and antisense DNA or RNA) sequences into an animal to
achieve expression of an albumin fusion protein of the invention.
This method requires a polynucleotide which codes for an albumin
fusion protein of the present invention operatively linked to a
promoter and any other genetic elements necessary for the
expression of the fusion protein by the target tissue. Such gene
therapy and delivery techniques are known in the art see, for
example, WO90/11092, which is herein incorporated by reference.
[0664] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) comprising a promoter operably
linked to a polynucleotide encoding an albumin fusion protein of
the present invention ex vivo, with the engineered cells then being
provided to a patient to be treated with the fusion protein of the
present invention. Such methods are well-known in the art. For
example, see Belldegrun et al., J. Natl. Cancer Inst. 85: 207-216
(1993); Ferrantini et al., Cancer Research 53: 1107-1112 (1993);
Ferrantini et al., J. Immunology 153: 4604-4615 (1994); Kaido at
al., Int. J. Cancer 60: 221-229 (1995); Ogura et al. Cancer
Research 50: 5102-5106 (1990); Santodonato et al., Human Gene
Therapy 7:1-10 (1996); Santodonato et al., Gene Therapy 4:1246-1255
(1997); and Zhang et al., Cancer Gene Therapy 3: 31-38 (1996)),
which are herein incorporated by reference. In one embodiment, the
cells which are engineered are arterial cells. The arterial cells
may be reintroduced into the patient through direct injection to
the artery, the tissues surrounding the artery, or through catheter
injection.
[0665] As discussed in more detail below, the polynucleotide
constructs can be delivered by any method that delivers injectable
materials to the cells of an animal, such as, injection into the
interstitial space of tissues (heart, muscle, skin, lung, liver,
and the like). The polynucleotide constructs may be delivered in a
pharmaceutically acceptable liquid or aqueous carrier.
[0666] In one embodiment, polynucleotides encoding the albumin
fusion proteins of the present invention is delivered as a naked
polynucleotide. The term "naked" polynucleotide, DNA or RNA refers
to sequences that are free from any delivery vehicle that acts to
assist, promote or facilitate entry into the cell, including viral
sequences, viral particles, liposome formulations, lipofectin or
precipitating agents and the like. However, polynucleotides
encoding the albumin fusion proteins of the present invention can
also be delivered in liposome formulations and lipofectin
formulations and the like can be prepared by methods well known to
those skilled in the art. Such methods are described, for example,
in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are
herein incorporated by reference.
[0667] The polynucleotide vector constructs used in the gene
therapy method are preferably constructs that will not integrate
into the host genome nor will they contain sequences that allow for
replication, Appropriate vectors include pWLNEO, pSV2CAT, pOG44,
pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL
available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2
available from Invitrogen. Other suitable vectors will be readily
apparent to the skilled artisan.
[0668] Any strong promoter known to those skilled in the art can be
used for driving the expression of the polynucleotide sequence.
Suitable promoters include adenoviral promoters, such as the
adenoviral major late promoter; or heterologous promoters, such as
the cromegalovirus (CMV) promoter; the respiratory syncytial virus
(RSV) promoter; inducible promoters, such as the MMT promoter, the
metallothionein promoter; heat shock promoters; the albumin
promoter; the ApoA1 promoter; human globin promoters; viral
thymidine kinase promoters, such as the Herpes Simplex thymidine
kinase promoter; retroviral LTRs; the b-actin promoter; and human
growth hormone promoters. The promoter also may be the native
promoter for the gene corresponding to the Therapeutic protein
portion of the albumin fusion proteins of the invention.
[0669] Unlike other gene therapy techniques, one major advantage of
introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be
introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
[0670] The polynucleotide construct can be delivered to the
interstitial space of tissues within the an animal, including of
muscle, skin, brain, lung, liver, spleen, bone marrow, thymus,
heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, eye, gland, and connective tissue. Interstitial space of
the tissues comprises the intercellular, fluid, mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers
in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that same matrix within connective tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space
occupied by the plasma of the circulation and the lymph fluid of
the lymphatic channels. Delivery to the interstitial space of
muscle tissue is preferred for the reasons discussed below. They
may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells are particularly competent in their ability to take up
and express polynucleotides.
[0671] For the naked nucleic acid sequence injection, an effective
dosage amount of DNA or RNA will be in the range of from about 0.05
mg/kg body weight to about 50 mg/kg body weight. Preferably the
dosage will be front about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration.
[0672] The preferred rotate of administration is by the parenteral
route of injection into the interstitial space of tissues. However,
other parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
DNA constructs can be delivered to arteries during angioplasty by
the catheter used in the procedure.
[0673] The naked polynucleotides are delivered by any method known
in the art, including, but not limited to, direct needle injection
at the delivery site, intravenous injection, topical
administration, catheter infusion, and so-called "gene guns". These
delivery methods are known in the art.
[0674] The constructs may also be delivered with delivery vehicles
such as viral sequences, viral particles, liposome formulations,
lipofectin, precipitating agents, etc. Such methods of delivery are
known in the art.
[0675] In certain embodiments, the polynucleotide constructs are
complexed in a liposome preparation. Liposomal preparations for use
in the instant invention include cationic (positively charged),
anionic (negatively charged) and neutral preparations. However,
cationic liposomes are particularly preferred because a tight
charge complex can be formed between the cationic liposome and the
polyanionic nucleic acid. Cationic liposomes have been shown to
mediate intracellular delivery of plasmid DNA (Feigner et al.,
Proc. Natl, Acad. Sci. USA 84:7413-7416 (1987), which is herein
incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad.
Sci. USA 86:6077-6081 (1989), which is herein incorporated by
reference); and purified transcription factors (Debs et al., J.
Biol. Chem. 265:10189-10192 (1990), which is herein incorporated by
reference), in functional form.
[0676] Cationic liposomes are readily available. For example,
N[1-2,3 dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are particularly useful and are available tinder the trademark
Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner
et al., Proc. Natl. Acad, Sci. USA 84:7413-7416 (1987), which is
herein incorporated by reference). Other commercially available
liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
[0677] Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g. PCT Publication No. WO 90/11092 (which is herein incorporated
by reference) for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Preparation of DOTMA liposomes is explained in the literature, see,
e.g., Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413-7416
(1987), which is herein incorporated by reference. Similar methods
can be used to prepare liposomes from other cationic lipid
materials.
[0678] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPC), dioleoylphosphatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0679] For example, commercially dioleoylphosphatidyl choline
(DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphosphatidyl ethanolamine (DOPE) can be used in various
combinations to make conventional liposomes, with or without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be prepared by drying 50 mg each of DOPG and DOPC under a stream of
nitrogen gas into a sonication vial. The sample is placed under a
vacuum pump overnight and is hydrated the following day with
deionized water. The sample is then sonicated for 2 hours in a
capped vial, using a Heat Systems model 350 sonicator equipped with
an inverted cup (bath type) probe at the maximum setting while the
bath is circulated at 15EC. Alternatively, negatively charged
vesicles can be prepared without sonication to produce
multilamellar vesicles or by extrusion through nucleopore membranes
to produce unilamellar vesicles of discrete size. Other methods are
known and available to those of skill in the art.
[0680] The liposomes can comprise multilamellar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs), with SUVs being preferred. The various liposome-nucleic
acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983),
101:512-527, which is herein incorporated by reference. For
example, MLVs containing nucleic acid can be prepared by depositing
a thin film of phospholipid on the walls of a glass tube and
subsequently hydrating with a solution of the material to be
encapsulated. SUVs are prepared by extended sonication of MLVs to
produce a homogeneous population of unilamellar liposomes. The
material to be entrapped is added to a suspension of preformed MLVs
and then sonicated. When using liposomes containing cationic
lipids, the dried lipid film is resuspended in an appropriate
solution such as sterile water or an isotonic buffer solution such
as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are
mixed directly with the DNA. The liposome and DNA form a very
stable complex due to binding of the positively charged liposomes
to the cationic DNA. SUVs find use with small nucleic acid
fragments. LUVs are prepared by a number of methods, well known in
the art. Commonly used methods include Ca.sup.2+-EDTA chelation
(Papahadjopoulos et al., Biochim. Biophys. Acta 394:483 (1975);
Wilson et al., Cell 17:77 (1979)); ether injection (Deamer et al.,
Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem.
Biophys, Res. Commun. 76:836 (1977); Fraley et al., Proc. Natl.
Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch et al.,
Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase
evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980);
Szoka et al., Proc. Natl. Acad. Sci. USA 75:145 (1978);
Schaefer-Ridder et al., Science 215:166 (1982)), which are herein
incorporated by reference.
[0681] Generally, the ratio of DNA to liposomes will be from about
10:1 to about 1:10. Preferably, the ration will be from about 5:1
to about 1:5. More preferably, the ration will be about 3:1 to
about 1:3. Still more preferably, the ratio will be about 1:1.
[0682] U.S. Pat. No. 5,676,954 (which is herein incorporated by
reference) reports on the injection of genetic material, complexed
with cationic liposomes carriers, into mice, U.S. Pat. Nos.
4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859, 5,703,055, and international publication no. WO 94/9469
(which are herein incorporated by reference) provide cationic
lipids for use in transfecting DNA into cells and mammals, U.S.
Pat. Nos. 5,589,646, 5,693,622, 5.580,859, 5,703,055, and
international publication no. WO 94/9469 provide methods for
delivering DNA-cationic lipid complexes to mammals.
[0683] In certain embodiments, cells are engineered, ex vivo or in
vivo, using a retroviral particle containing RNA which comprises a
sequence encoding an albumin fusion protein of the present
invention. Retroviruses from which the retroviral plasmid vectors
may be derived include, but are not limited to, Moloney Murine
Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey
Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus,
human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and
mammary tumor virus.
[0684] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X,
VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Human Gene Therapy 1:5-14 (1990), which is
incorporated herein by reference in its entirety. The vector may
transduce the packaging cells through any means known in the art.
Such means include, but are not limited to, electroporation, the
use of liposomes, and CaPO.sub.4 precipitation. In one alternative.
The retroviral plasmid vector may be encapsulated into a liposome,
or coupled to a lipid, and then administered to a host.
[0685] The producer cell line generates infectious retroviral
vector particles which include polynucleotide encoding an albumin
fusion protein of the present invention. Such retroviral vector
particles then may be employed, to transduce eukaryotic cells,
either in vitro or in vivo. The transduced eukaryotic cells will
express a fusion protein of the present invention.
[0686] In certain other embodiments, cells are engineered, ex vivo
or in vivo, with polynucleotide contained in an adenovirus vector.
Adenovirus can be manipulated such that it encodes and expresses
fusion protein of the present invention, and at the same time is
inactivated in terms of its ability to replicate in a normal lytic
viral life cycle. Adenovirus expression is achieved without
integration of the viral DNA into the host cell chromosome, thereby
alleviating concerns about insertional mutagenesis. Furthermore,
adenoviruses have been used as live enteric vaccines for many years
with an excellent safety profile (Schwartz et al. Ant. Rev. Respir.
Dis. 109:233-238 (1974)). Finally, adenovirus mediated gene
transfer has been demonstrated in a number or instances including
transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton
rats (Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et
al., Cell 68:143-155 (1992)). Furthermore, extensive studies to
attempt to establish adenovirus as a causative agent in human
cancer were uniformly negative (Green et al., Proc. Natl. Acad. Sci
USA 76:6606 (1979)).
[0687] Suitable adenoviral vectors useful in the present invention
are described, for example, in Kozarsky and Wilson, Curt. Opin.
Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155
(1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature
365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein
incorporated by reference. For example, the adenovirus vector Ad2
is useful and can be grown in human 293 cells. These cells contain
the EI region of adenovirus and constitutively express EIa and EIb,
which complement the defective adenoviruses by providing the
products of the genes deleted from the vector. In addition to Ad2,
other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also
useful in the present invention.
[0688] Preferably, the adenoviruses used in the present invention
are replication deficient. Replication deficient adenoviruses
require the aid of a helper virus and/or packaging cell line to
form infectious particles. The resulting virus is capable of
infecting cells and can express a polynucleotide of interest which
is operably linked to a promoter, but cannot replicate in most
cells. Replication deficient adenoviruses may be deleted in one or
more of all or a portion of the following genes: E1a, E1b, E3, E4,
E2a, or L1 through L5.
[0689] In certain other embodiments, the cells are engineered, ex
vivo or in vivo, rising an adeno-associated virus (AAV). AAVs are
naturally occurring defective viruses that require helper viruses
to produce infectious particles (Muzycaka, N., Cure Topics in
Microbiol. Immunol. 158:97 (1992)). It is also one of the few
viruses that may integrate its DNA into non-dividing cells. Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate, but space for exogenous DNA is limited to about 4.5
kb. Methods for producing and using such AAVs are known in the art.
See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
[0690] For example, an appropriate AAV vector for use in the
present invention will include all the sequences necessary for DNA
replication, encapsidation, and host-cell integration. The
polynucleotide construct is inserted into the AAV vector using
standard cloning methods, such as those found in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press
(1989). The recombinant AAV vector is then transfected into
packaging cells which are infected with a helper virus, using any
standard technique, including lipofection, electroporation, calcium
phosphate precipitation, etc. Appropriate helper viruses include
adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses. Once the packaging cells are transfected and infected,
they will produce infectious AAV viral particles which contain the
polynucleotide construct. These viral particles are then used to
transduce eukaryotic cells, either ex vivo or in vivo. The
transduced cells will contain the polynucleotide construct
integrated into its genome, and will express a fusion protein of
the invention.
[0691] Another method of gene therapy involves operably associating
heterologous control regions and endogenous polynucleotide
sequences (e.g. encoding a polypeptide of the present invention)
via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670,
issued Jun. 24, 1997; international Publication No. WO 96/2941,
published Sep. 26, 1996; International Publication No. WO 94/12650,
published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); and Zijistra et al., Nature 342:435-438
(1989), which are herein incorporated by reference. This method
involves the activation of a gene, which is present in the target
cells, but which is not normally expressed in the cells, or is
expressed at a lower level than desired.
[0692] Polynucleotide constructs are made, using standard
techniques known in the art, which contain the promoter with
targeting sequences flanking the promoter. Suitable promoters are
described herein. The targeting sequence is sufficiently
complementary to an endogenous sequence to permit homologous
recombination of the promoter-targeting sequence with the
endogenous sequence. The targeting sequence will be sufficiently
near the 5' end of the desired endogenous polynucleotide sequence
so the promoter will be operably linked to the endogenous sequence
upon homologous re-combination.
[0693] The promoter and the targeting sequences can be amplified
using PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter. The amplified promoter and
targeting sequences are digested and ligated together.
[0694] The promoter-targeting sequence construct is delivered to
the cells, either as naked polynucleotide, or in conjunction with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, whole viruses, lipofection,
precipitating agents, etc., described in more detail above. The
promoter-targeting sequence can be delivered by any method,
included direct needle injection, intravenous injection, topical
administration, catheter infusion, particle accelerators, etc. The
methods are described in more detail below.
[0695] The promoter-targeting sequence construct is taken up by
cells. Homologous recombination between the construct and the
endogenous sequence takes place, such that an endogenous sequence
is placed under the control of the promoter. The promoter then
drives the expression of the endogenous sequence.
[0696] The polynucleotide encoding an albumin fusion protein of the
present invention may contain a secretory signal sequence that
facilitates secretion of the protein, Typically, the signal
sequence is positioned in the coding region of the polynucleotide
to be expressed towards or at the 5' end of the coding region. The
signal sequence may be homologous or heterologous to the
polynucleotide of interest and may be homologous or heterologous to
the cells to be transfected. Additionally, the signal sequence may
be chemically synthesized using methods known in the art.
[0697] Any mode of administration of any of the above-described
polynucleotides constructs can be used so long as the mode results
in the expression of one or more molecules in an amount sufficient
to provide a therapeutic effect. This includes direct needle
injection, systemic injection, catheter infusion, biolistic
injectors, particle accelerators "gene guns"), gelfoam sponge
depots, other commercially available depot materials, osmotic pumps
(e.g., Alza minipumps), oral or suppositorial solid (tablet or
pill) pharmaceutical formulations, and decanting or topical
applications during surgery. For example, direct injection of naked
calcium phosphate-precipitated plasmid into rat liver and rat
spleen or a protein-coated plasmid into the portal vein has
resulted in gene expression of the foreign gene in the rat livers
(Kaneda et al., Science 243:375 (1989)).
[0698] A preferred method of local administration is by direct
injection. Preferably, an albumin fusion protein of the present
invention complexed with a delivery vehicle is administered by
direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries
refers to injecting the composition centimeters and preferably,
millimeters within arteries.
[0699] Another method of local administration is to contact a
polynucleotide construct of the present invention in or around a
surgical wound. For example, a patient can undergo surgery and the
polynucleotide construct cart be coated on the surface of tissue
inside the wound or the construct can be injected into areas of
tissue inside the wound.
[0700] Therapeutic compositions useful in systemic administration,
include fusion proteins of the present invention complexed to a
targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
Liposomes comprising ligands for targeting the vehicle to a
particular site. In specific embodiments, suitable delivery
vehicles for use with systemic administration comprise liposomes
comprising albumin fusion proteins of the invention for targeting
the vehicle to a particular site.
[0701] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad. Sci. USA 189:1127711281 (1992), which is
incorporated herein by reference). Oral delivery can be performed
by complexing a polynucleotide construct of the present invention
to a carrier capable of withstanding degradation by digestive
enzymes in the gut of an animal. Examples of such carriers, include
plastic capsules or tablets, such as those known in the art.
Topical delivery can be performed by mixing a polynucleotide
construct of the present invention with a lipophilic reagent (e.g.,
DMSO) that is capable of passing into the skin.
[0702] Determining an effective amount of substance to be delivered
can depend upon a number of factors including, for example, the
chemical structure and biological activity of the substance, the
age and weight of the animal, the precise condition requiring
treatment and its severity, and the route of administration. The
frequency, of treatments depends upon a number of factors, such as
the amount of polynucleotide constructs administered per dose, as
well as the health and history of the subject. The precise amount,
number of doses, and timing of doses will be determined by the
attending physician or veterinarian.
[0703] Albumin fusion proteins of the present invention can be
administered to any animal, preferably to mammals and birds.
Preferred mammals include humans, dogs, cats, mice, rats, rabbits
sheep, cattle, horses and pigs, with humans being particularly
preferred.
[0704] Biological Activities
[0705] Assays described herein or otherwise known in the art may be
applied or routinely modified TO test for one or more biological
activities (e.g., as described in Table 1, column 2, and in
Examples 28-35, for example, Example 28: [.sup.3H]-2-Deoxyglucose
Uptake Assay, Example 29: In vitro Assay of [.sup.3H]-Thymidine
Incorporation into Pancreatic Cell-lines, Example 30; Assaying for
Glycosuria, Example 31: Occurrence of Diabetes in NOD Mice, Example
32: Histological Examination of NOT.) Mice, Example 33: Pancreatic
Beta-Cell Transplantation Combination Therapy, Example 34: In vivo
Mouse Model of NIDDM, Example 35: In vitro H4Ile SEAP Reporter
Assays Establishing Involvement in Insulin Action) of the albumin
fusion proteins of the invention (including fragments and/or
variants of the albumin fusion proteins of the invention) and/or of
the biologically active and/or therapeutically active fragments
and/or variants of the Therapeutic protein portion of albumin
fusion proteins of the invention. If an albumin fusion protein
and/of therapeutic protein portion of an albumin fusion protein
exhibits an activity in a particular assay, it is likely that the
Therapeutic protein corresponding to the fusion protein may be
involved in the diseases associated with the biological activity.
Thus, the fusion protein could be used to treat the associated
disease or disorder.
[0706] The present invention encompasses methods of treating (e.g.,
ameliorating) or preventing a disease, disorder and/or a condition
associated with the disease or disorder, comprising administering
to a patient in which such treatment or prevention is desired an
albumin fusion protein of the invention. In preferred embodiments,
the present invention encompasses a method of treating or
preventing a disease or disorder listed in the "Preferred
Indication Y" column of Table 1, comprising administering to a
patient in which such treatment or prevention is desired an albumin
fusion protein of the invention that comprises a Therapeutic
protein portion corresponding to a Therapeutic protein (or fragment
or variant thereof) disclosed in the "Therapeutic Protein; X"
column of Table 1 (in the same row as the disease or disorder to be
treated is listed in the "Preferred Indication Y" column of Table
1) in an amount effective to treat or prevent the disease or
disorder.
[0707] In certain embodiments, an albumin fusion protein of the
present invention may be used to diagnose and/or prognose diseases
and/or disorders associated with the tissue(s) in which the gene
corresponding to the Therapeutic protein portion of the fusion
protein of the invention is expressed.
[0708] In another preferred embodiment, the "Therapeutic protein"
portion of an albumin fusion protein of the invention and/or an
albumin fusion protein of the invention can be used to treat (e.g.,
ameliorate) or prevent a metabolic/endocrine disease or disorder.
In a highly preferred embodiment, the metabolic/endocrine disease
or disorder is diabetes and/or a condition associated with
diabetes. As a non-limiting example, a "Therapeutic protein" may be
one that regulates glucose uptake by cells, that binds specifically
to a particular cell type (e.g., normal adipocytes, myotubes,
hepatocytes, and pancreatic beta cells of the Islet of Langerhans,
and/or abnormal (e.g., cancer cell or insulin-resistant adipocytes,
myotubes, and hepatocytes)), that enhances insulin sensitivity in
insulin-responsive tissues, and/or that regulates hepatic glucose
output, and therefore may be used to target a compound (drug, or
cytotoxic agent) to that cell type specifically.
[0709] Thus, the present invention encompasses a method of treating
(e.g., ameliorating) or preventing a metabolic/endocrine disorder
comprising administering to a patient in which such treatment or
prevention is desired an albumin fusion protein of the
invention.
[0710] Malfunctioning of any step(s) in insulin secretion and/or
action can lead to many metabolic/endocrine disorders (e.g., the
dysregulation of oxygen utilization, adipogenesis, glycogenesis,
lipogenesis, glucose uptake, protein synthesis, thermogenesis, and
maintenance of the basal metabolic rate) and is associated with
diseases and/or disorders that include, but are not limited to,
hyperinsulinemia, insulin resistance, insulin deficiency,
hyperglycemia, hyperlipidemia, hyperketonemia, and diabetes. In
preferred embodiments, an albumin fusion protein of the invention
is administered to a patient (preferably a human) to treat or
prevent an insulin related disease or disorder, and/or a condition
associated with an insulin related disease or disorder. In specific
embodiments and albumin fusion protein of the invention is
administered to treat or prevent a disease, disorder, or condition,
characterized by a state of insulin resistance. Disorders
characterized by insulin resistance that can be treated (e.g.,
ameliorated), prevented, diagnosed, and/or prognosed using a fusion
protein of the invention include, but are not limited to, NIDDM,
obesity, hypertension, hyperglycemia, heart disease, renal failure,
androgen excess, and liver cirrhosis or liver disease, injury
and/or a complication associated with transplantation.
[0711] In further, specific embodiments, a fusion protein of the
invention is administered to treat or prevent hyperinsulinemia or a
disorder or a condition associated with hyperinsulinemia.
[0712] In another embodiment, the invention provides a method of
increasing the sensitivity of a cell to insulin comprising
contacting a cell with an albumin fusion protein of the invention.
In one embodiment, this method is performed in vitro. In another
embodiment this method is performed in vitro. In specific
embodiments, the cell contacted according to this method is a liver
cell, an adipocyte, a kidney cell, a skin cell, a bone cell, or a
skeletal muscle cell.
[0713] In a preferred embodiment, the "Therapeutic protein" portion
of an albumin fusion protein of the invention and/or an albumin
fusion protein of the invention can be used to treat (e.g.,
ameliorate) or prevent diabetes mellitus and/or a condition
associated with diabetes mellitus. Thus, in a preferred embodiment,
the present invention encompasses a method of treating (e.g.,
ameliorating) or preventing diabetes mellitus and/or one or more
conditions associated with diabetes mellitus, comprising
administering to a patient in which such treatment or prevention is
desired an albumin fusion protein of the invention.
[0714] In another preferred embodiment, the present invention
encompasses e method of treating (e.g., ameliorating) or preventing
a condition associated with diabetes mellitus, comprising
administering to a patient in which such a treatment or prevention
is desired an albumin fusion protein of the invention. Conditions
that may be treated or prevented using an albumin fusion protein of
the invention include, but are not limited to, obesity,
cardiovascular disease (e.g., heart disease, atherosclerosis,
microvascular disease, hypertension, stroke, and/or other diseases
and disorders as described in the "Cardiovascular Disorders"
section below), blood vessel blockage, gangrene, impotence (e.g.,
due to diabetic neuropathy or blood vessel blockage), diabetic
retinopathy, diabetic nephropathy, kidney disease (e.g., renal
failure, nephropathy and/or other diseases and disorders as
described in the "Renal Disorders" section below), diabetic
neuropathy, nerve disease and nerve damage (e.g., due to diabetic
neuropathy), vision impairment (e.g., diabetic retinopathy,
cataract, and blindness), seizures, mental confusion, drowsiness,
nonketotic hyperglycemic-hyperosmolar coma, dyslipidemia (e.g.,
hyperlipidemia), hyperglycemia, hyperketonemia, ketoacidosis,
endocrine disorders (e.g., hyperinsulinemia, insulin insensitivity,
insulin resistance, and/or an endocrine disorder as described in
the "Endocrine Disorders" section below), ulcers, impaired wound
healing, infection (e.g., an infectious disease or disorder as
described in the "Infectious Diseases" section below, especially of
the urinary tract and skin), carpal tunnel syndrome and Dupuytren's
contracture, and/or an immune system disease or disorder (e.g.,
anemia, impaired white blood cell function, and/or an immune system
disease or disorder as described in the "Immune Activity" section
below).
[0715] In a most preferred embodiment, the "Therapeutic protein"
portion of an albumin fusion protein of the invention and/or an
albumin fusion protein of the invention can be used to treat (e.g.,
ameliorate) or prevent type II diabetes and/or a condition
associated with type II diabetes. Thus, in a preferred embodiment,
tire present invention encompasses a method of treating (e.g.,
ameliorating) or preventing type II diabetes mellitus and/or one or
more conditions associated with type II diabetes mellitus,
comprising administering to a patient in which such treatment or
prevention is desired an albumin fusion protein of the
invention.
[0716] In another preferred embodiment, the present invention
encompasses a method of treating (e.g., ameliorating) or preventing
a condition associated with type II diabetes mellitus, comprising
administering to a patient in which such a treatment or prevention
is desired an albumin fusion protein of the invention. Conditions
that may be treated or prevented using an albumin fusion protein of
the invention include, but are not limited to, obesity,
cardiovascular disease (e.g., heart disease, atherosclerosis,
microvascular disease, hypertension, stroke, and/or other diseases
and disorders as described in the "Cardiovascular Disorders"
section below), blood vessel blockage, gangrene, impotence (e.g.,
due to diabetic neuropathy or blood vessel blockage), diabetic
retinopathy, diabetic nephropathy, kidney disease (e.g., renal
failure, nephropathy and/or other diseases and disorders as
described in the "Renal Disorders" section below), diabetic
neuropathy, nerve disease and nerve damage (e.g., due to diabetic
neuropathy), vision impairment (e.g., diabetic retinopathy,
cataract, and blindness), seizures, mental confusion, drowsiness,
nonketotic hyperglycemic-hyperosmolar coma, dyslipidemia (e.g.,
hyperlipidemia), hyperglycemia, hyperketonemia, ketoacidosis,
endocrine disorders (e.g., hyperinsulinemia, insulin insensitivity,
insulin resistance, and/or an endocrine disorder as described in
the "Endocrine Disorders" section below), ulcers, impaired wound
healing, infection (e.g., an infectious disease or disorder as
described in the "Infectious Diseases" section below, especially of
the urinary tract and skin), carpal tunnel syndrome and Dupuytren's
contracture, and/or an immune system disease or disorder (e.g.,
anemia, impaired white blood cell function, and/or an immune system
disease or disorder as described in the "Immune Activity" section
below).
[0717] In another preferred embodiment, the "Therapeutic protein"
portion of an albumin fusion protein of the invention and/or an
albumin fusion protein of the invention can be used to treat (e.g.,
ameliorate) or prevent type I diabetes and/or a condition
associated with type I diabetes. Accordingly, in another preferred
embodiment, the present invention encompasses a method of treating
(e.g., ameliorating) or preventing type I diabetes mellitus and/or
one or more conditions associated with type I diabetes mellitus,
comprising administering to a patient in which such treatment or
prevention is desired an albumin fusion protein of the
invention.
[0718] In another preferred embodiment, the present invention
encompasses a method of treating (e.g., ameliorating) or preventing
a condition associated with type I diabetes mellitus, comprising
administering to a patient in which such a treatment or prevention
is desired an albumin fusion protein of the invention. Conditions
that may be treated or prevented using art albumin fusion protein
of the invention include, but are not limited to, obesity,
cardiovascular disease (e.g., heart disease, atherosclerosis,
microvascular disease, hypertension, stroke, and/or other diseases
and disorders as described in the "Cardiovascular Disorders"
section below), blood vessel blockage, gangrene, impotence (e.g.,
due to diabetic neuropathy or blood vessel blockage), diabetic
retinopathy, diabetic nephropathy, kidney disease (e.g., renal
failure, nephropathy and/or other diseases and disorders as
described in the "Renal Disorders" section below), diabetic
neuropathy, nerve disease and nerve damage (e.g., due to diabetic
neuropathy), vision impairment (e.g., diabetic retinopathy,
cataract, and blindness), seizures, mental confusion, drowsiness,
nonketotic hyperglycemic-hyperosmolar coma, dyslipidemia e.g.,
hyperlipidemia), hyperglycemia, hyperketonemia, ketoacidosis,
endocrine disorders (e.g., hyperinsulinemia, insulin insensitivity,
insulin resistance, and/or an endocrine disorder as described in
the "Endocrine Disorders" section below), ulcers, impaired wound
healing, infection (e.g., an infectious disease or disorder as
described in the "Infectious Diseases" section below, especially of
the urinary tract and skin), carpal tunnel syndrome and Dupuytren's
contracture, and/or an immune system disease or disorder (e.g.,
anemia, impaired white blood cell function, and/or an immune system
disease or disorder as described in the "Immune Activity" section
below).
[0719] In air additional embodiment, a fusion protein of the
invention is administered to a patient (preferably a human) to
modulate (e.g., increase) the effect of insulin on blood glucose
levels.
[0720] In a most preferred embodiment, an albumin fusion protein of
the invention is administered to a patient to modulate or maintain
glucose homeostasis.
[0721] In a preferred embodiment, the present invention provides a
method of increasing glucose uptake in a cell using a fusion
protein of the invention, comprising contacting a fusion protein of
the invention with the cell in which an increase in glucose uptake
is desired. In specific embodiments, the invention provides a
method of increasing glucose uptake into a cell using a fusion
protein of the invention in vitro. In other specific embodiments,
the invention provides a method of increasing glucose uptake into a
cell using a fusion protein of the invention in vivo. In preferred
embodiments, the invention provides a method of increasing glucose
uptake into a adipose cells. In other preferred embodiments, the
invention provides a method of increasing glucose uptake into a
skeletal cell. In other preferred embodiments, the invention
provides a method of increasing glucose uptake into a liver
cells
[0722] In a preferred embodiment, a fusion protein of the invention
is used to treat or prevent a disease and disorder associated with
aberrant glucose metabolism or glucose uptake into cells.
[0723] In another preferred embodiment, a fusion protein of the
invention is administered to a patient (preferably a human) to
regulate glucose metabolism. In a highly preferred embodiment, a
fusion protein of the invention, is administered to a patient
(preferably a human) to increase glucose metabolism.
[0724] In one embodiment, an albumin fusion protein of the
invention is administered to a patient (preferably a human) to
lower glucose production in liver and/or other cells.
[0725] In another embodiment, a fusion protein of the invention is
administered to a patient (preferably a human) to reduce
gluconeogenesis in liver and/or other cells.
[0726] In one embodiment, the invention provides a method of
decreasing glucose production of a cell comprising contacting a
cell with a fusion protein of the invention. In one embodiment,
this method is performed in vitro. In another embodiment this
method is performed in vitro. In specific embodiments, the cell
contacted according to this method is a liver cell, an adipocyte, a
kidney cell, or a muscle cell.
[0727] In another preferred embodiment, a fusion protein of the
invention is administered to a patient (preferably a human) to
treat or prevent a disease or disorder selected from the group:
glycogen storage disease (e.g., glycogenoses), hepatitis,
gallstones, cirrhosis of the liver, degenerative or necrotic liver
disease, alcoholic liver diseases, fibrosis, liver regeneration,
and cholesterol metabolism, and hepatocarcinomas.
[0728] In a further preferred embodiment, a fusion protein of the
invention is administered to a patient to treat or prevent
hyperglycemia and/or a condition associated with hyperglycemia.
Conditions associated with hyperglycemia that can be treated or
prevented using fusion proteins of the invention include, for
example, type II and/or type I diabetes mellitus, obesity, kidney
disease or impaired kidney function, nerve disease and/or damage
(e.g., mononeuropathy, polyneuropathy, and malfunctioning of the
autonomic nervous system) retinopathy, cataract, heart disease,
hypertension, atherosclerosis, stroke, gangrene (e.g., of the feet
and hands), impotence, infections, cataract, impaired white blood
cell function, Carpal tunnel syndrome, Dupuytren's contracture, and
diabetic ketoacidosis. In a further preferred embodiment, a fusion
protein of the invention is administered in combination (e.g.,
concomitantly or serially) with a bolus administration of another
diabetes Therapeutic (e.g., a bolus insulin treatment at time of
food consumption) to modulate post-prandial hyperglycemia.
[0729] In other embodiments, embodiments, a fusion protein of the
invention is administered to a patient to treat or prevent
dyslipidemia or a condition associated with dyslipidemia.
[0730] In other embodiments, embodiments, a fusion protein of the
invention is administered to a patient to treat or prevent
hyperlipidemia or a condition associated with hyperlipidemia.
[0731] In other embodiments, embodiments, a fusion protein of the
invention is administered to a patient to treat or prevent
hyperketonemia or a condition associated with hyperketonemia.
[0732] In a highly preferred embodiment, a fusion protein of the
invention is administered to a patient to treat or prevent obesity
or a condition associated with obesity.
[0733] In a preferred embodiment, a fusion protein of the invention
is administered to a patient to limit weight gain.
[0734] In another preferred embodiment, a fusion protein of the
invention is administered to a patient for the patient to lose
weight.
[0735] In other preferred embodiments, embodiments, a fusion
protein of the invention is administered to a patient to suppress
appetite.
[0736] In other preferred embodiments, a fusion protein of the
invention is administered to a patient to increase appetite.
[0737] In a further embodiment, the present invention provides a
method of activating the leptin receptor using a fusion protein of
the invention. In specific embodiments, the invention provides a
method of activating the leptin receptor using a fusion protein of
the invention in vitro. In other specific embodiments, the
invention provides a method of activating the leptin receptor using
a fusion protein of the invention in vivo.
[0738] In other preferred embodiments, a fusion protein of the
invention is administered to a patient to alter or regulate
nutritional partitioning in the patient. In one embodiment, an
albumin fusion protein of the invention is administered according
to this method to reduce fat mass. In another embodiment, an
albumin fusion protein of the invention is administered according
to this method to increase muscle mass.
[0739] In other preferred embodiments, a fusion protein of the
invention is administered to a patient to promote weight gain.
[0740] In another preferred embodiment, a fusion protein of the
invention is used to treat or prevent cardiovascular disease. In
one embodiment, a fusion protein of the invention is used to treat
or prevent heart disease. In another embodiment, a fusion protein
of the invention is used to treat or prevent atherosclerosis. In
another embodiment, a fusion protein of the invention is used to
treat or prevent hypertension or a condition associated with
hypertension. In another embodiment, a fusion protein of the
invention is used to treat or prevent coronary artery disease or a
condition associated with coronary artery disease.
[0741] In another embodiment, a fusion protein of the invention can
be used to treat or prevent neurological diseases, including but
not limited to, Parkinson's disease, Alzheimer's disease, and/or as
described herein under the section heading "Neural Activity and
Neurological Diseases".
[0742] In another embodiment, a fusion protein of the invention is
used to treat or prevent a neuropathy, neural injury, or a
condition associated with a neuropathy or neural injury.
Neuropathies that can be treated or prevented using a composition
of the invention include, but are not limited to, retinopathy,
autonomic neuropathy, parasympathetic neuropathy, and
polyneuropathy. In a preferred embodiment, a composition of the
invention is used to treat or prevent a parasympathetic neuropathy
or parasympathetic neural injury and/or a condition associated with
parasympathetic neuropathy or parasympathetic neural injury. In a
highly preferred embodiment, a composition of the invention is used
to treat or prevent a hepatic parasympathetic neuropathy or hepatic
parasympathetic neural injury, and/or a condition associated with a
hepatic parasympathetic neuropathy or hepatic parasympathetic
neural injury.
[0743] In another preferred embodiment, the present invention
provides a method of differentiating pancreatic ductal epithelial
cells into insulin secreting pancreatic beta cells. In specific
embodiments, the invention provides a method of differentiating
pancreatic ductal epithelial cells into insulin secreting
pancreatic beta cells using a fusion protein of the invention in
vitro. In other specific embodiments, the invention provides a
method of differentiating pancreatic ductal epithelial cells into
insulin secreting pancreatic beta cells using a fusion protein of
the invention in vivo. In still another preferred embodiment, the
present invention provides a method of inducing proliferation of
insulin secreting pancreatic beta cells using a fusion protein of
the invention. In specific embodiments, the invention provides a
method of inducing proliferation of insulin secreting pancreatic
beta cells using a fusion protein of the invention in vitro. In
other specific embodiments, the invention provides a method of
inducing proliferation of insulin secreting pancreatic beta cells
using a fusion protein of the invention in vivo.
[0744] In a further preferred embodiment, the present invention
provides a method of activating GSK3 kinase activity using a fusion
protein of the invention. In specific embodiments, the invention
provides a method of activating GSK3 kinase activity using a fusion
protein of the invention in vitro. In other specific embodiments,
the invention provides a method of activating GSK3 kinase activity
using a fusion protein of the invention in vivo.
[0745] The fusion proteins of the invention are useful in the
treatment (e.g., amelioration), prevention, diagnosis, and/or
detection of diseases and/or disorders associated with activities
that include, but are not limited to, prohormone activation in the
pancreatic, muscle, and fat tissues, cellular signaling in the
pancreatic, muscle, and fat tissues, cellular proliferation in the
pancreas, cellular differentiation of the pancreatic ductal
epithelial cells and the fibroblasts in the stromal-vasculature in
fat fissile, cell migration, and neurotransmitter activity.
[0746] In preferred embodiments, fusion proteins of the present
invention may be used in the diagnosis, prognosis, prevention
and/or treatment of diseases and/or disorders relating to diseases
and disorders of the endocrine system (see, for example, "Endocrine
Disorders" section below), the nervous system (see, for example,
"Neurological Disorders" section below), the immune system (see,
for example, "Immune Activity" section below), respiratory system
(see, for example, "Respiratory Disorders" section below),
cardiovascular system (see, for example, "Cardiovascular Disorders"
section below), reproductive system (see, for example,
"Reproductive System Disorders" section below) digestive system
(see, for example, "Gastrointestinal Disorders" section below),
diseases and/or disorders relating to cell proliferation (see, for
example, "Hyperproliferative Disorders" section below), and/or
diseases or disorders relating to the blood (see, for example,
"Blood-Related Disorders" section below).
[0747] In another embodiment, a fusion protein of the invention is
used to treat or prevent inflammatory and/or autoimmune disorders,
including but not limited to, lupus, scleroderma, dermatomyositis,
and/or as described herein under the section heading "Immune
Activity".
[0748] In another embodiment, a fusion protein of the invention is
used to neat or prevent of a disease, disorder, and/or condition
involving aberrant cellular proliferation, including but not
limited to a preneoplastic disorder (e.g., hyperplasia, metaplasia,
and dysplasia), a neoplastic disorder (e.g., cancer of the liver,
lung, and colon), and/or as described herein under the section
headings "Hyperproliferative Disorders" and "Diseases at the
Cellular Level".
[0749] In a further embodiment, a fusion protein of the invention
is used to promote wound healing or tissue regeneration, such as
described below under the section headings "Wound Healing and
Epithelial Cell Proliferation" and "Regeneration".
[0750] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is an insulin albumin fusion protein.
[0751] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a GLP1 albumin fusion protein.
[0752] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is an Exendin-4 albumin fusion protein.
[0753] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a HLDOU18 albumin fusion protein.
[0754] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a resistin albumin fusion proteins.
[0755] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a leptin albumin fusion proteins.
[0756] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a HCEIP80 albumin fusion protein.
[0757] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is an IGF1 albumin fusion protein.
[0758] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is an IFNa albumin fusion protein.
[0759] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a RegIV albumin fusion protein.
[0760] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a HDRMI62 albumin fusion protein.
[0761] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a IL-22 albumin fusion protein.
[0762] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a IL-22 albumin fusion protein.
[0763] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is Interferon beta albumin fusion protein.
[0764] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a TR6 albumin fusion protein.
[0765] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is TNFR2 albumin fusion protein.
[0766] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is ACE2 inhibitor albumin fusion protein.
[0767] In a further preferred embodiment, the albumin fission
protein administered according to the methods of the present
invention is an interferon hybrid albumin fusion protein.
[0768] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a HWHGZ51 albumin fusion protein.
[0769] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a C17 albumin fusion protein.
[0770] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a somatostatin albumin fusion protein.
[0771] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a HDALV07 albumin fusion protein.
[0772] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a C peptide albumin fusion protein.
[0773] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a Wnt10b albumin fusion protein.
[0774] In a further preferred embodiment, the albumin fusion
protein administered according to the methods of the present
invention is a CART albumin fusion protein.
[0775] The fusion proteins of the present invention can be used in
the diagnosis, prognosis, prevention and/or treatment of one or
more diseases, disorders and/or conditions of the endocrine system
(see, for example, "Endocrine Disorders" section below). In other
embodiments, a fusion protein of the present invention can be used
in the diagnosis, prognosis, prevention, and/or treatment of one or
more diseases, disorders and/or conditions of the digestive system
(see, for example, "Gastrointestinal Disorders" section below). In
other embodiments, a fusion protein of the present invention can be
used in the diagnosis, prognosis, prevention, and/or treatment of
one or more diseases, disorders and/or conditions of the nervous
system (see, for example, "Neurological Disorders" section below),
the immune system (see, for example, "Immune Activity" section
below), respiratory system (see, for example, "Respiratory
Disorders" section below), cardiovascular system (see, for example,
"Cardiovascular Disorders" section below), reproductive system
(see, for example, "Reproductive System Disorders" section below),
diseases, disorders, and/or conditions relating to cell
proliferation (see, for example, "Hyperproliferative Disorders"
section below), and/or diseases, disorders and/or conditions of the
blood (see, for example, "Blood-Related Disorders" section
below).
[0776] In certain embodiments, an albumin fusion protein of the
present invention may be used to diagnose and/or prognose diseases
and/or disorders associated with the tissue(s) in which the gene
corresponding to the Therapeutic protein portion of the fusion
protein of the invention is expressed.
[0777] Thus, fusion proteins of the invention and polynucleotides
encoding albumin fusion proteins of the invention are useful in the
diagnosis, detection and/or treatment of diseases and/or disorders
associated with activities that include, but are not limited to,
prohormone activation, neurotransmitter activity, cellular
signaling, cellular proliferation, cellular differentiation, and
cell migration.
[0778] More generally, fusion proteins of the invention and
polynucleotides encoding albumin fusion proteins of the invention
may be useful for the diagnosis, prognosis, prevention and/or
treatment of diseases and/or disorders associated with the
following systems.
[0779] Endocrine Disorders
[0780] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be used to treat, prevent, diagnose, and/or prognose disorders
and/or diseases related to hormone imbalance, and/or disorders or
diseases of the endocrine system.
[0781] Hormones secreted by the glands of the endocrine system
control physical growth, sexual function, metabolism, and other
functions. Disorders may be classified in two ways: disturbances in
the production of hormones, and the inability of tissues to respond
to hormones. The etiology of these hormone imbalance or endocrine
system diseases, disorders or conditions may be genetic, somatic,
such as cancer and some autoimmune diseases, acquired (e.g., by
chemotherapy, injury or toxins), or infectious. Moreover, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention can be used as a marker or
detector of a particular disease or disorder related to the
endocrine system and/or hormone imbalance.
[0782] Endocrine system and/or hormone imbalance and/or diseases
encompass disorders of uterine motility including, but not limited
to: complications with pregnancy and labor (e.g., pre-term labor,
post-term pregnancy, spontaneous abortion, and slow or stopped
labor): and disorders and/or diseases of the menstrual cycle (e.g.,
dysmenorrhea and endometriosis).
[0783] Endocrine system and/or hormone imbalance disorders and/or
diseases include disorders and/or diseases of the pancreas, such
as, for example, diabetes mellitus, diabetes insipidus, congenital
pancreatic agenesis, pheochromocytoma--islet cell tumor syndrome;
disorders and/or diseases of the adrenal glands such as, for
example, Addison's Disease, corticosteroid deficiency, virilizing
disease, hirsutism, Cushing's Syndrome, hyperaldosteronism,
pheochromocytoma; disorders and/or diseases of the pituitary gland,
such as, for example, hyperpituitarism, hypopituitarism, pituitary
dwarfism, pituitary adenoma, panhypopituitarism, acromegaly,
gigantism; disorders and/or diseases of the thyroid, including but
not limited to, hyperthyroidism, hypothyroidism, Plummer's disease,
Graves' disease (toxic diffuse goiter), toxic nodular goiter,
thyroiditis (Hashimoto's thyroiditis, subacute granulomatous
thyroiditis, and silent lymphocytic thyroiditis), Pendred's
syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone
coupling defect, thymic aplasia, Hurthle cell tumours of the
thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid
carcinoma; disorders and/or diseases of the parathyroid, such as,
for example, hyper parathyroidism, hypoparathyroidism; disorders
and/or diseases of the hypothalamus.
[0784] In addition, endocrine system and/or hormone imbalance
disorders and/or diseases may also include disorders and/or
diseases of the testes or ovaries, including cancer. Other
disorders and/or diseases of the testes or ovaries further include,
for example, ovarian cancer, polycystic ovary syndrome,
Klinefelter's syndrome, vanishing testes syndrome (bilateral
anorchia), congenital absence of Leydig's cells, cryptorchidism,
Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the
testis (benign), neoplasias of the testis and neo-testis.
[0785] Moreover, endocrine system and/or hormone imbalance
disorders and/or diseases may also include disorders and/or
diseases such as, for example, polyglandular deficiency syndromes,
pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and
disorders and/or cancers of endocrine tissues.
[0786] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention, may be used to diagnose, prognose, prevent,
and/or treat endocrine diseases and/or disorders associated with
the tissue(S) in which the Therapeutic protein corresponding to the
Therapeutic protein portion of the albumin protein of the invention
is expressed.
[0787] Gastrointestinal Disorders
[0788] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be used to treat, prevent, diagnose, and/or prognose
gastrointestinal disorders, including inflammatory diseases and/or
conditions, infections, cancers (e.g., intestinal neoplasms
(carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of
the small intestine, small bowl lymphoma)), and ulcers, such as
peptic ulcers.
[0789] Gastrointestinal disorders include dysphagia, odynophagia,
inflammation of the esophagus, peptic esophagitis, gastric reflux,
submucosal fibrosis and structuring. Mallory-Weiss lesions,
leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric
retention disorders, gastroenteritis, gastric atrophy,
gastric/stomach cancers, polyps of the stomach, autoimmune
disorders such as pernicious anemia, pyloric stenosis, gastritis
(bacterial, viral, eosinophilic, stress-induced, chronic erosive,
atrophic, plasma cell, and Menetrier's), and peritoneal diseases
chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric
lymphadenitis, mesenteric vascular occlusion, panniculitis,
neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess).
[0790] Gastrointestinal disorders also include disorders associated
with the small intestine, such as malabsorption syndromes,
distension, irritable bowel syndrome, sugar intolerance, celiac
disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's
disease, intestinal lymphangiectasia, Crohn's disease,
appendicitis, obstructions of the ileum, Meckel's diverticulum,
multiple diverticula, failure of complete rotation of the small and
large intestine, lymphoma, and bacterial and parasitic diseases
(such as Traveler's diarrhea, typhoid and paratyphoid, cholera,
infection by Roundworms (Ascariasis lumbricoides), Hookworms
(Ancylostoma duodenale), Threadworms (Enterobius vermicuraris),
Tapeworms (Taenia saginata, Echinococcus granulosus,
Diphyllobothrium spp., and T. solium).
[0791] Liver diseases and/or disorders include intrahepatic
cholestasis (alagille syndrome, biliary liver cirrhosis), fatty
liver (alcoholic fatty liver, reye syndrome), hepatic vein
thrombosis, hepatolentricular degeneration, hepatomegaly,
hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension
(esophageal and gastric varices), liver abscess (amebic liver
abscess), liver cirrhosis (alcoholic, biliary and experimental),
alcoholic liver diseases (fatty liver, hepatitis, cirrhosis),
parasitic (hepatic echinococcosis, fascioliasis, amebic liver
abscess), jaundice (hemolytic, hepatocellular, and cholestatic),
cholestasis, portal hypertension, liver enlargement, ascites,
hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis
(autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced),
toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B,
hepatitis C, hepatitis D, hepatitis E), Wilson's disease,
granulomatous hepatitis, secondary biliary cirrhosis, hepatic
encephalopathy, portal hypertension, varices, hepatic
encephalopathy, primary biliary cirrhosis, primary sclerosing
cholangitis, hepatocellular adenoma, hemangionias, bile stones,
liver failure (hepatic encephalopathy, acute liver failure), and
liver neoplasms (augiomyolipoma, calcified liver metastases, cystic
liver metastases, epithelial tumors, fibrolamellar hepatocarcinoma,
focal nodular hyperplasia, hepatic adenoma, hepatobiliary
cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma,
liver cancer, liver hemangioendothelioma, mesenchymal hamartoma,
mesenchymal tumors of liver, nodular regenerative hyperplasia,
benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver
disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal
tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma,
Hemangioma, Peliosis hepatis, Lipomas, inflammatory pseudotumor,
Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct
hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular
hyperplasia, Nodular regenerative hyperplasia)], malignant liver
tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma,
cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors
of blood vessels, angiosarcoma, Karposi's sarcoma,
hemangioendothelioma, other tumors, embryonal sarcoma,
fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma,
teratoma, carcinoid, squamous carcinoma, primary lymphomal]),
peliosis hepatis, erythrohepatic porphyria, hepatic porphyria
(acute intermittent porphyria, porphyria cutanea tarda), Zellweger
syndrome).
[0792] Pancreatic diseases and/or disorders include acute
pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis,
alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas,
cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic
neoplasms, islet-cell tumors, pancreoblastoma), and other
pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic
pseudocyst, pancreatic fistula, insufficiency)).
[0793] Gallbladder diseases include gallstones (cholelithiasis and
choledocholithiasis), postcholecystectomy syndrome, diverticulosis
of the gallbladder, acute cholecystitis, chronic cholecystitis,
bile duct tumors, and mucocele.
[0794] Diseases and/or disorders of the large intestine include
antibiotic-associated colitis, diverticulitis, ulcerative colitis,
acquired megacolon, abscesses, fungal and bacterial infections,
anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases
(colitis, colonic neoplasms [colon cancer, adenomatous colon polyps
(e.g., villous adenoma), colon carcinoma, colorectal cancer],
colonic diverticulitis, colonic diverticulosis, megacolon
[Hirschsprung disease, toxic megacolon]; sigmoid diseases
[proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease,
diarrhea (infantile diarrhea, dysentery), duodenal diseases
(duodenal neoplasms, duodenal obstruction, duodenal ulcer,
duodenitis), enteritis (enterocolitis), HIV enteropathy, deal
diseases (ileal neoplasms, ileitis), immune-proliferative small
intestinal disease, inflammatory bowel disease (ulcerative colitis,
Crohn's disease), intestinal atresia, parasitic diseases
(anisakiasis, balantidiasis, blastocystis infections,
cryptosporidiosis, dientarnoebiasis, amebic dysentery, giardiasis),
intestinal fistula (rectal fistula), intestinal neoplasms (cecal
neoplasms, colonic neoplasms, duodenal neoplasms, deal neoplasms,
intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal
obstruction (afferent loop syndrome, duodenal obstruction, impacted
feces, intestinal pseudo-obstruction [cecal volvulus],
inussusception), intestinal perforation, intestinal polyps (colonic
polyps, gardner syndrome, peutz-jeghers syndrome), jejunal diseases
(jejunal neoplasms), malabsorption syndromes (blind loop syndrome,
celiac disease, lactose intolerance, short bowl syndrome, tropical
sprue, whipple's disease), mesenteric vascular occlusion,
pneumatosis cystoides intestinalis, protein-losing enteropathies
(intestinal lymphagiectasis), rectal diseases (anus diseases, fecal
incontinence, hemorrhoids, proctitis, rectal fistula, rectal
prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic
esophagitis, hemorrhage, perforation, stomach ulcer,
Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping
syndrome), stomach diseases (e.g., achlorhydria, duodenogastric
reflux (bile reflux), gastric antral vascular ectasia, gastric
fistula, gastric outlet obstruction, gastritis (atrophic or
hypertrophic), gastroparesis, stomach dilatation, stomach
diverticulum, stomach neoplasms (gastric cancer, gastric polyps,
gastric adenocarcinoma, hyperplastic gastric polyp), stomach
rupture, stomach ulcer, stomach volvulus), tuberculosis,
visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum,
postoperative nausea and vomiting) and hemorrhagic colitis.
[0795] Further diseases and/or disorders of the gastrointestinal
system include biliary tract diseases, such as, gastroschisis,
fistula (e.g., biliary fistula, esophageal fistula, gastric
fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g.,
biliary tract neoplasms, esophageal neoplasms, such as
adenocarcinoma of the esophagus, esophageal squamous cell
carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such
as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the
pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and
peritoneal neoplasms), esophageal disease (e.g., bullous diseases,
candidiasis, glycogenic acanthosis, ulceration, barren esophagus
varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum),
fistula (e.g., tracheoesophageal fistula), motility disorders
(e.g., CREST syndrome, deglutition disorders, achalasia, spasm,
gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave
syndrome, Mallory-Weiss syndrome), stenosis, esophagitis,
diaphragmatic hernia (e.g., hiatal henna); gastrointestinal
diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk
virus infection), hemorrhage (e.g., hematemesis, melena, peptic
ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric
polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g.,
congenital diaphragmatic hernia, femoral hernia, inguinal hernia,
obturator hernia, umbilical hernia, ventral hernia), and intestinal
diseases (e.g., cecal diseases (appendicitis, cecal
neoplasms)).
[0796] Regeneration
[0797] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
can be used to differentiate, proliferate, and attract cells,
leading to the regeneration of tissues. (See, Science 276:59-87
(1997)). The regeneration of tissues could be used to repair,
replace, or protect tissue damaged by congenital defects, trauma
(wounds, burns, incisions, or ulcers), age, disease (e.g.
osteoporosis, osteocarthritis, periodontal disease, liver failure),
surgery, including cosmetic plastic surgery, fibrosis, reperfusion
injury, or systemic cytokine damage.
[0798] Tissues that could be regenerated using the present
invention include organs (e.g., pancreas, liver, intestine, kidney,
skin, endothelium), muscle (smooth, skeletal or cardiac),
vasculature (including vascular and lymphatics), nervous,
hematopoietic, and skeletal (bone, cartilage, tendon, and ligament)
tissue, Preferably, regeneration occurs without or decreased
scarring. Regeneration also may include angiogenesis.
[0799] Moreover, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may increase regeneration of tissues difficult to heal. For
example, increased tendon/ligament regeneration would quicken
recovery time after damage. Albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention could also be used prophylactically in an effort
to avoid damage. Specific diseases that could be treated include of
tendinitis, carpal tunnel syndrome, and other tendon or ligament
defects. A further example of tissue regeneration of non-healing
wounds includes pressure ulcers, ulcers associated with vascular
insufficiency, surgical, and traumatic wounds.
[0800] Similarly, nerve and brain tissue could also be regenerated
by using fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, to proliferate
and differentiate nerve cells. Diseases that could be treated using
this method include central and peripheral nervous system diseases,
neuropathies, or mechanical and traumatic disorders (e.g. spinal
cord disorders, head trauma, cerebrovascular disease, and stoke).
Specifically, diseases associated with peripheral nerve injuries,
peripheral neuropathy (e.g., resulting from chemotherapy or other
medical therapies), localized neuropathies, and central nervous
system diseases (e.g. Alzheimer's disease, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome), could all be treated using the albumin fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention.
[0801] Wound Healing and Epithelial Cell Proliferation
[0802] In accordance with yet is further aspect of the present
invention, there is provided a process for utilizing fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, for therapeutic purposes, for
example, to stimulate epithelial cell proliferation and basal
keratinocytes for the purpose of wound healing, and to stimulate
hair follicle production and healing of dermal wounds. Albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention, may be clinically useful
in stimulating wound healing including surgical wounds, excisional
wounds, deep wounds involving damage of the dermis and epidermis,
eye tissue wounds, dental tissue wounds, oral cavity wounds,
diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers,
venous stasis ulcers, burns resulting from heat exposure or
chemicals, and other abnormal wound healing conditions such as
uremia, malnutrition, vitamin deficiencies and complications
associated with systemic treatment with steroids, radiation therapy
and antineoplastic drugs and antimetabolites. Albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, could be used to promote dermal
reestablishment subsequent to dermal loss
[0803] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could be used to increase the adherence of skin grafts to a wound
bed and to stimulate re-epithelialization from the wound bed. The
following are types of grafts that fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, could be used to increase adherence to a wound bed;
autografts, artificial skin, allografts, autodermic graft,
autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone
graft, brephoplastic grafts, cubs graft, delayed graft, dermic
graft, epidermic graft, fascia graft, full thickness graft,
heterologous graft, xenograft, homologous graft, hyperplastic
graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch
graft, omenpal graft, patch graft, pedicle graft, penetrating
graft, split skin graft, thick split graft. Albumin fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention, can be used to promote skin strength and
to improve the appearance of aged skin.
[0804] It is believed that fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
will also produce changes in hepatocyte proliferation, and
epithelial cell proliferation in the lung, breast, pancreas,
stomach, small intestine, and large intestine. Albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, could promote proliferation of
epithelial cells such as sebocytes, hair follicles, hepatocytes,
type II pneumocytes, mucin-producing goblet cells, and other
epithelial cells and their progenitors contained within the skin,
lung, liver, and gastrointestinal tract. Albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention, may promote proliferation of endothelial
cells, keratinocytes, and basal keratinocytes.
[0805] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could also be used to reduce the side effects of gut toxicity that
result from radiation, chemotherapy treatments or viral infections.
Albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, may have a
cytoprotective effect on the small intestine mucosa. Albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, may also stimulate healing of
mucositis (mouth ulcers) that result from chemotherapy and viral
infections.
[0806] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could further be used in full regeneration of skin in full and
partial thickness skin defects, including burns, (i.e.,
repopulation of hair follicles, sweat glands, and sebaceous
glands), treatment of other skin defects such as psoriasis. Albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention, could be used to treat
epidermolysis bullosa, a defect in adherence of the epidermis to
the underlying dermis which results in frequent, open and painful
blisters by accelerating reepithclialization of these lesions.
Albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, could also be
used to treat gastric and duodena ulcers and help heal by scar
formation of the mucosal lining and regeneration of glandular
mucosa and duodenal mucosal lining more rapidly. Inflammatory bowel
diseases, such as Crohn's disease and ulcerative colitis, are
diseases which result in destruction of the mucosal surface of the
small or large intestine, respectively. Thus, fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention, could be used to promote the resurfacing
of the mucosal surface to aid more rapid healing and to prevent
progression of inflammatory bowel disease. Treatment with fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, is expected to have a significant
effect on the production of mucus throughout the gastrointestinal
tract and could be used to protect the intestinal mucosa from
injurious substances that are ingested or following surgery.
Albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, could be used to
treat diseases associate with the tinder expression.
[0807] Moreover, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could be used to prevent and heal damage to the lungs due to
various pathological states. Albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention, which could stimulate proliferation and
differentiation and promote the repair of alveoli and brochiolar
epithelium to prevent or treat acute or chronic lung damage. For
example, emphysema, which results in the progressive loss of
aveoli, and inhalation injuries, i.e., resulting from smoke
inhalation and burns, that cause necrosis of the bronchiolar
epithelium and alveoli could be effectively treated using
polynucleotides or polypeptides, agonists or antagonists of the
present invention. Also fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could be used to stimulate the proliferation of and differentiation
of type II pneumocytes, which may help treat or prevent disease
such as hyaline membrane diseases, such as infant respiratory
distress syndrome and bronchopulmonary displasia, in premature
infants.
[0808] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could stimulate the proliferation and differentiation of
hepatocytes and, thus, could be used to alleviate or treat liver
diseases and pathologies such as fulminant liver failure caused by
cirrhosis, liver damage caused by viral hepatitis and toxic
substances (i.e., acetaminophen, carbon tetrachloride and other
hepatotoxins known in the art).
[0809] In addition, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
could be used treat or prevent the onset of diabetes mellitus. In
patients with newly diagnosed Types I and II diabetes, where some
islet cell function remains, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, could be used to maintain the islet function so as to
alleviate, delay or prevent permanent manifestation of the disease.
Also, fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, could be used as
an auxiliary in islet cell transplantation to improve or promote
islet cell function.
[0810] Diseases at the Cellular Level
[0811] Diseases associated with increased cell survival or the
inhibition of apoptosis that could be treated, prevented,
diagnosed, and/or prognosed using fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, include cancers (such as follicular lymphomas,
carcinomas with p53 mutations, and hormone-dependent tumors,
including, but not limited to colon cancer, cardiac tumors,
pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung
cancer, intestinal cancer, testicular cancer, stomach cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma,
adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian cancer); autoimmune disorders (such as, multiple sclerosis,
Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis,
Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomemlonephritis and rheumatoid
arthritis) and viral infections (such as herpes viruses, pox
viruses and adenoviruses), inflammation, graft v. host disease,
acute graft rejection, and chronic graft rejection.
[0812] In preferred embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention are used to inhibit growth, progression, and/or metasis
of cancers, in particular those listed above.
[0813] Additional diseases or conditions associated with increased
cell survival that could be treated or detected by fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention include, but are not limited to,
progression, and/or metastases of malignancies and related
disorders such as leukemia (including acute leukemias (e.g., acute
lymphocytic leukemia, acute myelocytic leukemia (including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia)),
polycythemia vera, lymphomas (e.g., Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including,
but not limited to, sarcomas and carcinomas such as fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0814] Diseases associated with increased apoptosis that could be
treated, prevented, diagnosed, and/or prognesed using fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, include, but are not limited to,
AIDS; neurodegenerative disorders (such as Alzheimer's disease,
Parkinson's disease, Amyotrophic lateral sclerosis. Retinitis
pigmentosa, Cerebellar degeneration and brain tumor or prior
associated disease); autoimmune disorders (such as, multiple
sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis) myelodysplastic syndromes (such as
aplastic anemia), graft v. host disease, ischemic injury such as
that caused by myocardial infarction, stroke and reperfusion
injury), liver injury (e.g., hepatitis related liver injury,
ischemia/reperfusion injury, cholestosis (bile duct injury) and
liver cancer); toxin-induced liver disease (such as that caused by
alcohol), septic shock, cachexia and anorexia.
[0815] Renal Disorders
[0816] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be used to treat, prevent, diagnose, and/or prognose disorders
of the renal system. Renal disorders which can be diagnosed,
prognosed, prevented, and/or treated with compositions of the
invention include, but are not limited to, kidney failure,
nephritis, blood vessel disorders of kidney, metabolic and
congenital kidney disorders, urinary disorders of the kidney,
autoimmune disorders, sclerosis and necrosis, electrolyte
imbalance, and kidney cancers.
[0817] Kidney diseases which can be diagnosed, prognosed,
prevented, and/or treated with compositions of the invention
include, but are not limited to, acute kidney failure, chronic
kidney failure, atheroembolic renal failure, end-stage renal
disease, inflammatory diseases of the kidney (e.g. acute
glomerulonephritis, postinfectious glomerulonephritis, rapidly
progressive glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis, familial nephrotic syndrome,
membranoproliferative glomerulonephritis I and II, mesangial
proliferative glomerulonephritis, chronic glomerulonephritis, acute
tubulointerstitial nephritis, chronic tubulointerstitial nephritis,
acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis,
lupus nephritis, chronic nephritis, interstitial nephritis, and
post-streptococcal glomerulonepluitis), blood vessel disorders of
the kidneys (e.g., kidney infarction, atheroembolic kidney disease,
cortical necrosis, malignant nephrosclerosis, renal vein
thrombosis, renal underperfusion, renal retinopathy, renal
ischemia-reperfusion, renal artery embolism, and renal artery
stenosis), and kidney disorders resulting form urinary tract
disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal
lithiasis, nephrolithiasis), reflux nephropathy, urinary tract
infections, urinary retention, and acute or chronic unilateral
obstructive uropathy.)
[0818] In addition, compositions of the invention can be used to
diagnose, prognose, prevent, and/or treat metabolic and congenital
disorders of the kidney (e.g., uremia, renal amyloidosis, renal
osteodystrophy, renal tubular acidosis, renal glycosuria,
nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome,
renal fibrocystic osteosis (renal rickets), Hartnup disease,
Banter's syndrome, Liddle's syndrome, polycystic kidney disease,
medullary cystic disease, medullary sponge kidney, Alport's
syndrome, nail-patella syndrome, congenital nephrotic syndrome,
CRUSH syndrome, horseshoe kidney, diabetic nepluopathy, nephrogenic
diabetes insipidus, analgesic nephropathy, kidney stones, and
membranous nephropathy), and autoimmune disorders of the kidney
(e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome,
IgA nephropathy, and IgM mesangial proliferative
glomerulonephritis).
[0819] Compositions of the invention can also be used to diagnose,
prognose, prevent, and/or treat sclerotic or necrotic disorders of
the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal
segmental glomerulosclerosis (FSGS), necrotizing
glomerulonephritis, and renal papillary necrosis), cancers of the
kidney (e.g., nephroma, hypernephroma, nephroblastoma, renal cell
cancer, transitional cell cancer, renal adenocarcinoma, squamous
cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g.,
nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria,
hyponatremia, hypematremia, hypokalemia, hyperkalemia,
hypocalcemia, hypercalcemia, hypophosphatemia, and
hyperphosphatemia).
[0820] Compositions of the invention may be administered using any
method known in the art, including, but not limited to, direct
needle injection at the delivery site, intravenous injection,
topical administration, catheter infusion, biolistic injectors,
particle accelerators, gelfoam sponge depots, other commercially
available depot materials, osmotic pumps, oral or suppositorial
solid pharmaceutical formulations, decanting or topical
applications during surgery, aerosol delivery. Such methods are
known in the art. Compositions of the invention may be administered
as part of a Therapeutic, described in more detail below. Methods
of delivering polynucleotides of the invention are described in
more detail herein.
[0821] Neural Activity and Neurological Diseases
[0822] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used for the diagnosis and/or treatment of diseases,
disorders, damage or injury of the brain and/or nervous system.
Nervous system disorders that can be treated with the compositions
of the invention (e.g., fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention),
include, but are not limited to, nervous system injuries, and
diseases or disorders which result in either a disconnection of
axons, a diminution or degeneration of neurons, or demyelination.
Nervous system lesions which may be treated in a patient (including
human and non-human mammalian patients) according to the methods of
the invention, include but are not limited to, the following
lesions of either the central (including spinal cord, brain) or
peripheral nervous systems: (1) ischemic lesions, in which a lack
of oxygen in a portion of the nervous system results in neuronal
injury or death, including cerebral infarction or ischemia, or
spinal cord infarction or ischemia; (2) traumatic lesions,
including lesions caused by physical injury or associated with
surgery, for example, lesions which sever a portion of the nervous
system, or compression injuries; (3) malignant lesions, in which a
portion of the nervous system is destroyed or injured by malignant
tissue which is either a nervous system associated malignancy or a
malignancy derived from non-nervous system tissue; (4) infectious
lesions, in which a portion of the nervous system is destroyed or
injured as a result of infection, for example, by an abscess or
associated with infection by human immunodeficiency virus, herpes
zoster, or herpes simplex virus or with Lyme disease, tuberculosis,
or syphilis; (5) degenerative lesions, in which a portion of the
nervous system is destroyed or injured as a result of a
degenerative process including but not limited to, degeneration
associated with Parkinson's disease, Alzheimer's disease,
Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6)
lesions associated with nutritional diseases or disorders, in which
a portion of the nervous system is destroyed or injured by a
nutritional disorder or disorder of metabolism including, but not
limited to, vitamin B12 deficiency, folic acid deficiency. Wernicke
disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease
(primary degeneration of the corpus callosum), and alcoholic
cerebellar degeneration; (7) neurological lesions associated with
systemic diseases including, but not limited to, diabetes (diabetic
neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma,
or sarcoidosis; (8) lesions caused by toxic substances including
alcohol, lead, or particular neurotoxins; and (9) demyelinated
lesions in which a portion of the nervous system is destroyed or
injured by a demyelinating disease including, but not limited to,
multiple sclerosis, human immunodeficiency virus-associated
myelopathy, transverse myelopathy or various etiologies,
progressive multifocal leukoencephalopathy, and central pontine
myelinolysis.
[0823] In one embodiment, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used to protect neural cells from the damaging
effects of hypoxia. In a further preferred embodiment, the albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention are used to protect neural
cells from the damaging effects of cerebral hypoxia. According to
this embodiment, the compositions of the invention are used to
treat or prevent neural cell injury associated with cerebral
hypoxia. In one non-exclusive aspect of this embodiment, the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, are used to
treat or prevent neural cell injury associated with cerebral
ischemia. In another non-exclusive aspect of this embodiment, the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention are used to treat
or prevent neural cell injury associated with cerebral
infarction.
[0824] In another preferred embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to treat or prevent neural cell
injury associated with a stroke. In a specific embodiment, albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention are used to treat or
prevent cerebral neural cell injury associated with a stroke.
[0825] In another preferred embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to treat or prevent neural cell
injury associated with a heart attack. In a specific embodiment,
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention are used to treat
or prevent cerebral neural cell injury associated with a heart
attack.
[0826] The compositions of the invention which are useful for
treating or preventing a nervous system disorder may be selected by
testing for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, compositions of the invention which elicit any of the
following effects may be useful according to the invention: (1)
increased survival time of neurons in culture either in the
presence or absence of hypoxia or hypoxic conditions; (2) increased
sprouting of neurons in culture or in vivo; (3) increased
production of a neuron-associated molecule in culture or in vivo,
e.g., choline acetyltransferase or acetylcholinesterase with
respect to motor neurons; or (4) decreased symptoms of neuron
dysfunction in vivo, Such effects may be measured by any method
known in the art. In preferred, non-limiting embodiments, increased
survival of neurons may routinely be measured using a method set
forth herein or otherwise known in the art, such as, for example,
in Zhang et al., Proc Natl Acad Sci USA 97:3637-42 (2000) or in
Arakawa et al., J. Neurosci. 10:3507-15 (1990); increased sprouting
of neurons may be detected by methods known in the art, such as,
for example, the methods set forth in Pestronk et al., Exp.
Neural., 70:65-82 (1980), or Brown et al., Ann. Rev. Neurosci.,
4:17-42 (1981); increased production of neuron-associated molecules
may be measured by bioassay, enzymatic assay, antibody binding,
Northern blot assay, etc., using techniques known in the art and
depending on the molecule to be measured; and motor neuron
dysfunction may be measured by assessing the physical manifestation
of motor neuron disorder, e.g., weakness, motor neuron conduction
velocity, or functional disability.
[0827] In specific embodiments, motor neuron disorders that may be
treated according to the invention include, but are not limited to,
disorders such as infarction, infection, exposure to toxin, trauma,
surgical damage, degenerative disease or malignancy that may affect
motor neurons as well as other components of the nervous system, as
well as disorders that selectively affect neurons such as
amyotrophic lateral sclerosis, and including, but not limited to,
progressive spinal muscular atrophy, progressive bulbar palsy,
primary lateral sclerosis, infantile and juvenile muscular atrophy,
progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Chareoe-Marie-Tooth Disease).
[0828] Further, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may play a role in neuronal survival; synapse formation;
conductance; neural differentiation, etc. Thus, compositions of the
invention (including fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention)
may be used to diagnose and/or treat or prevent diseases or
disorders associated with these roles, including, but not limited
to, learning and/or cognition disorders. The compositions of the
invention may also be useful in the treatment or prevention of
neurodegenerative disease states and/or behavioural disorders. Such
neurodegenerative disease states and/or behavioral disorders
include, but are not limited to, Alzheimer's Disease, Parkinson's
Disease, Huntington's Disease, Tourette Syndrome, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, compositions of the invention
may also play a role in the treatment, prevention and/or detection
of developmental disorders associated with the developing embryo,
or sexually-linked disorders.
[0829] Additionally, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be useful in protecting neural cells from diseases, damage,
disorders, or injury, associated with cerebrovascular disorders
including, but not limited to, carotid artery diseases (e.g.,
carotid artery thrombosis, carotid stenosis, or Moyamoya Disease),
cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,
cerebral arteriosclerosis, cerebral arteriovenous malformations,
cerebral artery diseases, cerebral embolism and thrombosis (e.g.,
carotid artery thrombosis, sinus thrombosis, of Wallenberg's
Syndrome), cerebral hemorrhage (e.g., epidural or subdural
hematoma, or subarachnoid hemorrhage), cerebral infarction,
cerebral ischemia (e.g., transient cerebral ischemia, Subclavian
Steal Syndrome, or vertebrobasilar insufficiency), vascular
dementia (e.g., multi-infarct), leukomalacia, periventricular, and
vascular headache (e.g., cluster headache or migraines).
[0830] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, for therapeutic purposes, for
example, to stimulate neurological cell proliferation and/or
differentiation. Therefore, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to treat and/or detect neurologic diseases. Moreover,
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention, can be used as a marker
or detector of a particular nervous system disease or disorder.
[0831] Examples of neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, brain diseases, such as metabolic brain diseases which
includes phenylketonuria such as maternal phenylketonuria, pyruvate
carboxylase deficiency, pyruvate dehydrogenase complex deficiency,
Wernicke's Encephalopathy, brain edema, brain neoplasms such as
cerebellar neoplasms which include infratentorial neoplasms,
cerebral ventricle neoplasms such as choroid plexus neoplasms,
hypothalamic neoplasms, supratentorial neoplasms, canavan disease,
cerebellar diseases such as cerebellar ataxia which include
spinocerebellar degeneration such as ataxia telangiectasia,
cerebellar dyssynergia, Friederich's Ataxia, Machado-Joseph
Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as
infratentorial neoplasms, diffuse cerebral sclerosis such as
encephalitis periaxialis, globoid cell leukodystrophy,
metachromatic leukodystrophy and subacute sclerosing
panencephalitis.
[0832] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include cerebrovascular disorders (such as carotid artery diseases
which include carotid artery thrombosis, carotid stenosis and
Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm,
cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous
malformations, cerebral artery diseases, cerebral embolism and
thrombosis such as carotid artery thrombosis, sinus thrombosis and
Wallenberg's Syndrome, cerebral hemorrhage such as epidural
hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral
infarction, cerebral ischemia such as transient cerebral ischemia,
Subclavian Steal Syndrome and vertebrobasilar insufficiency,
vascular dementia such as multi-infarct dementia, periventricular
leukomalacia, vascular headache such as cluster headache and
migraine.
[0833] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include dementia such as AIDS Dementia Complex, presenile dementia
such as Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile
dementia such as Alzheimer's Disease and progressive supranuclear
palsy, vascular dementia such as multi-infarct dementia,
encephalitis which include encephalitis periaxialis, viral
encephalitis such as epidemic encephalitis, Japanese Encephalitis,
St. Louis Encephalitis, tick-borne encephalitis and West Nile
Fever, acute disseminated encephalomyelitis, meningoencephalitis
such as uveomeningoencephalitic syndrome, Postencephalitic
Parkinson Disease and subacute sclerosing panencephalitis,
encephalomalacia such as periventricular leukomalacia, epilepsy
such as generalized epilepsy which includes infantile spasms,
absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome,
tonic-clonic epilepsy, partial epilepsy such as complex partial
epilepsy, frontal lobe epilepsy and temporal lobe epilepsy,
post-traumatic epilepsy, status epilepticus such as Epilepsia
Partialis Continua, and Hallervorden-Spatz Syndrome.
[0834] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include hydrocephalus such as Dandy-Walker Syndrome and normal
pressure hydrocephalus, hypothalamic diseases such as hypothalamic
neoplasms, cerebral malaria, narcolepsy which includes cataplexy,
bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome, Reye's
Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial
tuberculoma and Zellweger Syndrome, central nervous system
infections such as AIDS Dementia Complex, Brain Abscess, subdural
empyema, encephalomyelitis such as Equine Encephalomyelitis,
Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhagic
Encephalomyelitis, Visna, and cerebral malaria.
[0835] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include meningitis such as arachnoiditis, aseptic meningtitis such
as viral meningtitis which includes lymphocytic choriomeningitis,
Bacterial meningtitis which includes Haemophilus Meningtitis,
Listeria Meningtitis. Meningococcal Meningitis such as
Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and
meningeal tuberculosis, fungal meningitis such as Cryptococcal
Meningtitis, subdural effusion, meningoencephalitis such as
uvemeningoencephalitic syndrome, myelitis such as transverse
myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which
includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion
diseases (such as Creutzfeldt-Jakob Syndrome. Bovine Spongiform
Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and
cerebral toxoplasmosis.
[0836] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include central nervous system neoplasms such as brain neoplasms
that include cerebellar neoplasms such as infratentorial neoplasms,
cerebral ventricle neoplasms such as choroid plexus neoplasms,
hypothalamic neoplasms and supratentorial neoplasms, meningeal
neoplasms, spinal cord neoplasms which include epidural neoplasms,
demyelinating diseases such as Canavan Diseases, diffuse cerebral
sceloris which includes adrenoleukodystrophy, encephalitis
periaxialis, globoid cell leukodystrophy, diffuse cerebral
sclerosis such as metachromatic leukodystrophy, allergic
encephalomyelitis, necrotizing hemorrhagic encephalomyelitis,
progressive multifocal leukoencephalopaihy, multiple sclerosis,
central pontine myelinolysis, transverse myelitis, neuromyelitis
optica, Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High
Pressure Nervous Syndrome, Meningism, spinal cord diseases such as
amyotonia congenita, amyotrophic lateral sclerosis, spinal muscular
atrophy such as Werdnig-Hoffmann Disease, spinal cord compression,
spinal cord neoplasms such as epidural neoplasms, syringomyelia,
Tabes Dorsalis, Stiff-Man Syndrome, mental retardation such as
Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome, Down
Syndrome, Gangliosidoses such as gangliosidoses G(M1), Sandhoff
Disease, Tay-Sachs Disease, Hannon Disease, homocystinuria,
Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup
Urine Disease, mucolipidosis such as fucosidosis, neuronal
ceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria
such as maternal phenylketonuria, Prader-Willi Syndrome, Rett
Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR
Syndrome, nervous system abnormalities such as holoprosencephaly,
neural tube defects such as anencephaly which includes
hydrangencephaly, Arnold-Chairi Deformity, encephalocele,
meningocele, meningomyelocele, spinal dysraphism such as spina
bifida cystica and spina bifida occulta.
[0837] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include hereditary motor and sensory neuropathies which include
Charcot-Marie Disease, Hereditary optic atrophy, Refsum's Disease,
hereditary spastic paraplegia. Werdnig-Hoffmann Disease, Hereditary
Sensory and Autonomic Neuropathies such as Congenital Analgesia and
Familial Dysautonomia, Neurologic manifestations (such as agnosia
that include Gerstmann's Syndrome, Amnesia such as retrograde
amnesia, apraxia, neurogenic bladder, cataplexy, communicative
disorders such as hearing disorders that includes deafness, partial
hearing loss, loudness recruitment and tinnitus, language disorders
such as aphasia which include agraphia, anomia, broca aphasia, and
Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language
development disorders, speech disorders such as aphasia which
includes anomia, broca aphasia and Wernicke Aphasia, articulation
disorders, communicative disorders such as speech disorders which
include dysarthria, echolalia, mutism and stuttering, voice
disorders such as aphonia and hoarseness, decerebrate state,
delirium, fasciculation, hallucinations, meningism, movement
disorders such as angelman syndrome, ataxia, athetosis, chorea,
dystonia, hypokinesia, muscle hypotonia, myoclonus, tic,
torticollis and tremor, muscle hypertonia such as muscle rigidity
such as stiff man syndrome, muscle spasticity, paralysis such as
facial paralysis which includes Herpes Zoster Oriel's;
Gastroparesis, Hemiplegia, ophthalmoplegia such as diplopia,
Duane's Syndrome, Horner's Syndrome, Chronic progressive external
ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis, Tropical
Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome,
quadriplegia, respiratory paralysis and vocal cord paralysis,
paresis, phantom limb, taste disorders such as ageusia and
dysgeusia, vision disorders such as amblyopia, blindness, color
vision defects, diplopia, hemianopsia, scotoma and subnormal
vision, sleep disorders such as hypersomnia which includes
Kleine-Levin Syndrome, insomnia, and somnambulism, spasm such as
trismus, unconsciousness such as coma, persistent vegetative state
and syncope and vertigo, neuromuscular diseases such as amyotonia
congenita, amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic
Syndrome, motor neuron disease, muscular atrophy such as spinal
muscular atrophy, Charcot-Marie Disease and Werdnig-Hoffmann
Disease, Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia
Gravis, Myotonia Atrophica, Myotonia Cerifenita, Nemaline Myopathy,
Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical
Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous
system diseases such as acrodynia, amyloid neuropathies, autonomic
nervous system diseases such as Adie's Syndrome, Barre-Lieou
Syndrome, Familial Dysautonomia, Horner's Syndrome, Reflex
Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve
Diseases such as Acoustic Nerve Diseases such as Acoustic Neuroma
which includes Neurofibromatosis 2, Facial Nerve Diseases such as
Facial Neuralgia.Melkersson-Rosenthal Syndrome, ocular motility
disorders which includes amblyopia, nystagmus, oculomotor nerve
paralysis, ophthalmoplegia such as Duane's Syndrome, Horner's
Syndrome, Chronic Progressive External Ophthalmoplegia which
includes Kearns Syndrome, Strabismus such as Esotropia and
Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such as
Optic Atrophy which includes Hereditary Optic Atrophy, Optic Disk
Drusen, Optic Neuritis such as Neuromyelitis Optics, Papilledema,
Trigeminal Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases
such as Neuromyelitis Optica and Swayback, and Diabetic
neuropathies such as diabetic foot.
[0838] Additional neurologic diseases which can be treated or
detected with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include nerve compression syndromes such as carpal tunnel syndrome,
tarsal tunnel syndrome, thoracic outlet syndrome such as cervical
rib syndrome, ulnar nerve compression syndrome, neuralgia such as
causalgia, cervico-brachial neuralgia, facial neuralgia and
trigeminal neuralgia, neuritis such as experimental allergic
neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and
radiculities such as polyradiculitis, hereditary motor and sensory
neuropathies such as Charcot-Marie Disease, Hereditary Optic
Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and
Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic
Neuropathies which include Congenital Analgesia and Familial
Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and
Tetany).
[0839] Immune Activity
[0840] Albumin fusion proteins of the invention and polynucleotides
encoding albumin fusion proteins of the invention may be useful in
treating, preventing, diagnosing and/or prognosing diseases,
disorders, and/or conditions of the immune system, by, for example,
activating or inhibiting the proliferation, differentiation, or
mobilization (chemotaxis) of immune cells. Immune cells develop
through a process called hematopoiesis, producing myeloid
(platelets, red blood cells, neutrophils, and macrophages) and
lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
The etiology of these immune diseases, disorders, and/or conditions
may be genetic, somatic, such as cancer and some autoimmune
diseases, acquired (e.g., by chemotherapy or toxins), or
infectious. Moreover, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
can be used as a marker or detector of a particular immune system
disease or disorder.
[0841] In another embodiment, a fusion protein of the invention
and/or polynucleotide encoding an albumin fusion protein of the
invention, may be used to treat diseases and disorders of the
immune system and/or to inhibit or enhance an immune response
generated by cells associated with the tissue(s) in which the
polypeptide of the invention is expressed.
[0842] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in treating, preventing, diagnosing, and/or
prognosing immunodeficiencies, including both congenital and
acquired immunodeficiencies. Examples of B cell immunodeficiencies
in which immunoglobulin levels B cell function and/or B cell
numbers are decreased include X-linked agammaglobulinemia (Bruton's
disease), X-finked infantile agammaglobulinemia, X-linked
immunodeficiency with hyper IgM, non X-linked immunodeficiency with
hyper IgM, X-linked Lymphoproliferative syndrome (XLP),
agammaglobulinemia including congenital and acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,
unspecified hypogammaglobulinemia, recessive agammaglobulinemia
(Swiss type). Selective IgM deficiency, selective IgA deficiency,
selective IgG subclass deficiencies, IgG subclass deficiency (with
or without IgA deficiency), Ig deficiency with increased IgM, IgG
and IgA deficiency with increased IgM, antibody deficiency with
normal or elevated Igs, Ig heavy chain deletions, kappa chain
deficiency, B cell lymphoproliferative disorder (BLPD), common
variable immunodeficiency (CVID), common variable immunodeficiency
(CVI) (acquired), and transient hypogammaglobulinemia of
infancy.
[0843] In specific a specific embodiment, ataxia-telangiectasia or
conditions associated with ataxia-telangiectasia are treated,
prevented, diagnosed, and/or prognosing using the fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention.
[0844] Examples of congenital immunodeficiencies in which T cell
and/or B cell function and/or number is decreased include, but are
not limited to: DiGeorge anomaly, severe combined
immunodeficiencies (SCID) (including, but not limited to, X-linked
SCID, autosomal recessive SCID, adenosine deaminase deficiency,
purine nucleoside phosphorylase (PNP) deficiency, Class if MHC
deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome,
and ataxia telangiectasia), thymic hypoplasia, third and fourth
pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous
candidiasis, natural killer cell deficiency (NK), idiopathic CD4+
T-lymphocytopenia, immunodeficiency with predominant T cell defect
(unspecified), and unspecified immunodeficiency of cell mediated
immunity.
[0845] In a specific embodiment, DiGeorge anomaly or conditions
associated with DiGeorge anomaly are treated, prevented, diagnosed,
and/or prognosed using fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the
invention.
[0846] Other immunodeficiencies that may be treated, prevented,
diagnosed, and/or prognosed using fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, include, but are not limited to, chronic granulomatous
disease, Chediak-Higashi syndrome, myeloperoxidase deficiency,
leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked
lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency,
complement component deficiencies (including C1, C2, C3, C4, C5,
C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic
alymphoplasia-aplasia, immunodeficiency with thymoma, severe
congenital leukopenia, dysplasia with immunodeficiency, neonatal
neutropenia, short limbed dwarfism and Nezelof syndrome-combined
immunodeficiency with Igs.
[0847] In a preferred embodiment, the immunodeficiencies and/or
conditions associated with the immunodeficiencies recited above are
treated, prevented, diagnosed and/or prognosed using fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention.
[0848] In a preferred embodiment fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention could be used as an agent to boost immunoresponsiveness
among immunodeficient individuals. In specific embodiments, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention could be used as an agent to boost
immunoresponsiveness among B cell and/or T cell immunodeficient
individuals.
[0849] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in treating, preventing, diagnosing and/or prognosing
autoimmune disorders. Many autoimmune disorders result from
inappropriate recognition of self as foreign material by immune
cells. This inappropriate recognition results in an immune response
leading to the destruction of the host tissue. Therefore, the
administration of fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
that can inhibit an immune response, particularly the
proliferation, differentiation, or chemotaxis of T-cells, may be an
effective therapy in preventing autoimmune disorders.
[0850] Autoimmune diseases or disorders that may be treated,
prevented, diagnosed and/or prognosed lay fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention include, but are not limited to, one or more of
the following: systemic lupus erythematosus, rheumatoid arthritis,
ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis,
Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic
anemia, thrombocytopenia, autoimmune thrombocytopenia purpura,
autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia
purpura, purpura (e.g., Henloch-Scoenlein purpura),
autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris,
myasthenia gravis, Grave's disease (hypenhyroldism), and
insulin-resistant diabetes mellitus.
[0851] Additional disorders that are likely to have an autoimmune
component that may be treated, prevented, and/or diagnosed with the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention include, but are
not limited to, type II collagen-induced arthritis,
antiphospholipid syndrome, dermatitis, allergic encephalomyelitis,
myocarditis, relapsing polychondritis, rheumatic heart disease,
neuritis, uveitis ophthalmia, Polyendocrinopathies, Reiter's
Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation,
autism, Guillain-Barre Syndrome, insulin dependent diabetes
mellitus, and autoimmune inflammatory eye disorders.
[0852] Additional disorders that are likely to have an autoimmune
component that may be treated, prevented, diagnosed and/or
prognosed with the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, but am not limited to, scleroderma with anti-collagen
antibodies (often characterized, e.g., by nucleolar and other
nuclear antibodies), mixed connective tissue disease (often
characterized, e.g., by antibodies to extractable nuclear antigens
(e.g., ribonucleoprotein)), polymyositis (often characterized,
e.g., by nonhistone ANA), pernicious anemia (often characterized,
e.g., by antiparietal cell, microsomes, and intrinsic factor
antibodies), idiopathic Addison's disease (often characterized,
e.g., by humoral and cell-mediated adrenal cytotoxicity,
infertility (often characterized, e.g., by antispermatozoal
antibodies), glomerulonephritis (often characterized, e.g., by
glomerular basement membrane antibodies or immune complexes),
bullous pemphigoid (often characterized, e.g., by IgG and
complement in basement membrane), Sjogren's syndrome (often
characterized, e.g. by multiple tissue antibodies, and/or a
specific nonhistone ANA (SS-B)), diabetes mellitus (often
characterized, e.g., by cell-mediated and humoral islet cell
antibodies), and adrenergic drug resistance (including adrenergic
drug resistance with asthma or cystic fibrosis) (often
characterized, e.g., by beta-adrenergic receptor antibodies).
[0853] Additional disorders that may have an autoimmune component
that may be treated, prevented, diagnosed and/or prognosed with the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention include, but are
not limited to, chronic active hepatitis (often characterized,
e.g., by smooth muscle antibodies), primary biliary cirrhosis
(often characterized, e.g., by mitochondria antibodies), other
endocrine gland failure (often characterized, e.g., by specific
tissue antibodies in some cases), vitiligo (often characterized,
e.g., by melanocyte antibodies), vasculitis (often characterized,
by Ig and complement in vessel walls and/or low serum complement),
post-MI (often characterized, e.g., by myocardial antibodies),
cardiotomy syndrome (often characterized, e.g., by myocardial
antibodies), urticaria (often characterized, e.g., by IgG and IgM
antibodies to IgE), atopic dermatitis (often characterized, e.g.,
by IgG and IgM antibodies to IgE), asthma (often characterized,
e.g., by IgG and IgM antibodies to IgE), and many other
inflammatory, granulomatous, degenerative, and atrophic
disorders.
[0854] In a preferred embodiment, the autoimmune diseases and
disorders and/or condition's associated with the diseases and
disorders recited above are treated, prevented, diagnosed and/or
prognosed using for example, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention. In a specific preferred embodiment, rheumatoid arthritis
is treated, prevented, and/or diagnosed using fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention.
[0855] In another specific preferred embodiment, systemic lupus
erythematosus is treated, prevented, and/or diagnosed using fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention. In another specific preferred
embodiment, idiopathic thrombocytopenia purpura is treated,
prevented, and/or diagnosed using fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention.
[0856] In another specific preferred embodiment IgA nephropathy is
treated, prevented, and/or diagnosed using fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention.
[0857] In a preferred embodiment, the autoimmune diseases and
disorders and/or conditions associated with the diseases and
disorders recited above are treated, prevented, diagnosed and/or
prognosed using fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the
invention.
[0858] In preferred embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention are used as a immunosuppressive agent(s).
[0859] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in treating, preventing, prognosing, and/or
diagnosing diseases, disorders, and/or conditions of hematopoietic
cells. Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
could be used to increase differentiation and proliferation of
hematopoietic cells, including the pluripotent stem cells, in an
effort to treat or prevent those diseases, disorders, and/or
conditions associated with a decrease in certain (or many) types
hematopoietic cells, including but not limited to, leukopenia,
nentropenia, anemia, and thrombocytopenia. Alternatively, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention could be used to increase
differentiation and proliferation of hematopoietic cells, including
the pluripotent stem cells, in an effort to treat or prevent those
diseases, disorders, and/or conditions associated with an increase
in certain (or many) types of hematopoietic cells, including but
not limited to, histiocytosi.
[0860] Allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated, prevented, diagnosed and/or prognosed using fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention. Moreover, these molecules can be
used to treat, prevent, prognose, and/or diagnose anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group
incompatibility.
[0861] Additionally, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be used to neat, prevent, diagnose and/or prognose IgE-mediated
allergic reactions. Such allergic reactions include, but are not
limited to, asthma, rhinitis, and eczema. In specific embodiments,
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention may be used to modulate.
IgE concentrations in vitro or in vivo.
[0862] Moreover, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
have uses in the diagnosis, prognosis, prevention, and/or treatment
of inflammatory conditions. For example, since fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may inhibit the activation, proliferation
and/or differentiation of cells involved in an inflammatory
response, these molecules can be used to prevent and/or treat
chronic and acute inflammatory conditions. Such inflammatory
conditions include, but are not limited to, for example,
inflammation associated with infection (e.g., septic shock, sepsis,
or systemic inflammatory response syndrome), ischemia-reperfusion
injury, endotoxin lethality, complement-mediated hyperacute
rejection, nephritis, cytokine or chemokine induced lung injury,
inflammatory bowel disease, Crohn's disease, over production of
cytokines (e.g., TNF respiratory disorders (e.g., asthma and
allergy); gastrointestinal disorders (e.g., inflammatory bowel
disease); cancers (e.g., gastric, ovarian, lung, bladder, liver,
and breast); CNS disorders (e.g., multiple sclerosis; ischemic
brain injury and/or stroke, traumatic brain injury,
neurodegenerative disorders (e.g., Parkinson's disease and
Alzheimer's disease); AIDS-related dementia; and prion disease);
cardiovascular disorders (e.g., atherosclerosis, myocarditis,
cardiovascular disease, and cardiopulmonary bypass complications);
as well as many additional diseases, conditions, and disorders that
are characterized by inflammation (e.g., hepatitis, rheumatoid
arthritis, gout, trauma, pancreatitis, sarcoidosis, dermatitis,
renal ischemia-reperfusion injury, Grave's disease, systemic lupus
erythematosus, diabetes mellitus, and allogenic transplant
rejection).
[0863] Because inflammation is a fundamental defense mechanism,
inflammatory disorders can effect virtually any tissue of the body.
Accordingly, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
have uses in the treatment of tissue-specific inflammatory
disorders, including, but not limited to, adrenalitis, alveolitis,
angiocholecystitis, appendicitis, balanitis, blepharitis,
bronchitis, bursitis, carditis, cellulitis, cervicitis,
cholecystitis, chorditis, cochlitis, colitis, conjunctivitis,
cystitis, dermatitis, diverticulitis, encephalitis, endocarditis,
esophagitis, eustachitis, fibrositis, folliculitis, gastritis,
gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis,
labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis,
meningitis, metritis, mucitis, myocarditis, myosititis, myringitis,
nephritis, neuritis, orchitis, osteochondritis, otitis,
pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis,
poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis,
salpingitis, selertitis, sclerochoroiditis, serotitis, sinusitis,
spondylitis, steatitis, stomatitis, synovitis, syringitis,
tendonitis, tonsillitis, urethritis, and vaginitis.
[0864] In specific embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, are useful to diagnose, prognose, prevent, and/or treat
organ transplant rejections and graft-versus-host disease. Organ
rejection occurs by host immune cell destruction of the
transplanted tissue through an immune response. Similarly, an
immune response is also involved in GVHD, but, in this case, the
foreign transplanted immune cells destroy the host tissues.
Polypeptides, antibodies, or polynucleotides of the invention,
and/or agonists or antagonists thereof, that inhibit an immune
response, particularly the activation, proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing organ rejection or GVHD. In specific
embodiments, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
that inhibit an immune response, particularly the activation,
proliferation, differentiation, or chemotaxis of T-cells, may be an
effective therapy in preventing experimental allergic and
hyperacute xenograft rejection.
[0865] In other embodiments, fusion proteins of the invention
and/in polynucleotides encoding albumin fusion proteins of the
invention, are useful to diagnose, prognose, prevent, and/or treat
immune complex diseases, including, but not limited to, serum
sickness, post streptococcal glomerulonephritis, polyarteritis
nodosa, and immune complex-induced vasculitis.
[0866] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
can be used to treat, detect, and/or prevent infectious agents. For
example, by increasing the immune response, particularly increasing
the proliferation activation and/or differentiation of B and/or T
cells, infectious diseases may be treated, detected, and/or
prevented. The immune response may be increased by either enhancing
an existing immune response, or by initiating a new immune
response. Alternatively, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may also directly inhibit the infectious agent (refer to section of
application listing infectious agents, etc), without necessarily
eliciting an immune response.
[0867] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used as a vaccine adjuvant that enhances
immune responsiveness to an antigen. In a specific embodiment,
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention are used as an
adjuvant to enhance tumor-specific immune responses.
[0868] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an adjuvant to enhance
anti-viral immune responses. Anti-viral immune responses that may
be enhanced using the compositions of the invention as an adjuvant,
include virus and virus associated diseases or symptoms described
herein or otherwise known in the art. In specific embodiments, the
compositions of the invention are used as an adjuvant to enhance an
immune response to a virus, disease, or symptom selected from the
group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis
(e.g., hepatitis B). In another specific embodiment, the
compositions of the invention are used as an adjuvant to enhance an
immune response to a virus, disease, or symptom selected from the
group consisting of: HIV/AIDS, respiratory syncytial virus. Dengue,
rotavirus, Japanese B encephalitis, influenza A and B,
parainfluenza, measles, cytomegalovirus, rabies, Junin,
Chikungunya, Rift Valley Fever, herpes simplex, and yellow
fever.
[0869] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an adjuvant to enhance
anti-bacterial or anti-fungal immune responses. Anti-bacterial or
anti-fungal immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include bacteria or
fungus and bacteria or fungus associated diseases or symptoms
described herein or otherwise known in the art. In specific
embodiments, the compositions of the invention are used as an
adjuvant to enhance an immune response to a bacteria or fungus,
disease, or symptom selected from the group consisting of: tetanus,
Diphtheria, botulism, and meningitis type B.
[0870] In another specific embodiment, the compositions of the
invention are used as an adjuvant to enhance an immune response to
a bacteria or fungus, disease, or symptom selected from the group
consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella
typhi, Salmonella paratyphi, Aleisseria meningitidis, Streptococcus
pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic
Escherichia coli, Enterohemorrhagic E. coli, and Borrelia
burgdorferi.
[0871] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an adjuvant to enhance
anti-parasitic immune responses. Anti-parasitic immune responses
that may be enhanced using the compositions of the invention as an
adjuvant, include parasite and parasite associated diseases or
symptoms described herein or otherwise known in the art. In
specific embodiments, the compositions of the invention are used as
an adjuvant to enhance an immune response to a parasite. In another
specific embodiment, the compositions of the invention are used as
an adjuvant to enhance an immune response to Plasmodium (malaria)
or Leishmania.
[0872] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may also be employed to treat infectious
diseases including silicosis, sarcoidosis, and idiopathic pulmonary
fibrosis; for example, by preventing the recruitment and activation
of mononuclear phagocytes.
[0873] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an antigen for the generation
of antibodies to inhibit or enhance immune mediated responses
against polypeptides of the invention.
[0874] In one embodiment, albumin fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention are administered to an animal (e.g., mouse, rat, rabbit,
hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse,
cow, sheep, dog, cat, non-human primate, and human, most preferably
human) to boost the immune system to produce increased quantities
of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce
higher affinity antibody production and immunoglobulin class
switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an
immune response.
[0875] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a stimulator of B cell
responsiveness to pathogens.
[0876] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an activator of T cells.
[0877] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent that elevates the
immune status of an individual prior to their receipt of
immunosuppressive therapies.
[0878] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to induce higher
affinity antibodies.
[0879] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to increase serum
immunoglobulin concentrations.
[0880] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to accelerate
recovery of immunocompromised individuals.
[0881] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to boost
immunoresponsiveness among aged populations and/or neonates.
[0882] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an immune system enhancer
prior to, during, or after bone marrow transplant and/or other
transplants (e.g., allogeneic or xenogeneic organ transplantation).
With respect to transplantation, compositions of the invention may
be administered prior to, concomitant with, and/or after
transplantation. In a specific embodiment, compositions of the
invention are administered after transplantation, prior to the
beginning of recovery of T-cell populations. In another specific
embodiment, compositions of the invention are first administered
after transplantation after the beginning of recovery of T cell
populations, but prior to full recovery of B cell populations.
[0883] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to boost
immunoresponsiveness among individuals having an acquired loss of B
cell function. Conditions resulting in an acquired loss of B cell
function that may be treated (e.g., ameliorated) by administering
an albumin fusion protein of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, include, but are
not limited to, HIV Infection, AIDS, bone mallow transplant, and B
cell chronic lymphocytic leukemia (CLL).
[0884] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to boost
immunoresponsiveness among individuals having a temporary immune
deficiency. Conditions resulting in a temporary immune deficiency
that may be treated (e.g., ameliorated) by administering an albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention, include, but are not
limited to, recovery from viral infections (e.g., influenza),
conditions associated with malnutrition, recovery from infectious
mononucleosis, or conditions associated with stress, recovery from
measles, recovery from blood transfusion, and recovery from
surgery.
[0885] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a regulator of antigen
presentation by monocytes, dendritic cells, and/or B-cells. In one
embodiment, albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
enhance antigen presentation or antagonize antigen presentation in
vitro or in vivo. Moreover, in related embodiments, this
enhancement or antagonism of antigen presentation may be useful as
an anti-tumor treatment or to modulate the immune system.
[0886] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as an agent to direct an
individual's immune system towards development of a humoral
response (i.e., TH2) as opposed to a TH1 cellular response.
[0887] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a means to induce tumor
proliferation and thus make it, more susceptible to anti-neoplastic
agents. For example, multiple myeloma is a slowly dividing disease
and is thus refractory to virtually all anti-neoplastic regimens.
If these cells were forced to proliferate more rapidly their
susceptibility profile would likely change.
[0888] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a stimulator of B cell
production in pathologies such as AIDS, chronic lymphocyte disorder
and/or Common Variable Immundificiency.
[0889] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a therapy for generation
and/or regeneration of lymphoid tissues following surgery, trauma
or genetic defect. In another specific embodiment, albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention are used in the pretreatment of
bone marrow samples prior to transplant.
[0890] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a gene-based therapy for
genetically inherited disorders resulting in
immuno-incompetence/immunodeficiency such as observed among SCID
patients.
[0891] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a means of activating
monocytes/macrophages to defend against parasitic diseases that
effect monocytes such as Leishmania.
[0892] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a means of regulating
secreted cytokines that are elicited by polypeptides of the
invention.
[0893] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used in one or more of the applications
described herein, as they may apply to veterinary medicine.
[0894] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a means of blocking various
aspects of immune responses to foreign agents or self. Examples of
diseases or conditions in which blocking of certain aspects of
immune responses may be desired include autoimmune disorders such
as lupus, and arthritis, as well as immunoresponsiveness to skin
allergies, inflammation, bowel disease, injury and
diseases/disorders associated with pathogens.
[0895] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a therapy for preventing the
B cell proliferation and Ig secretion associated with autoimmune
diseases such as idiopathic thrombocytopenic purpura, systemic
lupus erythematosus and multiple sclerosis.
[0896] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention are used as a inhibitor of
B and/or T cell migration in endothelial cells. This activity
disrupts tissue architecture or cognate responses and is useful,
for example in disrupting immune responses, and blocking
sepsis.
[0897] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a therapy for chronic
hypergammaglobulinemia evident in such diseases as monoclonal
gammopathy of undetermined significance (MGUS), Waldenstrom's
disease, related idiopathic monoclonal gammopathies, and
plasmacytomas.
[0898] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be employed for instance to inhibit
polypeptide chemotaxis and activation of macrophages and their
precursors, and of neutrophils, basophils, B lymphocytes and same
T-cell subsets, e.g., activated and CD8 cytotoxic T cells and
natural killer cells, in certain autoimmune and chronic
inflammatory and infective diseases. Examples of autoimmune
diseases are described herein and include multiple sclerosis, and
insulin-dependent diabetes.
[0899] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may also be employed to treat idiopathic hyper-eosinophilic
syndrome by, for example, preventing eosinophil production and
migration.
[0900] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to enhance or inhibit complement
mediated cell lysis.
[0901] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to enhance or inhibit antibody
dependent cellular cytotoxicity.
[0902] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may also be employed for treating
atherosclerosis, for example, by preventing monocyte infiltration
in the artery wall.
[0903] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be employed to treat adult
respiratory distress syndrome (ARDS).
[0904] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be useful for stimulating wound and
tissue repair, stimulating angiogenesis, and/or stimulating the
repair of vascular or lymphatic diseases or disorders.
Additionally, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to stimulate the regeneration of mucosal surfaces.
[0905] In a specific embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used to diagnose, prognose, treat, and/or
prevent a disorder characterized by primary or acquired
immunodeficiency, deficient serum immunoglobulin production,
recurrent infections, and/or immune system dysfunction. Moreover,
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention may be used to treat or
prevent infections of the joints, hones, skin, and/or parotid
glands, blood-borne infections (e.g. sepsis, meningitis, septic
arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those
disclosed herein), inflammatory disorders, and malignancies, and/or
any disease or disorder or condition associated with these
infections, diseases, disorders and/or malignancies) including, but
not limited to, CV ID, other primary immune deficiencies, HIV
disease, CLL, recurrent bronchitis, sinusitis, otitis media,
conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster
(e.g., severe herpes luster), and/or pneumocystis carnii, Other
diseases and disorders that may be prevented, diagnosed, prognosed,
and/or treated with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, but are not limited to. HIV infection, HTLV-BLV infection,
lymphopenia, phagocyte bactericidal dysfunction anemia,
thrombocytopenia, and hemoglobinuria.
[0906] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used to treat, and/or diagnose an individual
having common variable immunodeficiency disease ("CVID"; also known
as "acquired agammaglobulinemia" and "acquired
hypogammaglobulinemia") or a subset of this disease.
[0907] In a specific embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be used to diagnose, prognose, prevent, and/or
treat cancers or neoplasms including immune cell or immune
tissue-related cancers or neoplasms. Examples of cancers or
neoplasms that may be prevented, diagnosed, or treated by fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention include, but are not limited to,
acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's
disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL)
Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma,
Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and
disorders described in the section entitled "Hyperproliferative
Disorders" elsewhere herein.
[0908] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a therapy for decreasing
cellular proliferation of Large B-cell Lymphomas.
[0909] In another specific embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used as a means of decreasing the
involvement of B cells and Ig associated with Chronic Myelogenous
Leukemia.
[0910] In specific embodiments, the compositions of the invention
are used as an agent to boost immunoresponsiveness among B cell
immunodeficient individuals, such as, for example, an individual
who has undergone a partial or complete splenectomy.
[0911] Blood-Related Disorders
[0912] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to modulate hemostatic (the stopping of bleeding) or
thrombolytic (clot dissolving) activity. For example, by increasing
hemostatic or thrombolytic activity, fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention could be used to treat or prevent blood
coagulation diseases, disorders, and/or conditions (e.g.,
afibrinogenemia, factor deficiencies, hemophilia), blood platelet
diseases, disorders, and/or conditions (e.g., thrombocytopenia), or
wounds resulting from trauma, surgery, or other causes.
Alternatively, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
that can decrease hemostatic or thrombolytic activity could be used
to inhibit or dissolve clotting. These molecules could be important
in the treatment or prevention of heart attacks (infarction),
strokes, or scarring.
[0913] In specific embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be used to prevent, diagnose, prognose, and/or
treat thrombosis, arterial thrombosis, venous thrombosis,
thromboembolism, pulmonary embolism, atherosclerosis, myocardial
infarction, transient ischemic attack, unstable angina. In specific
embodiments, the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used for the prevention of occulsion of saphenous grafts,
for reducing the risk of periprocedural thrombosis as might
accompany angioplasty, procedures, for reducing the risk of stroke
in patients with atrial fibrillation including nonrheumatic atrial
fibrillation, for reducing the risk of embolism associated with
mechanical heart valves and or mitral valves disease. Other uses
for the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
include, but are not limited to, the prevention of occlusions in
extracorporeal devices (e.g., intravascular cannulas, vascular
access shunts in hemodialysis patients, hemodialysis machines, and
cardiopulmonary bypass machines).
[0914] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention, may be used to prevent, diagnose, prognose,
and/or treat diseases and disorders of the blood and/or blood
forming organs associated with the tissue(s) in which the
polypeptide of the invention is expressed.
[0915] The fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention may be used to
modulate hematopoietic activity (the formation of blood cells). For
example, the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to increase the quantity of all or subsets of blood
cells, such as, for example, erythrocytes, lymphocytes (B or T
cells), myeloid cells (e.g., basophils, eosinophils, neutrophils,
mast cells, macrophages) and platelets. The ability to decrease the
quantity of blood cells or subsets of blood cells may be useful in
the prevention, detection, diagnosis and/or treatment of anemias
and leukopenias described below. Alternatively, the albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention may be used to decrease the
quantity of all or subsets of blood cells, such as, for example,
erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g.,
basophils, eosinophils, neutrophils, mast cells, macrophages) and
platelets. The ability to decrease the quantity of blood cells or
subsets of blood cells may be useful in the prevention, detection,
diagnosis and/or treatment of leukocytoses, such as, for example
eosinophilia.
[0916] The fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention may be used to
prevent, treat, or diagnose blood dyscrasia.
[0917] Anemias are conditions in which the number of red blood
cells or amount of hemoglobin (the protein that carries oxygen) in
them is below normal. Anemia may be caused by excessive bleeding,
decreased red blood cell production, or increased red blood cell
destruction (hemolysis). The albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in treating, preventing, and/or
diagnosing anemias. Anemias that may be treated prevented or
diagnosed by the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include iron deficiency anemia, hypochromic anemia, microcytic
anemia, chlorosis, hereditary sideroblastic anemia, idiopathic
acquired sideroblastic anemia, red cell aplasia, megaloblastic
anemia (e.g., pernicious anemia, (vitamin B12 deficiency) and folic
acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g.,
autoimmune helolytic anemia, microangiopathic hemolytic anemia, and
paroxysmal nocturnal hemoglobinuria). The albumin fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be useful in treating, preventing,
and/or diagnosing anemias associated with diseases including but
not limited to, anemias associated with systemic lupus
erythematosus, cancers, lymphomas, chronic renal disease, and
enlarged spleens. The albumin fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention may be useful in treating, preventing, and/or diagnosing
anemias arising from drug treatments such as anemias associated
with methyldopa, dapsone, and/or sulfadrugs. Additionally, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention may be useful in treating,
preventing, and/or diagnosing anemias associated with abnormal red
blood cell architecture including but not limited to, hereditary
spherocytosis, hereditary elliptocytosis, glucose-6-phosphate
dehydrogenase deficiency, and sickle cell anemia.
[0918] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in treating, preventing, and/or diagnosing hemoglobin
abnormalities, (e.g., those associated with sickle cell anemia,
hemoglobin C disease, hemoglobin S-C disease, and hemoglobin E
disease). Additionally, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating thalassemias, including, but not
limited to, major and minor forms of alpha-thalassemia and
beta-thalassemia.
[0919] In another embodiment, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating bleeding disorders including, but not
limited to, thrombocytopenia (e.g., idiopathic thrombocytopenic
purpura, and thrombotic thrombocytopenic purpura), Von Willebrand's
disease, hereditary platelet disorders (e.g., storage pool disease
such as Chediak-Higashi and Hermansky-Pudiak syndromes, thromboxane
A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome),
hemolytic-uremic syndrome, hemophelias such as hemophelia A or
Factor VH deficiency and Christmas disease or Factor IX deficiency,
Hereditary Hemorhhagic Telangiectsia, also known as
Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein
purpura) and disseminated intravascular coagulation.
[0920] The effect of the albumin fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention on the clotting time of blood may be monitored using any
of the clotting tests known in the art including, but not limited
to, whole blood partial thromboplastin time (PIT), the activated
partial thromboplastin time (aPTT), the activated clotting time
(ACT), the recalcified activated clotting time, or the Lee-White
Clotting time.
[0921] Several diseases and a variety of drugs can cause platelet
dysfunction. Thus, in a specific embodiment, the albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention may be useful in diagnosing,
prognosing, preventing, and/or treating acquired platelet
dysfunction such as platelet dysfunction accompanying kidney
failure, leukemia, multiple myeloma, cirrhosis of the liver, and
systemic lupus erythematosus as well as platelet dysfunction
associated with drug treatments, including treatment with aspirin,
ticlopidine, nonsteroidal anti-inflammatory drugs (used for
arthritis, pain, and sprains), and penicillin in high doses.
[0922] In another embodiment, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating diseases and disorders characterized by
or associated with increased or decreased numbers of white blood
cells. Leukopenia occurs when the number of white blood cells
decreases below normal. Leukopenias include, but are not limited
to, neutropenia and lymphocytopenia. An increase in the number of
white blood cells compared to normal is known as leukocytosis. The
body generates increased numbers of white blood cells during
infection. Thus, leukocytosis may simply be a normal physiological
parameter that reflects infection. Alternatively, leukocytosis may
be an indicator of injury or other disease such as cancer.
Leokocytoses, include but are not limited to, eosinophilia, and
accumulations of macrophages. In specific embodiments, the albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention may be useful in
diagnosing; prognosing, preventing, and/or treating leukopenia. In
other specific embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating leukocytosis.
[0923] Leukopenia may be a generalized decreased in all types of
white blood cells, or may be a specific depletion of particular
types of white blood cells. Thus, in specific embodiments, the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention may be useful in
diagnosing, prognosing, preventing, and/or treating decreases in
neutrophil numbers, known as neutropenia. Neutropenias that may be
diagnosed, prognosed, prevented, and/or treated by the albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention include, but are not
limited to, infantile genetic agranulocytosis, familial
neutropenia, cyclic neutropenia, neutropenias resulting from or
associated with dietary deficiencies (e.g., vitamin B12 deficiency
or folic acid deficiency), neutropenias resulting from or
associated with drug treatments (e.g., antibiotic regimens such as
penicillin treatment, sulfonamide treatment, anticoagulant
treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer
chemotherapy), and neutropenias resulting from increased neutrophil
destruction that may occur in association with some bacterial or
viral infections, allergic disorders, autoimmune diseases,
conditions in which an individual has an enlarged spleen (e.g.,
Felty syndrome, malaria and sarcoidosis), and some drug treatment
regimens.
[0924] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in diagnosing, prognosing, preventing, and/or
treating lymphocytopenias (decreased numbers of B and/or T
lymphocytes), including, but not limited to, lymphocytopenias
resulting from OT associated with stress, drug treatments (e.g.,
drug treatment with corticosteroids, cancer chemotherapies, and/or
radiation therapies), AIDS infection and/or other diseases such as,
for example, cancer, rheumatoid arthritis, systemic lupus
erythematosus, chronic infections, some viral infections and/or
hereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich
Syndrome, severe combined immunodeficiency, ataxia
telangiectsia).
[0925] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in diagnosing, prognosing, preventing, and/or
treating diseases and disorders associated with macrophage numbers
and/or macrophage function including, but not limited to, Gaucher's
disease, Niemann-Pick disease, Letterer-Siwe disease and
Hand-Schuller-Christian disease.
[0926] In another embodiment, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating diseases and disorders associated with
eosinophil numbers and/or eosinophil function including, but not
limited to, idiopathic hypereosinophilic syndrome,
eosinophilia-myalgia syndrome, and Hand-Schuller-Christian
disease.
[0927] In yet another embodiment, the albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating leukemias and lymphomas including, but
not limited to, acute lymphocytic (lymphpblastic) leukemia (ALL),
acute myeloid (myelocytic, myelogenous, myeloblastic, or
myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., B
cell leukemias, T cell leukemias, Sezary syndrome, and Hairy cell
leukemia), chronic myelocytic (myeloid, myelogenous, or
granulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma,
Burkitt's lymphoma, and mycosis fungoides.
[0928] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in diagnosing, prognosing,
preventing, and/or treating diseases and disorders of plasma cells
including, but not limited to, plasma cell dyscrasias, monoclonal
gammaopathies, monoclonal gammopathies of undetermined
significance, multiple myeloma, macroglobulinemia, Waldenstrom's
macroglobulinemia, cryoglobulinemic, and Raynaud's phenomenon.
[0929] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in treating, preventing, and/or
diagnosing myeloproliferative disorders, including but not limited
to, polycythemia vera, relative polycythemia, secondary
polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myclod
metaplasia, thrombocythemia, (including both primary and secondary
thrombocythemia) and chronic myelocytic leukemia.
[0930] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful as a treatment prior to surgery, to
increase blood cell production.
[0931] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful as an agent to enhance the
migration, phagocytosis, superoxide production, antibody dependent
cellular cytotoxicity of neutrophils, eosinophils and
macrophages.
[0932] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful as an agent to increase the number
of stem cells in circulation prior to stem cells pheresis. In
another specific embodiment, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful as an agent to increase the nut
ulcer of stem cells in circulation prior to platelet pheresis.
[0933] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful as an agent to increase cytokine
production.
[0934] In other embodiments, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be useful in preventing, diagnosing, and/or
treating primary hematopoietic disorders.
[0935] Hyperproliferative Disorders
[0936] In certain embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention can be used to treat or detect hyperproliferative
disorders, including neoplasms. Albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may inhibit the proliferation of the disorder
through direct or indirect interactions. Alternatively, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention may proliferate other cells which
can inhibit the hyperproliferative disorder.
[0937] For example, by increasing an immune response, particularly
increasing antigenic qualities of the hyperproliferative disorder
or by proliferating, differentiating, or mobilizing T-cells,
hyperproliferative disorders can be treated. This immune response
may be increased by either enhancing an existing immune response,
or by initiating a new immune response. Alternatively, decreasing
an immune response may also be a method of treating
hyperproliferative disorders, such as a chemotherapeutic agent.
[0938] Examples of hyperproliferative disorders that can be treated
or detected by fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, but are not limited to neoplasms located in the colon,
abdomen, bone, breast, digestive system, liver, pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous
(central and peripheral), lymphatic system, pelvis, skin, soft
tissue, spleen, thorax, and urogenital tract.
[0939] Similarly, other hyperproliferative disorders can also be
treated or detected by fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention.
Examples of such hyperproliferative disorders include, but are not
limited to: Acute Childhood Lymphoblastic Leukemia, Acute
lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid
Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular
Cancer, Adult (Primary) Liver Cancer. Adult Acute Lymphocytic
Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease,
Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult
Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft
Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies,
Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone
Cancer, Brain Stern Glioma, Brain Tumors, Breast Cancer, Cancer of
the Renal Pelvis and Ureter, Central Nervous System (Primary)
Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma,
Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)
Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood
Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia,
Childhood Brain Stern Glioma, Childhood Cerebellar Astrocytoma,
Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell
Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma,
Childhood Hypothalamic and Visual Pathway Glioma, Childhood
Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood
Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial
Primitive Neuroectodermal Tumors, Childhood Primary-Liver Cancer,
Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma,
Childhood Visual Pathway and Hypothalamic Glioma, Chronic
Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer,
Cutaneous T-Cell Lymphoma. Endocrine Pancreas islet Cell Carcinoma,
Endometrial Cancer. Ependymoma, Epithelial Cancer, Esophageal
Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic
Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor,
Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer,
Gaucher's Disease, Gallbladder Cancer. Gastric Cancer,
Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ
Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia,
Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease,
Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer,
Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma,
Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer,
Laryngeal Cancer, Lip and Oral Cavity Cancer, liver Cancer, Lung
Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male
Breast Cancer, Malignant Mesothelioma, Malignant Thymoma,
Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary
Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,
Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple
Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous
Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal
Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer,
Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma
Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic
Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant
Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,
Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian
Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant
Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura,
Parathyroid Career, Penile Cancer, Pheochromocytoma, Pituitary
Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central
Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer,
Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,
Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck
Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal
and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and
Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic
Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer.
Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's
Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[0940] In another preferred embodiment, albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to diagnose, prognose, prevent,
and/or treat premalignant conditions and to prevent progression to
a neoplastic or malignant state, including but not limited to those
disorders described above, Such uses are indicated in conditions
known or suspected of preceding progression to neoplasia or cancer,
in particular, where non-neoplastic cell growth consisting of
hyperplasia, metaplasia, or most particularly, dysplasia has
occurred (for review of such abnormal growth conditions, see
Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders
Co., Philadelphia, pp. 68-79.)
[0941] Hyperplasia is a form of controlled cell proliferation,
involving an increase in cell number in a tissue or organ, without
significant alteration in structure or function. Hyperplastic
disorders which can be diagnosed, prognosed, prevented, and/or
treated with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, but are not limited to, angiofollicular mediastinal lymph
node hyperplasia, angiolymphoid hyperplasia with eosinophilia,
atypical melanocytic hyperplasia, basal cell hyperplasia, benign
giant lymph node hyperplasia, cementum hyperplasia, congenital
adrenal hyperplasia, congenital sebaceous hyperplasia, cystic
hyperplasia, cystic hyperplasia of the breast, denture hyperplasia,
ductal hyperplasia, endometrial hyperplasia, fibromuscular
hyperplasia, focal epithelial hyperplasia, gingival hyperplasia,
inflammatory fibrous hyperplasia, inflammatory papillary
hyperplasia, intravascular papillary endothelial hyperplasia,
nodular hyperplasia of prostate, nodular regenerative hyperplasia,
pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia,
and verrucous hyperplasia.
[0942] Metaplasia is a form of controlled cell growth in which one
type of adult or fully differentiated cell substitutes for another
type of adult cell. Metaplastic disorders which can be diagnosed,
prognosed, prevented, and/or treated with fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention include, but are not limited to, agnogenic myeloid
metaplasia, apocrine metaplasia, atypical metaplasia,
autoparenchymatous metaplasia, connective tissue metaplasia,
epithelial metaplasia, intestinal metaplasia, metaplastic anemia,
metaplastic ossification, metaplastic polyps, myeloid metaplasia,
primary myeloid metaplasia, secondary myeloid metaplasia, squamous
metaplasia, squamous metaplasia of amnion, and symptomatic myeloid
metaplasia.
[0943] Dysplasia is frequently a forerunner of cancer, and is found
mainly in the epithelia; it is the most disorderly form of
non-neoplastic cell growth, involving a loss in individual cell
uniformity and in the architectural orientation of cells.
Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism. Dysplasia characteristically
occurs where there exists chronic irritation or inflammation.
Dysplastic disorders which can be diagnosed, prognosed, prevented,
and/or treated with fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
include, but are not limited to, anhidrotic ectodermal dysplasia,
anterofacial dysplasia, asphyxiating thoracic dysplasia,
atriodigital dysplasia, bronchopulmonary dysplasia, cerebral
dysplasia, cervical dysplasia, chondroectodermal dysplasia,
cleidocranial dysplasia, congenital ectodermal dysplasia,
craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial
dysplasia, ectodermal dysplasia, enamel dysplasia,
encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,
epithelial dysplasia, faciodigitogenital dysplasia, familial
fibrous dysplasia of jaws, familial white folded dysplasia,
fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous
dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal
dysplasia, hypohidrotic ectodermal dysplasia, lvmphopenic thymic
dysplasia, mammary dysplasia, mandibulufacial dysplasia,
metaphysial dysplasia, Mondini dysplasia, monostotic fibrous
dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia,
oculoauriculovertebral dysplasia, oculodentodigital dysplasia,
oculoverrehral dysplasia, odontogenic dysplasia,
ophthalmomandibulomelic dysplasia, periapical cemental dysplasia,
polyostotic fibrous dysplasia, pseudoachondroplastic
spondyloepiphysial dysplasia, retinal dysplasia, septo-optic
dysplasia, spondyloepiphysial dysplasia, and ventriculoradial
dysplasia.
[0944] Additional pre-neoplastic disorders which can be diagnosed,
prognosed, prevented, and/or treated with fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention include, but are not limited to, benign
dysproliferative disorders (e.g., benign tumors, fibrocystic
conditions, tissue hypertrophy, intestinal polyps, colon polyps,
and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease,
Farmer's Skin, solar cheilitis, and solar keratosis.
[0945] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention, may be used to diagnose and/or prognose disorders
associated with the tissue(s) in which the polypeptide of the
invention is expressed.
[0946] In another embodiment, albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention conjugated to a toxin or a radioactive isotope, as
described herein, may be used to treat cancers and neoplasms,
including, but not limited to, those described herein. In a further
preferred embodiment, albumin fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention conjugated to a toxin or a radioactive isotope, as
described herein, may be used to treat acute myelogenous
leukemia.
[0947] Additionally, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may affect apoptosis, and therefore, would be useful in treating a
number of diseases associated with increased cell survival or the
inhibition of apoptosis. For example, diseases associated with
increased cell survival or the inhibition of apoptosis that could
be diagnosed, prognosed, prevented, and/or treated by
polynucleotides, polypeptides, and/or agonists or antagonists of
the invention, include cancers (such as follicular lymphomas,
carcinomas with p53 mutations, and hormone-dependent tumors,
including, but not limited to colon cancer, cardiac tumors,
pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung
cancer, intestinal cancer, testicular cancer, stomach cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma,
adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian cancer); autoimmune disorders such as, multiple sclerosis,
Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis,
Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erytheutatosus and immune-related glomerulonephritis and rheumatoid
arthritis) and viral infections (such as herpes viruses, pox
viruses and adenoviruses), inflammation, graft v. host disease,
acute graft rejection, and chronic graft rejection.
[0948] In preferred embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention are used to inhibit growth, progression, and/or
metastasis of cancers, in particular those listed above.
[0949] Additional diseases or conditions associated with increased
cell survival that could be diagnosed, prognosed, prevented, and/or
treated by fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention, include, but are
not limited to, progression, and/or metastases of malignancies and
related disorders such as leukemia (including acute leukemias
(e.g., acute lymphocytic leukemia, acute myelocytic leukemia
(including myeloblastic, promyelocytic, myelomonocytic, monocytic,
and erythroleukemia)) and chronic leukemias (e.g., chronic
myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease
and non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including,
but not limited to, sarcomas and carcinomas such as fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, emangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0950] Diseases associated with increased apoptosis that could be
diagnosed, prognosed, prevented, and/or treated by fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention, include AIDS; neurodegenerative
disorders (such as Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar
degeneration and brain tumor or prior associated disease);
autoimmune disorders (such as, multiple sclerosis, Sjogren's
syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's
disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid
arthritis) myelodysplastic syndromes (such as aplastic anemia),
graft v. host disease, ischemic injury (such as that caused by
myocardial infarction, stroke and reperfusion injury), liver injury
(e.g., hepatitis related liver injury, ischemia/reperfusion injury,
cholestosis (bile duct injury) and liver cancer); toxin-induced
liver disease (such as that caused by alcohol), septic shock,
cachexia and anorexia.
[0951] Hyperproliferative diseases and/or disorders that could be
diagnosed, prognosed, prevented, and/or treated by fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention, include, but are not limited to,
neoplasms located in the liver, abdomen, bone, breast, digestive
system, pancreas, peritoneum, endocrine glands (adrenal,
parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye,
head and neck, nervous system (central and peripheral), lymphatic
system, pelvis, skin, soft tissue, spleen, thorax, and urogenital
tract.
[0952] Similarly, other hyperproliferative disorders can also be
diagnosed, prognosed, prevented, and/or treated by fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention. Examples of such hyperproliferative
disorders include, but are not limited to: hypergammaglobulinemia,
lymphoproliferative disorders, paraproteinemias, purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia,
Gaucher's Disease, histiocytosis, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[0953] Another preferred embodiment utilizes polynucleotides
encoding albumin fusion proteins of the invention to inhibit
aberrant cellular division, by gene therapy using the present
invention, and/or protein fusions or fragments thereof.
[0954] Thus, the present invention provides a method for treating
cell proliferative disorders by inserting into an abnormally
proliferating cell a polynucleotide encoding an albumin fusion
protein of the present invention, wherein said polynucleotide
represses said expression.
[0955] Another embodiment of the present invention provides a
method of treating cell-proliferative disorders in individuals
comprising administration of one or more active gene copies of the
present invention to an abnormally proliferating cell or cells. In
a preferred embodiment, polynucleotides of the present invention is
a DNA construct comprising a recombinant expression vector
effective in expressing a DNA sequence encoding said
polynucleotides. In another preferred embodiment of the present
invention, the DNA construct encoding the fusion protein of the
present invention is inserted into cells to be treated utilizing a
retrovirus, or more preferably an adenoviral vector (See G J.
Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated
by reference). In a most preferred embodiment, the viral vector is
defective and will not transform non-proliferating cells, only
proliferating cells. Moreover, in a preferred embodiment, the
polynucleotides of the present invention inserted into
proliferating cells either alone, or in combination with or fused
to other polynucleotides, can then be modulated via an external
stimulus (i.e., magnetic, specific small molecule, chemical, or
drug administration, etc.), which acts upon the promoter upstream
of said polynucleotides to induce expression of the encoded protein
product. As such the beneficial therapeutic affect of the present
invention may be expressly modulated (i.e., to increase, decrease,
or inhibit expression of the present invention) based upon said
external stimulus.
[0956] polynucleotides of the present invention may be useful in
repressing expression of oncogenic genes or antigens. By
"repressing expression of the oncogenic genes" is intended the
suppression of the transcription of the gene, the degradation of
the gene transcript (pre-message RNA), the inhibition of splicing,
the destruction of the messenger RNA, the prevention of the
post-translational modifications of the protein, the destruction of
the protein, or the inhibition of the normal function of the
protein.
[0957] For local administration to abnormally proliferating cells,
polynucleotides of the present invention may be administered by any
method known to those of skill in the art including, but not
limited to transfection, electroporation, microinjection of cells,
or in vehicles such as liposomes, lipofectin, or as naked
polynucleotides, or any other method described throughout the
specification. The polynucleotide of the present invention may be
delivered by known gene delivery systems such as, but not limited
to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hooke,
Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci.
U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.
Cell. Biol. 5:3403 (1985) or other efficient DNA delivery systems
(Yates et al., Nature 313:812 (1985)) known to those skilled in the
art. These references are exemplary only and are hereby
incorporated by reference. In order to specifically deliver or
transfect cells which are abnormally proliferating and spare
non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as described in the art and elsewhere herein) delivery
system known to those of skill in the art. Since host DNA
replication is required for retroviral DNA to integrate and the
retrovirus will be unable to self replicate due to the lack of the
retrovirus genes needed for its life cycle. Utilizing such a
retroviral delivery system for polynucleotides of the present
invention will target said gene and constructs to abnormally
proliferating cells and will spare the non-dividing normal
cells.
[0958] The polynucleotides of the present invention may be
delivered directly to cell proliferative disorder/disease sites in
internal organs, body cavities and the like by use of imaging
devices used to guide an injecting needle directly to the disease
site. Tire polynucleotides of the present invention may also be
administered to disease sites at the time of surgical
intervention.
[0959] By "cell proliferative disease" is meant any human or animal
disease or disorder, affecting any one or any combination of
organs, cavities, or body parts, which is characterized by single
or multiple local abnormal proliferations of cells, groups of
cells, or tissues, whether benign or malignant.
[0960] Any amount of the polynucleotides of the present invention
may be administered as long as it has a biologically inhibiting
effect on the proliferation of the treated cells. Moreover, it is
possible to administer more than one of the polynucleotide of the
present invention simultaneously to the same site. By "biologically
inhibiting" is meant partial or total growth inhibition as well as
decreases in the rate of proliferation or growth of the cells. The
biologically inhibitory dose may be determined by assessing the
effects of the polynucleotides of the present invention on target
malignant or abnormally proliferating cell growth in tissue
culture, tumor growth in animals and cell cultures, or any other
method known to one of ordinary skill in the art.
[0961] Moreover, fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
of the present invention are useful in inhibiting the angiogenesis
of proliferative cells or tissues, either alone, as a protein
fusion, or in combination with other polypeptides directly or
indirectly, as described elsewhere herein. In a most preferred
embodiment, said anti-angiogenesis effect may be achieved
indirectly, for example, through the inhibition of hematopoietic,
tumor-specific cells, such as tumor-associated macrophages (See
Joseph I B, et al. J. Natl Cancer Inst. 90(21):1648-53 (1998),
which is hereby incorporated by reference).
[0962] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in inhibiting proliferative cells or tissues through
the induction of apoptosis. These fusion proteins and/or
polynucleotides may act either directly, or indirectly to induce
apoptosis of proliferative cells and tissues, for example in the
activation of a death-domain receptor, such as minor necrosis
factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related
apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See
Schulze-Osthoff K, Eur J Biochem 254(3):439-59 (1998), which is
hereby incorporated by reference). Moreover, in another preferred
embodiment of the present invention, these fusion proteins and/or
polynucleotides may induce apoptosis through other mechanisms, such
as in the activation of other proteins which will activate
apoptosis, or through stimulating the expression of these proteins,
either alone or in combination with small molecule drugs or
adjuvants, such as apoptonin, galectins, thioredoxins,
anti-inflammatory proteins (See for example, Mutat Res
400(1-2):447-55 (1998), Med Hypotheses. 50(5):423-33 (1998), Chem
Biol Interact. April 24;111-112:23-34 (1998), 3 Mol Med.
76(6):402-12 (1998), Int J Tissue React; 20(1):3-15 (1998), which
are all hereby incorporated by reference).
[0963] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
are useful in inhibiting the metastasis of proliferative cells or
tissues. Inhibition may occur as a direct result of administering
these albumin fusion proteins and/or polynucleotides, or
indirectly, such as activating the expression of proteins known to
inhibit metastasis, for example alpha 4 integrins, (See, Curr Top
Microbiol Immunol 1998; 231:125-41, which is hereby incorporated by
reference). Such therapeutic affects of the present invention may
be achieved either alone, or in combination with small molecule
drugs or adjuvants.
[0964] In another embodiment, the invention provides a method of
delivering compositions containing the albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention to targeted cells expressing the a
polypeptide bound by, that binds to, or associates with an albumin
fusion protein of the invention. Albumin fusion proteins of the
invention may be associated with heterologous polypeptides,
heterologous nucleic acids, toxins, or prodrugs via hydrophobic,
hydrophilic, ionic and/or covalent interactions.
[0965] Albumin fusion proteins of the invention are useful in
enhancing the immunogenicity and/or antigenicity of proliferating
cells or tissues, either directly, such as would occur if the
albumin fusion proteins of the invention `vaccinated` the immune
response to respond to proliferative antigens and immunogens, or
indirectly, such as in activating the expression of proteins known
to enhance the immune response (e.g. chemokines to said antigens
and immunogens.
[0966] Cardiovascular Disorders
[0967] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
may be used to treat, prevent, diagnose, and/or prognose
cardiovascular disorders, including, but not limited to, peripheral
artery disease, such as limb ischemia.
[0968] Cardiovascular disorders include, but are not limited to,
cardiovascular abnormalities, such as arterio-arterial fistula,
arteriovenous fistula, cerebral arteriovenous malformations,
congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
Congenital heart defects include, but are not limited to, aortic
coarctation, ear triatriatum, coronary vessel anomalies, crisscross
heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,
Eisenmenger complex, hypoplastic left heart syndrome, levocardia,
tetralogy of tailor, transposition of great vessels, double outlet
right ventricle, tricuspid atresia, persistent truncus arteriosus,
and heart septal defects, such as aortopulmonary septal defect,
endocardial cushion defects, Lutembacher's Syndrome, trilogy of
Fallot, ventricular heart septal defects.
[0969] Cardiovascular disorders also include, but are not limited
to, heart disease, such as arrhythmias, carcinoid heart disease,
high cardiac output, low cardiac output, cardiac tamponade,
endocarditis (including bacterial), heart aneurysm, cardiac arrest,
congestive heart failure, congestive cardiomyopathy, paroxysmal
dyspnea, cardiac edema, heart hypertrophy, congestive
cardiomyopathy, left ventricular hypertrophy, right ventricular
hypertrophy, post-infarction heart rupture, ventricular septal
rupture, heart valve diseases, myocardial diseases, myocardial
ischemia, pericardial effusion, pericarditis (including
constrictive and tuberculous), pneumopericardium,
postpericardiotomy syndrome, pulmonary heart disease, rheumatic
heart disease, ventricular dysfunction, hyperemia, cardiovascular
pregnancy complications, Scimitar Syndrome, cardiovascular
syphilis, and cardiovascular tuberculosis.
[0970] Arrhythmias include, but are not limited to, sinus
arrhythmia, atrial fibrillation, atrial flutter, bradycardia,
extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine
Syndrome, Mahaim-type pre-excitation syndrome,
Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias,
and ventricular fibrillation. Tachycardias include paroxysmal
tachycardia, supraventricular tachycardia, accelerated
idioventricular rhythm, atrioventricular nodal reentry tachycardia,
ample atrial tachycardia, ectopic junctional tachycardia,
sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades
de Pointes, and ventricular tachycardia.
[0971] Heart valve diseases include, but are not limited to, aortic
valve insufficiency, aortic valve stenosis, hear murmurs, aortic
valve prolapse, mitral valve prolapse, tricuspid valve prolapse,
mitral valve insufficiency, mitral valve stenosis, pulmonary
atresia, pulmonary valve insufficiency, pulmonary valve stenosis,
tricuspid atresia, tricuspid valve insufficiency, and tricuspid
valve stenosis.
[0972] Myocardial diseases include, but are not limited to,
alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic
cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular
stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy,
endocardial fibroelastosis, endomyocardial fibrosis, Kearns
Syndrome, myocardial reperfusion injury, and myocarditis.
[0973] Myocardial ischemias include, but are not limited to,
coronary disease, such as angina pectoris, coronary aneurysm,
coronary arteriosclerosis, coronary thrombosis, coronary vasospasm,
myocardial infarction and myocardial stunning.
[0974] Cardiovascular diseases also include vascular diseases such
as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,
Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome,
Sturge-Weber Syndrome, angioneurotic edema, aortic diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular disorders, diabetic angiopathies, diabetic
retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids,
hepatic veno-occlusive disease, hypertension, hypotension,
ischemia, peripheral vascular diseases, phlebitis, pulmonary
veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal
vein occlusion, Scimitar syndrome, superior vena cava syndrome,
telangiectasia, atacia telangiectasia, hereditary hemorrhagic
telangiectasia, varicocele, varicose veins, varicose ulcer,
vasculitis, and venous insufficiency.
[0975] Aneurysms include, but are not limited to, dissecting
aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms,
aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart
aneurysms, and iliac aneurysms.
[0976] Arterial occlusive diseases include, but are not limited to,
arteriosclerosis, intermittent claudication, carotid stenosis,
fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya
disease, renal artery obstruction, retinal artery occlusion, and
thromboangiitis obliterans.
[0977] Cerebrovascular disorders include, but are not limited to,
carotid artery diseases, cerebral amyloid angiopathy, cerebral
aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral
arteriovenous malformation, cerebral artery diseases, cerebral
embolism and thrombosis, carotid artery thrombosis, sinus
thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural
hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral
infarction, cerebral ischemia (including transient), subclavian
steal syndrome, periventricular leukomalacia, vascular headache,
cluster headache, migraine, and vertebrobasilar insufficiency.
[0978] Embolisms include, but are not limited to, air embolisms,
amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome,
fat embolisms, pulmonary embolisms, and thromoboembolisms.
Thrombosis include, but are not limited to, coronary thrombosis,
hepatic vein thrombosis, retinal vein occlusion, carotid artery
thrombosis, sinus thrombosis. Wallenberg's syndrome, and
thrombophlebitis.
[0979] Ischemic disorders include, but are not limited to, cerebral
ischemia, ischemic colitis, compartment syndromes, anterior
compartment syndrome, myocardial ischemia, reperfusion injuries,
and peripheral limb ischemia. Vasculitis includes, but is not
limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss
Syndrome, mucocutaneous lymph node syndrome, thromboangiitis
obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura,
allergic cutaneous vasculitis, and Wegener's granulomatosis.
[0980] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be administered using any method known in the art, including,
but not limited to, direct needle injection at the delivery site,
intravenous injection, topical administration, catheter infusion,
biolistic injectors, particle accelerators, gelfoam sponge depots,
other commercially available depot materials, osmotic pumps, oral
or suppositorial solid pharmaceutical formulations, decanting or
topical applications during surgery, aerosol delivery. Such methods
are known in the art. Methods of delivering polynucleotides are
described in more detail herein.
[0981] Respiratory Disorders
[0982] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used to treat, prevent, diagnose, and/or prognose diseases
and/or disorders of the respiratory system.
[0983] Diseases and disorders of the respiratory system include,
but we not limited to, nasal vestibulitis, nonallergic rhinitis
(e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis,
vasomotor rhinitis), nasal polyps, and sinusitis, juvenile
angiofibromas, cancer of the nose and juvenile papillomas, vocal
cord polyps, nodules (singer's nodules), contact ulcers, vocal cord
paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial),
tonsillitis, tonsillar cellulitis, parapharyngeal abscess,
laryngitis, laryngoceles, and throat cancers (e.g., cancer of the
nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g.,
squamous cell carcinoma, small cell (oat cell) carcinoma, large
cell carcinoma, and adenocarcinoma), allergic disorders
(eosinophilic pneumonia, hypersensitivity pneumonitis (e.g.,
extrinsic allergic alveolitis, allergic interstitial pneumonitis,
organic dust pneumoconiosis, allergic bronchopulmonary
aspergillosis, asthma, Wegener's granulomatosis (granulomatous
vasculitis), Goodpasture's syndrome)), pneumonia (e.g., bacterial
pneumonia (e.g., Streptococcus pneumoniae (pneumoncoccal
pneumonia), Staphylococcus aureus (staphylococcal pneumonia),
Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and
Pseudomos spp.), Mycoplasma pneumoniae pneumonia, Heamphilas
influeuzae pneumonia, Legionella pneumophila (Legionnaires'
disease), and Chlamydia psittaci (Psittacosis)), and viral
pneumonia (e.g., influenza, chickenpox (varicella).
[0984] Additional diseases and disorders of the respiratory system
include, but are not limited to bronchiolitis, polio
(poliomyelitis), croup, respiratory syncytial viral infection,
mumps, erythema infectiosum (fifth disease), roseola infantum,
progressive rubella panencephalitis, german measles, and subacute
sclerosing panencephalitis), fungal pneumonia (e.g.,
Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal
infections in people with severely suppressed immune systems (e.g.,
cryptococcosis, caused by Cryptococcus neoformans; aspergillosis,
caused by Aspergillus spp.; candidiasis, caused by Candida; and
mucormycosis)). Pneumocystis carinii (pneumocystis pneumonia),
atypical pneumonias (e.g., Mycoplasmas and Chlamydia spp.),
opportunistic infection pneumonia, nosocomial pneumonia, chemical
pneumonitis, and aspiration pneumonia, pleural disorders (e.g.,
pleurisy, pleural effusion, and pneumothorax (e.g., simple
spontaneous pneumothorax, complicated spontaneous pneumothorax,
tension pneumothorax)), obstructive airway diseases (e.g., asthma,
chronic obstructive pulmonary disease (COPD), emphysema, chronic or
acute bronchitis), occupational lung diseases (e.g., silicosis,
black lung (coat workers' pneumoconiosis), asbestosis, berylliosis,
occupational asthma, byssinosis, and benign pneumoconioses),
Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g.,
fibrosing alveolitis, usual interstitial pneumonia), idiopathic
pulmonary fibrosis, desquamative interstitial pneumonia, lymphoid
interstitial pneumonia, histiocytosis X (e.g., Letterer-Siwe
disease, Hand-Schuller-Christian disease, eosinophilic granuloma),
idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary
alveolar proteinosis), Acute respiratory distress syndrome (also
called, e.g., adult respiratory distress syndrome), edema,
pulmonary embolism, bronchitis (e.g., viral, bacterial),
bronchiectasis, atelectasis, lung abscess (caused by, e.g.,
Staphylococcus nurses or Legionella pneumophila), and cystic
fibrosis.
[0985] Anti-Angiogenesis Activity
[0986] The naturally occurring balance between endogenous
stimulators and inhibitors of angiogenesis is one in which
inhibitory influences predominate. Rastinejad et al., Cell
56:345-355 (1989). In those rare instances in which
neovascularization occurs under normal physiological conditions,
such as wound healing, organ regeneration, embryonic development,
and female reproductive processes, angiogenesis is stringently
regulated and spatially and temporally delimited. Under conditions
of pathological angiogenesis such as that characterizing solid
tumor growth, these regulator controls fail. Unregulated
angiogenesis becomes pathologic and sustains progression of many
neoplastic and non-neoplastic diseases. A number of serious
diseases are dominated by abnormal neovascularization including
solid tumor growth and metastases, arthritis, some types of eye
disorders, and psoriasis. See, e.g., reviews by Moses et al.,
Biotech. 9:630-634 (1991); Folkman er at, N. Engl. J. Med.,
333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401411
(1985); Folkman, Advances in Cancer Research, eds. Klein and
Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am.
J. Opthalmol. 94:715-743 (1982); and Folkman et al. Science
221:719-725 (1983). In a number of pathological conditions, the
process of angiogenesis contributes to the disease state, For
example, significant data have accumulated which suggest that the
growth of solid tumors is dependent on angiogenesis. Folkman and
Klagshrun, Science 235:442-447 (1987).
[0987] The present invention provides for treatment of diseases or
disorders associated with neovascularization by administration of
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention. Malignant and metastatic
conditions which can be treated with the polynucleotides and
polypeptides, or agonists or antagonists of the invention include,
but are not limited to, malignancies, solid tumors, and cancers
described herein and otherwise known in the art (for a review of
such disorders, see Fishman er of Medicine. 2d Ed., J. B.
Lippincott Co Philadelphia (1985)). Thus, the present invention
provides a method of treating an angiogenesis-related disease
and/or disorder, comprising administering to an individual in need
thereof a therapeutically effective amount of an albumin fusion
protein of the invention and/or polynucleotides encoding an albumin
fusion protein of the invention. For example, fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be utilized in a variety of
additional methods in order to therapeutically treat a cancer or
tumor. Cancers which may be treated with fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention include, but are not limited to solid tumors,
including prostate, lung, breast, ovarian, stomach, pancreas,
larynx, esophagus, testes, liver, parotid, biliary tract, colon,
rectum, cervix, uterus, endometrium, kidney, bladder, thyroid
cancer; primary tumors and metastases; melanomas; glioblastoma;
Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer;
colorectal cancer; advanced malignancies; and blood born tumors
such as leukemias. For example, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention may be delivered topically, in order to treat cancers
such as skin cancer, head and neck tumors, breast tumors, and
Kaposi's sarcoma.
[0988] Within yet other aspects, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention may be utilized to treat superficial forms of bladder
cancer by, for example, intravesical administration. Albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention may be delivered directly into the
tumor, or near the tumor site, via injection or a catheter. Of
course, as the artisan of ordinary skill will appreciate, the
appropriate mode of administration will vary according to the
cancer to be treated. Other modes of delivery are discussed
herein.
[0989] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful in treating other disorders, besides cancers, which
involve angiogenesis. These disorders include, but are not limited
to: benign tumors, for example hemangiomas, acoustic neuron/as,
neurofibromas, trachomas, and pyogenic granulomas; artheroscleric
plaques; ocular angiogenic diseases, for example, diabetic
retinopathy, retinopathy of prematurity, macular degeneration,
corneal graft rejection, neovascular glaucoma, retrolental
fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia
(abnormal blood vessel growth) of the eye; rheumatoid arthritis;
psoriasis; delayed wound healing; endometriosis; vasculogenesis;
granulations; hypertrophic scars (keloids); nonunion fractures;
scleroderma; trachoma, vascular adhesions; myocardial angiogenesis;
coronary collaterals; cerebral collaterals; arteriovenous
malformations; ischemic limb angiogenesis; Osler-Webber Syndrome;
plaque neovascularization; telangiectasia; hemophiliac joints;
angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's
disease; and atherosclerosis.
[0990] Far example, within one aspect of the present invention
methods are provided for treating hypertrophic scars and keloids,
comprising the step of administering albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention to a hypertrophic scar or keloid.
[0991] Within one embodiment of the present invention fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention are directly injected into a
hypertrophic scar Of keloid, in order to prevent the progression of
these lesions. This therapy is of particular value in the
prophylactic treatment of conditions which are known to result in
the development of hypertrophic scars and keloids (e.g., burns),
and is preferably initiated after the proliferative phase has had
time to progress (approximately 14 days after the initial injury),
but before hypertrophic scar or keloid development. As noted above,
the present invention also provides methods for treating
neovascular diseases of the eye, including for example, corneal
neovascularization, neovascular glaucoma, proliferative diabetic
retinopathy, retrolental fibroplasia and macular degeneration.
[0992] Moreover, Ocular disorders associated with
neovascularization which can be treated with the albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention include, but are not limited to:
neovascular glaucoma, diabetic retinopathy, retinoblastoma,
retrolental fibroplasia, uveitis, retinopathy of prematurity
macular degeneration, corneal graft neovascularization, as well as
other eye inflammatory diseases, ocular tumors and diseases
associated with choroidal or iris neovascularization. See, e.g.,
reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1973) and
Gartner et al., Surv. Ophthal. 22:291-312 (1978).
[0993] Thus, within one aspect of the present invention methods are
provided for treating neovascular diseases of the eye such as
corneal neovascularization (including corneal graft
neovascularization), comprising the step of administering to a
patient a therapeutically effective amount of a compound (e.g.,
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention) to the cornea, such that
the formation of blood vessels is inhibited. Briefly, the cornea is
a tissue which normally lacks blood vessels. In certain
pathological conditions however, capillaries may extend into the
cornea from the pericorneal vascular plexus of the limbus. When the
cornea becomes vascularized, it also becomes clouded, resulting in
a decline in the patient's visual acuity. Visual loss may become
complete if the cornea completely opacitates. A wide variety of
disorders can result in corneal neovascularization, including for
example, corneal infections (e.g., trachoma, herpes simplex
keratitis, leishmaniasis and onchocerciasis), immunological
processes (e.g., graft rejection and Stevens-Johnson's syndrome),
alkali burns, trauma, inflammation (of any cause), toxic and
nutritional deficiency states, and as a complication of wearing
contact lenses.
[0994] Within particularly preferred embodiments of the invention,
may be prepared for topical administration in saline (combined with
any of the preservatives and antimicrobial agents commonly used in
ocular preparations), and administered in eyedrop form. The
solution or suspension may be prepared in its pure form and
administered several times daily. Alternatively, anti-angiogenic
compositions, prepared as described above, may also be administered
directly to the cornea. Within preferred embodiments, the
anti-angiogenic composition is prepared with a muco-adhesive
polymer which binds to cornea. Within further embodiments, the anti
angiogenic factors or anti-angiogenic compositions may be utilized
as an adjunct to conventional steroid therapy. Topical therapy may
also be useful prophylactically in corneal lesions which are known
to have a high probability of inducing an angiogenic response (such
as chemical burns). In these instances the treatment, likely in
combination with steroids, may be instituted immediately to help
prevent subsequent complications.
[0995] Within other embodiments, the compounds described above may
be injected directly into the corneal stroma by an ophthalmologist
under microscopic guidance. The preferred site of injection may
vary with the morphology of the individual lesion, but the goal of
the administration would be to place the composition at the
advancing front of the vasculature (i.e., interspersed between the
blood vessels and the normal cornea). In most cases this would
involve perilimbic corneal injection to "protect" the cornea from
the advancing blood vessels. This method may also be utilized
shortly after a corneal insult in order to prophylactically prevent
corneal neovascularization. In this situation the material could be
injected in the perilimbic cornea interspersed between the corneal
lesion and its undesired potential limbic blood supply. Such
methods may also be utilized in a similar fashion to prevent
capillary invasion of transplanted corneas. In a sustained-release
form injections might only be required 2-3 times per year. A
steroid could also be added to the injection solution to reduce
inflammation resulting from the injection itself.
[0996] Within another aspect of the present invention, methods are
provided for treating neovascular glaucoma, comprising the step of
administering to a patient a therapeutically effective amount of an
albumin fusion protein of the invention and/or polynucleotides
encoding an albumin fusion protein of the invention to the eye,
such that the formation of blood vessels is inhibited. In one
embodiment, the compound may be administered topically to the eye
in order to treat early forms of neovascular glaucoma. Within other
embodiments, the compound may be implanted by injection into the
region of the anterior chamber angle. Within other embodiments, the
compound may also be placed in any location such that the compound
is continuously released into the aqueous humor. Within another
aspect of the present invention, methods are provided for treating
proliferative diabetic retinopathy, comprising the step of
administering to a patient a therapeutically effective amount can
albumin fusion protein of the invention and/or polynucleotides
encoding an albumin fusion protein of the invention to the eyes,
such that the formation of blood vessels is inhibited.
[0997] Within particularly preferred embodiments of the invention,
proliferative diabetic retinopathy may be treated by injection into
the aqueous humor or the vitreous, in order to increase the local
concentration of the polynucleotide, polypeptide, antagonist and/or
agonist in the retina. Preferably, this treatment should be
initiated prior to the acquisition of severe disease requiring
photocoagulation.
[0998] Within another aspect of the present invention, methods are
provided for treating retrolental fibroplasia, comprising the step
of administering to a patient a therapeutically effective amount of
an albumin fusion protein of the invention and/or polynucleotides
encoding an albumin fusion protein of the invention to the eye,
such that the formation of blood vessels is inhibited. The compound
may be administered topically, via intravitreous injection and/or
via intraocular implants.
[0999] Additionally, disorders which can be treated with fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention include, but are not limited to,
hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic
plaques, delayed wound healing, granulations, hemophilic joints,
hypertrophic scars, nonunion fractures, Osier-Weber syndrome,
pyogenic granuloma, scleroderma, trachoma, and vascular
adhesions.
[1000] Moreover, disorders and/or states, which can be treated,
prevented, diagnosed, and/or prognosed with the albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention of the invention include, but are
not limited to, solid tumors, blood born tumors such as leukemias,
tumor metastasis, Kaposi's sarcoma, benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular
angiogenic diseases, for example, diabetic retinopathy, retinopathy
of prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rebeosis,
retinoblastoma, and uvietis, delayed wound healing, endometriosis,
vascluogenesis, granulations, hypertrophic scars (keloids),
nonunion fractures, scleroderma, trachoma, vascular adhesions,
myocardial angiogenesis, coronary collaterals, cerebral
collaterals, arteriovenous malformations, ischemic limb
angiogenesis, Osler-Webber Syndrome, plaque neovascularization,
telangiectasia, hemophiliac joints, angiofibroma fibromuscular
dysplasia, wound granulation, Crohn's disease, atherosclerosis,
birth control agent by preventing vascularization required for
embryo implantation controlling menstruation, diseases that have
angiogenesis as a pathologic consequence such as cat scratch
disease (Rochele minalia quintosa), ulcers (Helicobacter pylori),
Bartonellosis and bacillary angiomatosis.
[1001] In one aspect of the birth control method, an amount of the
compound sufficient to block embryo implantation is administered
before or after intercourse and fertilization have occurred, thus
providing an effective method of birth control, possibly a "morning
after" method. Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may also be used in controlling menstruation or administered as
either a peritoneal lavage fluid or for peritoneal implantation in
the treatment of endometriosis.
[1002] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be incorporated into surgical sutures in order to prevent
stitch granulomas.
[1003] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be utilized in a wide variety of surgical procedures. For
example, within one aspect of the present invention a compositions
(in the form of, for example, a spray or film) may be utilized to
coat or spray an area prior to removal of a rumor, in order to
isolate normal surrounding tissues from malignant tissue, and/or to
prevent the spread of disease to surrounding tissues. Within other
aspects of the present invention, compositions (e.g., in the form
of a spray) may be delivered via endoscopic procedures in order to
coat tumors, or inhibit angiogenesis in a desired locale. Within
yet other aspects of the present invention, surgical meshes which
have been coated with anti-angiogenic compositions of the present
invention may be utilized in any procedure wherein a surgical mesh
might be utilized. For example, within one embodiment of the
invention a surgical mesh laden with an anti-angiogenic composition
may be utilized during abdominal cancer resection surgery (e.g.,
subsequent to colon resection) in order to provide support to the
structure, and to release an amount of the anti-angiogenic
factor.
[1004] Within further aspects of the present invention, methods are
provided for treating tumor excision sites, comprising
administering albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
to the resection margins of a tumor subsequent to excision, such
that the local recurrence of cancer and the formation of new blood
vessels at the site is inhibited. Within one embodiment of the
invention, the anti-angiogenic compound is administered directly to
the tumor excision site (e.g., applied by swabbing, brushing or
otherwise coating the resection margins of the tumor with the
anti-angiogenic compound). Alternatively, the anti-angiogenic
compounds may be incorporated into known surgical pastes prior to
administration. Within particularly preferred embodiments of the
invention, the anti-angiogenic compounds are applied after hepatic
resections for malignancy, and after neurosurgical operations.
[1005] Within one aspect of the present invention, fusion proteins
of the invention and/or polynucleotides encoding albumin fusion
proteins of the invention may be administered to the resection
margin of a wide variety of tumors, including for example, breast,
colon, brain and hepatic tumors. For example, within one embodiment
of the invention, anti-angiogenic compounds may be administered to
the site of a neurological tumor subsequent to excision, such that
the formation of new blood vessels at the site are inhibited.
[1006] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may also be administered along with other anti-angiogenic factors.
Representative examples of other ami-angiogenic factors include:
Anti-Invasive Factor, retinoic acid and derivatives thereof,
paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1,
Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator
Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms
of the lighter "d group" transition metals.
[1007] Lighter "d group" transition metals include, for example,
vanadium, molybdenum, tungsten, titanium, niobium, and tantalum
species. Such transition metal species may form transition metal
complexes. Suitable complexes of the above-mentioned transition
metal species include oxo transition metal complexes.
[1008] Representative examples of vanadium complexes include oxo
vanadium complexes such as vanadate and vanadyl complexes. Suitable
vanadate complexes include metavanadate and orthovanadate complexes
such as, for example, ammonium metavanadate, sodium metavanadate,
and sodium orthovanadate. Suitable vanadyl complexes include, for
example, vanadyl acetylacetonate and vanadyl sulfate including
vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
[1009] Representative examples of tungsten and molybdenum complexes
also include oxo complexes. Suitable oxo tungsten complexes include
tungstate and tungsten oxide complexes. Suitable tungstate
complexes include ammonium tungstate, calcium tungstate, sodium
tungstate dihydrate, and tungstic acid, Suitable tungsten oxides
include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and
molybdenyl complexes. Suitable molybdate complexes include ammonium
molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium molybdate and its hydrates. Suitable molybdenum oxides
include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic
acid. Suitable molybdenyl complexes include, for example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes include hydroxo derivatives derived from, for example,
glycerol, tartaric acid, and sugars.
[1010] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include platelet factor 4; protamine
sulphate; sulphated chitin derivatives (prepared from queen crab
shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated
Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this
compound may be enhanced by the presence of steroids such as
estrogen, and tamoxifen citrate); Staurosporine; modulators of
matrix metabolism, including for example, proline analogs,
cishydroxyproline, d,l-3,4-dehydroproline, Thiaproline,
alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl
5-(4-pyridinyl) 2(3H)-oxazolone; Methotrexate; Mitoxantrone;
Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et
al., J. Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson
et al., Biochem J. 286:475-480, (1992)); Cyclodextrin
Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (higher et
al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987));
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol.
Chem. 262(4):1659-1664, (1987)); Bisantrene (National Cancer
Institute); Lobenzarit disodium
(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA";
Takeuchi et al., Agents Actions 36:312-316, (1992)); Thalidomide;
Angostatic steroid; AGM-1470; carboxynaminolmidazole; and
metalloproteinase inhibitors such as BB94.
[1011] Reproductive System Disorders
[1012] The albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be used for the diagnosis, treatment, or prevention of diseases
and/or disorders of the reproductive system. Reproductive system
disorders that can be treated by the compositions of the invention,
include, but are not limited to, reproductive system injuries,
infections, neoplastic disorders, congenital defects, and diseases
or disorders which result in infertility, complications with
pregnancy, labor, or parturition, and postpartum difficulties.
[1013] Reproductive system disorders and/or diseases include
diseases and/or disorders of the testes, including testicular
atrophy, testicular feminization, cryptorchism (unilateral and
bilateral), anorchia, ectopic testis, epididymitis and orchids
(typically resulting from infections such as, for example,
gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion,
vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell
carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors,
and teratomas), stromal tumors (e.g., Leydig cell tumors),
hydrocele, hematocele, varicocele, spermatocele, inguinal hernia,
and disorders of sperm production (e.g., immotile cilia syndrome,
aspermia, asthenozoospermia, azoospermia, oligospermia, and
teratozoospermia).
[1014] Reproductive system disorders also include disorders of the
prostate gland, such as acute non-bacterial prostatitis, chronic
non-bacterial prostatitis, acute bacterial prostatitis, chronic
bacterial prostatitis, prostatodystonia, prostatosis, granulomatous
prostatitis, malacoplakia, benign prostatic hypertrophy or
hyperplasia, and prostate neoplastic disorders, including
adenocarcinomas, transitional cell carcinomas, ductal carcinomas,
and squamous cell carcinomas.
[1015] Additionally, the compositions of the invention may be
useful in the diagnosis, treatment, and/or prevention of disorders
or diseases of the penis and urethra, including inflammatory
disorders, such as balanoposthitis, balanitis xerotica obliterans,
phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea,
non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV,
AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and
pearly penile papules; urethral abnormalities, such as hypospadias,
epispadias, and phimosis; premalignant lesions, including
Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant
condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile
cancers, including squamous cell carcinomas, carcinoma in situ,
verrucous carcinoma, and disseminated penile carcinoma; urethral
neoplastic disorders, including penile urethral carcinoma,
bulbomembranous urethral carcinoma, and prostatic urethral
carcinoma; and erectile disorders, such as priapism, Peyronie's
disease, erectile dysfunction, and impotence.
[1016] Moreover, diseases and/or disorders of the vas deferens
include vasculititis and CRAVD (congenital bilateral absence of the
vas deferens); additionally, the albumin fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be used in the diagnosis, treatment, and/or
prevention of diseases and/or disorders of the seminal vesicles,
including hydatid disease, congenital chloride diarrhea, and
polycystic kidney disease.
[1017] Other disorders and/or diseases of the male reproductive
system include, for example, Klinefelter's syndrome, Young's
syndrome, premature ejaculation, diabetes mellitus, cystic
fibrosis, Kartagener's syndrome, high fever, multiple sclerosis,
and gynecomastia.
[1018] Further, the polynucleotides, fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may be used in the diagnosis, treatment, and/or
prevention of diseases and/or disorders of the vagina and vulva,
including bacterial vaginosis, candida vaginitis, herpes simplex
virus, chancroid, granuloma inguinale, lymphogranuloma venereum,
scabies, human papillomavirus, vaginal trauma, vulvar trauma,
adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis,
condyloma acuminatum, syphilis, molluscum contagiosum, atrophic
vaginitis, Paget's disease, lichen sclerosus, lichen planus,
vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis,
vulvar vestibulitis, and neoplastic disorders, such as squamous
cell hyperplasia, clear cell carcinoma, basal cell carcinoma,
melanomas, cancer of Bartholin's gland, and vulvar intraepithelial
neoplasia.
[1019] Disorders and/or diseases of the uterus include
dysmenorrhea, retroverted uterus, endometriosis, fibroids,
adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome,
hydatidiform moles, Asherman's syndrome, premature menopause,
precocious puberty, uterine polyps, dysfunctional uterine bleeding
(e.g., due to aberrant hormonal signals), and neoplastic disorders,
such as adenocarcinomas, keiomyrisarcomas, and sarcomas.
Additionally, the albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may be useful as a marker or detector of, as well as in the
diagnosis, treatment, and/or prevention of congenital uterine
abnormalities, such as bicomuate uterus, septate uterus, simple
unicornuate uterus, unicornuate uterus with a noncavitary
rudimentary horn, unicornuate uterus with a non-communicating
cavitary rudimentary horn, unicornuate uterus with a communicating
cavitary horn, arcuate uterus, uterine didelfus, and T-shaped
uterus.
[1020] Ovarian diseases and/or disorders include anovulation,
polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian
cysts, ovarian hypofunction, ovarian insensitivity to
gonadotropins, ovarian overproduction of androgens, right ovarian
vein syndrome, amenorrhea, hirutism, and ovarian cancer (including,
but not limited to, primary and secondary cancerous growth,
Sertoli-Leydig tumors, endometriod carcinoma of the ovary, ovarian
papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma,
and Ovarian Krukenberg tumors).
[1021] Cervical diseases and/or disorders include cervicitis,
chronic cervicitis, mucopurulent cervicitis, cervical dysplasia,
cervical polyps, Nabothian cysts, cervical erosion, cervical
incompetence, and cervical neoplasms (including, for example,
cervical carcinoma, squamous metaplasia, squamous cell carcinoma,
adenosquamous cell neoplasia, and columnar cell neoplasia).
[1022] Additionally, diseases and/or disorders of the reproductive
system include disorders and/or diseases of pregnancy, including
miscarriage and stillbirth, such as early abortion, late abortion,
spontaneous abortion, induced abortion, therapeutic abortion,
threatened abortion, missed abortion, incomplete abortion, complete
abortion, habitual abortion, missed abortion, and septic abortion;
ectopic pregnancy, anemia. Rh incompatibility, vaginal bleeding
during pregnancy, gestational diabetes, intrauterine growth
retardation, polyhydramnios. HELLP syndrome, abruptio placentae,
placenta previa, hyperemesis, preeclampsia, eclampsia, herpes
gestationis, and urticaria of pregnancy. Additionally, the albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention may be used in the
diagnosis, treatment, and/or prevention of diseases that can
complicate pregnancy, including heart disease, heart failure,
rheumatic heart disease, congenital heart disease, mitral valve
prolapse, high blood pressure, anemia, kidney disease, infectious
disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious
hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes
mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's
thyroiditis, chronic active hepatitis, cirrhosis of the liver,
primary biliary cirrhosis, asthma, systemic lupus eryematosis,
rheumatoid arthritis, myasthenia gravis, idiopathic
thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder
disorders, and obstruction of the intestine.
[1023] Complications associated with labor and parturition include
premature rupture of the membranes, pre-term labor, post-term
pregnancy, postmaturity, labor that progresses too slowly, fetal
distress (e.g., abnormal heart rate (fetal or maternal), breathing
problems, and abnormal fetal position), shoulder dystocia,
prolapsed umbilical cord, amniotic fluid embolism, and aberrant
uterine bleeding.
[1024] Further, diseases and/or disorders of the postdelivery
period, including endometritis, myometritis, parametritis,
peritonitis, pelvic thrombophlebitis, pulmonary embolism,
endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis,
cystitis, postpartum hemorrhage, and inverted uterus.
[1025] Other disorders and/or diseases of the female reproductive
system that may be diagnosed, treated, and/or prevented by the
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention include, for
example, Turner's syndrome, pseudohermaphroditism, premenstrual
syndrome, pelvic inflammatory disease, pelvic congestion (vascular
engorgement), frigidity, anorgasmia, dyspareunia, ruptured
fallopian tube, and Mittelschmerz.
[1026] Infectious Disease
[1027] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
can be used to treat or detect infectious agents. For example, by
increasing the immune response, particularly increasing the
proliferation and differentiation of B and/or T cells, infectious
diseases may be treated. The immune response may be increased by
either enhancing an existing immune response, or by initiating a
new immune response. Alternatively, fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may also directly inhibit the infectious agent,
without necessarily eliciting an immune response.
[1028] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated or detected by
albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention. Examples of
viruses, include, but are not limited to Examples of viruses,
include, but are not limited to the following DNA and RNA viruses
and viral Arbovirus, Adenoviridae, Arenaviridae, Arterivirus,
Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae,
Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae
(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes
Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,
Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,
Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,
Parvoviridae, Picornaviridae, Poxyiridae (such as Smallpox or
Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I,
HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses
falling within these families can cause a variety of diseases or
symptoms, including, but not limited to: arthritis, bronchiolitis,
respiratory syncytial virus, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A,
B, C, B, Chronic Active, Delta), Japanese B encephalitis, Junin,
Chikungunya, Rift Valley fever, yellow fever, meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's
Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,
sexually transmitted diseases, skin diseases (e.g., Kaposi's,
warts), and viremia. Albumin fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention, can be used to treat or detect any of these symptoms or
diseases. In specific embodiments, fusion proteins of the invention
and/or polynucleotides encoding albumin fusion proteins of the
invention are used to treat: meningitis, Dengue, EBV, and/or
hepatitis (e.g., hepatitis B). In an additional specific embodiment
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention are used to treat patients
nonresponsive to one or more other commercially available hepatitis
vaccines. In a further specific embodiment fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention are used to treat AIDS.
[1029] Similarly, bacterial and fungal agents that can cause
disease or symptoms and that can be treated or detected by albumin
fusion proteins of the invention and/or polynucleotides encoding
albumin fusion proteins of the invention include, but not limited
to, the following Gram-Negative and Gram-positive bacteria,
bacterial families, and fungi: Actinomyces (e.g., Norcardia),
Acinetobacter, Cryptococcus neaformums, Aspergillus, Bacillaceae
(e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroides
fragilis) Blastomycosis, Bordetella, Borrelia (e.g., Borrelia
burgdorferi), Brucella, Candidia, Carnpylobacter, Chlamydia,
Clostridium (e.g., Clostridium botulinum, Clostridium dificile,
Clostridium perfringens, Clostridium tetani), Coccidioides,
Corynebacterium (e.g., Corynebacterium diptheriue), Cryptococcus,
Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and
Enterohemorrhagic E. coli), Enterobacter (e.g. Enterobacter
aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g.,
Salmonella typhi, Salmonella enteritidis, Salmonella typhi),
Serratia, Yersinia, Shigella), Erysipelothrix, Fleemophilus (e.g.,
Haemophilus influenza type E3), Helicobacter, Legionella (e.g.,
Legionella pneumophila), Leptospira, Listeda Listeria
monocyrogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium
leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio
cholerae), Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria
meningitidis), Pasteurellacea, Proteus, Pseudomonas (Pseudomonas
aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp.,
Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus
(e.g., Staphylococcus aureus), Meningiococcus, Pneumococcus and
Streptococcus (e.g., Streptococcus pneumoniae and (loops A, B, and
C Streptococci), and Ureaplasmas. These bacterial, parasitic, and
fungal families can cause diseases or symptoms, including, but not
limited to: antibiotic-resistant infections, bacteremia,
endocarditis, septicemia, eye infections (e.g., conjunctivitis),
uveitis, tuberculosis, gingivitis, bacterial diarrhea,
opportunistic infections (e.g., AIDS related infections),
paronychia, prosthesis-related infections, dental canes, Reiter's
Disease, respiratory tract infections, such as Whooping Cough or
Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery,
paratyphoid fever, food poisoning, Legionella disease, chronic and
acute inflammation, erythema, yeast infections, typhoid, pneumonia,
gonorrhea, meningitis (e.g., mengitis types A and B), chlamydia,
syphilis, diphtheria, leprosy, brucellosis, peptic ulcers, anthrax,
spontaneous abortions, birth defects, pneumonia, lung infections,
ear infections, deafness, blindness, lethargy, malaise, vomiting,
chronic diarrhea, Crohn's disease, colitis, vaginosis, sterility,
pelvic inflammatory diseases, candidiasis, paratuberculosis,
tuberculosis, lupus, botulism, gangrene, tetanus, impetigo,
Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract
infections, wound infections, noscomial infections. Albumin fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention, can be used to treat or detect
any of these symptoms or diseases. In specific embodiments, fusion
proteins of the invention and/or polynucleotides encoding albumin
fusion proteins of the invention are used to treat: tetanus,
diptheria, botulism, and/or meningitis type B.
[1030] Moreover, parasitic agents causing disease or symptoms that
can be treated, prevented, and/or diagnosed by fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention include, but not limited to, the
following families or class: Amebiasis, Babesiosis, Coccidiosis,
Cryptosporidiosis, Dientamoebiasis, Dourine, Ectnparasitic,
Clardias, Helminthiasis, Leishmaniasis, Schistisoma, Theileriasis,
Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans
(e.g., Plasmodium virax, Plasmodium fakiparium, Plasmodium malariae
and Plasmodium ovate). These parasites can cause a variety of
diseases or symptoms, including, but not bruited to: Scabies,
Trombiculiasis, eye infections, intestinal disease (e.g.,
dysentery, giardiasis), liven disease, lung disease, opportunistic
infections (e.g., AIDS related), malaria, pregnancy complications,
and toxoplasmosis. Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention,
can be used to treat, prevent, and/or diagnose any of these
symptoms or diseases. In specific embodiments, fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention are used to treat, prevent, and/or
diagnose malaria.
[1031] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
could either be by administering an effective amount of an albumin
fusion protein of the invention to the patient, or by removing
cells from the patient, supplying the cells with a polynucleotide
of the present invention, and returning the engineered cells to the
patient (ex vivo therapy). Moreover, the albumin fusion proteins of
the invention and/or polynucleotides encoding albumin fusion
proteins of the invention can be used as an antigen in a vaccine to
raise an immune response against infectious disease.
[1032] Chemotaxis
[1033] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may have chemotaxis activity. A chemotaxic molecule attracts or
mobilizes cells (e.g., monocytes, fibroblasts, neutrophils,
T-cells, mast cells, eosinophils, epithelial and/or endothelial
cells) to a particular site in the body, such as inflammation,
infection, or site of hyperproliferation. The mobilized cells can
then fight off and/or heal the particular trauma or
abnormality.
[1034] Albumin fusion proteins of the invention and/or
polynucleotides encoding albumin fusion proteins of the invention
may increase chemotaxic activity of particular cells. These
chemotactic molecules can then be used to treat inflammation,
infection, hyperproliferative disorders, or any immune system
disorder by increasing the number of cells targeted to a particular
location in the body. For example, chemotaxic molecules can be used
to treat wounds and other trauma to tissues by attracting immune
cells to the injured location. Chemotactic molecules of the present
invention can also attract fibroblasts, which can be used to treat
wounds.
[1035] It is also contemplated that fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention may inhibit chemotactic activity. These molecules
could also be used to treat disorders. Thus, fusion proteins of the
invention and/or polynucleotides encoding albumin fusion proteins
of the invention could be used as an inhibitor of chemotaxis.
[1036] Binding Activity
[1037] Albumin fusion proteins of the invention may be used to
screen for molecules that bind to the Therapeutic protein portion
of the fusion protein or for molecules to which the Therapeutic
protein portion of the fusion protein binds. The binding of the
fusion protein and the molecule may activate (agonist), increase,
inhibit (antagonist), or decrease activity of the fusion protein or
the molecule bound. Examples of such molecules include antibodies,
oligonucleotides, proteins (e.g., receptors), or small
molecules.
[1038] Preferably, the molecule is closely related to the natural
ligand of the Therapeutic protein portion of the fusion protein of
the invention, e.g., a fragment of the ligand, or a natural
substrate, a ligand, a structural or functional mimetic. (See,
Coligan et al., Current Protocols in immunology 1(2):Chapter 5
(1991)). Similarly, the molecule can be closely related to the
natural receptor to which the Therapeutic protein portion of an
albumin fusion protein of the invention binds, or at least, a
fragment of the receptor capable of being bound by the Therapeutic
protein portion of an albumin fusion protein of the invention
(e.g., active site). In either case, the molecule can be rationally
designed using known techniques.
[1039] Preferably, the screening for these molecules involves
producing appropriate cells which express the albumin fusion
proteins of the invention. Preferred cells include cells from
mammals, yeast, Drosophila, or E. coli.
[1040] The assay may simply test binding of a candidate compound to
an albumin fusion protein of the invention, wherein binding is
detected by a label, or in an assay involving competition with a
labeled competitor. Further, the assay may test whether the
candidate compound results in a signal generated by binding to the
fusion protein.
[1041] Alternatively, the assay can be carried out using cell-free
preparations, fusion protein/molecule affixed to a solid support,
chemical libraries, or natural product mixtures. The assay may also
simply comprise the steps of mixing a candidate compound with a
solution containing an albumin fusion protein, measuring fusion
protein/molecule activity or binding, and comparing the fusion
protein/molecule activity or binding to a standard.
[1042] Preferably, an ELISA assay can measure fusion protein level
or activity in a sample (e.g., biological sample) using a
monoclonal or polyclonal antibody. The antibody can measure fusion
protein level or activity by either binding, directly or
indirectly, to the albumin fusion protein or by competing with the
albumin fusion protein fin a substrate.
[1043] Additionally, the receptor to which a Therapeutic protein
portion of an albumin fusion protein of the invention hinds can be
identified by numerous methods known to those of skill in the art,
for example, ligand panning and FACS sorting (Coligan, et al.,
Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example,
in eases wherein the Therapeutic protein portion of the fusion
protein corresponds to FGF, expression cloning may be employed
wherein polyadenylated RNA is prepared from a cell responsive to
the albumin fusion protein, for example, NIH3T3 cells which are
known to contain multiple receptors for the FGF family proteins,
and SC-3 cells, and a cDNA library created from this RNA is divided
into pools and used to transfect COS cells or other cells that are
not responsive to the albumin fusion protein. Transfected cells
which are grown on glass slides are exposed to the albumin fusion
protein of the present invention, after they have been labeled. The
albumin fusion proteins can be labeled by a variety of means
including iodination or inclusion of a recognition site for a
site-specific protein kinase.
[1044] Following fixation and incubation, the slides are subjected
to auto-radiographic analysis. Positive pools are identified and
sub-pools are prepared and re-transfected using an iterative
sub-pooling and re-screening process, eventually yielding a single
clones that encodes the putative receptor.
[1045] As an alternative approach for receptor identification, a
labeled albumin fusion protein can be photoaffinity linked with
cell membrane or extract preparations that express the receptor
molecule for the Therapeutic protein component of an albumin fusion
protein of the invention, the linked material may be resolved by
PAGE analysis and exposed to X-ray film. The labeled complex
containing the receptors of the fusion protein can be excised,
resolved into peptide fragments, and subjected to protein
microsequencing. The amino acid sequence obtained from
microsequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
genes encoding the putative receptors.
[1046] Moreover, the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon shuffling (collectively referred to as
"DNA shuffling") may be employed to modulate the activities of the
fusion protein, and/or Therapeutic protein portion or albumin
component of an albumin fusion protein of the present invention,
thereby effectively generating agonists and antagonists of an
albumin fusion protein of the present invention, See generally,
U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and
5,837,458, and Patten. P. A. et. al., Curr. Opinion Biotechnol.
8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82
(1998); Hanson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and
Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998);
each of these patents and publications are hereby incorporated by
reference). In one embodiment, alteration of polynucleotides
encoding albumin fusion proteins of the invention and thus, the
albumin fusion proteins encoded thereby, may be achieved by DNA
shuffling. DNA shuffling involves the assembly of two or more DNA
segments into a desired molecule by homologous, or site-specific,
recombination. In another embodiment, polynucleotides encoding
albumin fusion proteins of the invention and thus, the albumin
fusion proteins encoded thereby, may be altered by being subjected
to random mutagenesis by error-prone PCR, random nucleotide
insertion or other methods prior to recombination. In another
embodiment, one or more components, motifs, sections, pans,
domains, fragments, etc., of an albumin fusion protein of the
present invention may be recombined with one or more components,
motifs, sections, pans, domains, fragments, etc. of one or more
heterologous molecules. In preferred embodiments, the heterologous
molecules are family members. In further preferred embodiments, the
heterologous molecule is a growth factor such as, for example,
platelet-derived growth factor (PDGF), insulin-like growth factor
(IGF-I), transforming growth factor (TGF)-alpha, epidermal growth
factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone
morphogenetic protein (BMP)-2, BMP-5, BMP-6, BMP-7, activins A and
B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth
differentiation factors (GDFs), nodal, MIS, inhibin-alpha,
TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived
neurotrophic factor (GDNF).
[1047] Other preferred fragments are biologically active fragments
of the Therapeutic protein portion and/or albumin component of the
albumin fusion proteins of the present invention. Biologically
active fragments are those exhibiting activity similar, but not
necessarily identical, to an activity of a Therapeutic protein
portion and/or albumin component of the albumin fusion proteins of
the present invention. The biological activity of the fragments may
include all improved desired activity, or a decreased undesirable
activity.
[1048] Additionally, this invention provides a method of screening
compounds to identify those which modulate the action of an albumin
fusion protein of the present invention. An example of such an
assay comprises combining a mammalian fibroblast cell, an albumin
fusion protein of the present invention, and the compound to be
screened and .sup.3[H] thymidine under cell culture conditions
where the fibroblast cell would normally proliferate. A control
assay may be performed in the absence of the compound to be
screened and compared to the amount of fibroblast proliferation in
the presence of the compound to determine if the compound
stimulates proliferation by determining the uptake of .sup.3-[H]
thymidine in each case. The amount of fibroblast cell proliferation
is measured by liquid scintillation chromatography which measures
the incorporation of .sup.3-[H] thymidine. Both agonist and
antagonist compounds may be identified by this procedure.
[1049] In another method, a mammalian cell or membrane preparation
expressing a receptor for the Therapeutic protein component of a
fusion protein of the invention is incubated with a labeled fusion
protein of the present invention in the presence of the compound.
The ability of the compound to enhance or block this interaction
could then be measured. Alternatively, the response of a known
second messenger system following interaction of a compound to be
screened and the receptor is measured and the ability of the
compound to bind to the receptor and elicit a second messenger
response is measured to determine if the compound is a potential
fusion protein. Such second messenger systems include but are not
limited to, cAMP guanylate cyclase, ion channels or
phosphoinositide hydrolysis.
[1050] All of these above assays can be used as diagnostic or
prognostic markers. The molecules discovered using these assays can
be used to treat disease or to bring about a particular result in a
patient (e.g., blood vessel growth) by activating or inhibiting the
fusion protein/molecule. Moreover, the assays can discover agents
which may inhibit or enhance the production of the albumin fusion
proteins of the invention from suitably manipulated cells or
tissues.
[1051] Therefore, the invention includes a method of identifying
compounds which bind to an albumin fusion protein of the invention
comprising the steps of: (a) incubating a candidate binding
compound with an albumin fusion protein of the present invention;
and (b) determining if binding has occurred. Moreover, the
invention includes a method of identifying agonists/antagonists
comprising the steps of: (a) incubating a candidate compound with
an albumin fusion protein of the present invention, (b) assaying a
biological activity, and (b) determining if a biological activity
of the fusion protein has been altered.
[1052] Targeted Delivery
[1053] In another embodiment, the invention provides a method of
delivering compositions to targeted cells expressing a receptor for
a component of an albumin fusion protein of the invention.
[1054] As discussed herein, fusion proteins of the invention may be
associated with heterologous polypeptides, heterologous nucleic
acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic
and/or covalent interactions. In one embodiment, the invention
provides a method for the specific delivery of compositions of the
invention to cells by administering fusion proteins of the
invention (including antibodies) that are associated with
heterologous polypeptides or nucleic acids. In one example, the
invention provides a method for delivering a Therapeutic protein
into the targeted cell. In another example, the invention provides
a method for delivering a single stranded nucleic acid (e.g.,
antisense or ribozymes) or double stranded nucleic acid (e.g., DNA
that can integrate into the cell's genome or replicate episomally
and that can be transcribed) into the targeted cell.
[1055] In another embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering an albumin fusion protein of the invention
(e.g., polypeptides of the invention or antibodies of the
invention) in association with toxins or cytotoxic prodrugs.
[1056] By "toxin" is meant compounds that bind and activate
endogenous cytotoxic effector systems, radioisotopes, holotoxins,
modified toxins, catalytic subunits of toxins, or any molecules or
enzymes not normally present in or on the surface of a cell that
under defined conditions cause the cell's death. Toxins that may be
used according to the methods of the invention include, but are not
limited to, radioisotopes known in the art, compounds such as, for
example, antibodies (or complement fixing containing portions
thereof) that bind an inherent or induced endogenous cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha
toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin,
saporin, momordin, gelonin, pokeweed antiviral protein, alpha
sarcin and cholera toxin. By "cytotoxic prodrug" is meant a
non-toxic compound that is converted by an enzyme, normally present
in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may
be used according to the methods of the invention include, but are
not limited to, glutamyl derivatives of benzoic acid mustard
alkylating agent, phosphate derivatives of etoposide or mitomycin
C, cytosine, arabinoside, daunonibisin, and phenoxyacetamide
derivatives of dexorubicin.
[1057] Drug Screening
[1058] Further contemplated is the use of the albumin fusion
proteins of the present invention, or the polynucleotides encoding
these fusion proteins, to screen for molecules which modify the
activities of the albumin fusion protein of the present invention
or proteins corresponding to the Therapeutic protein portion of the
albumin fusion protein. Such a method would include contacting the
fusion protein with a selected compound(s) suspected of having
antagonist or agonist activity, and assaying the activity of the
fusion protein following binding.
[1059] This invention is particularly useful for screening
therapeutic compounds by using the albumin fusion proteins of the
present invention, or binding fragments thereof, in any of a
variety of drug screening techniques. The albumin fusion protein
employed in such a test may be affixed to a solid support,
expressed on a cell surface, free in solution, or located
intracellularly. One method of drug screening utilizes eukaryotic
or prokaryotic host cells which are stably transformed with
recombinant nucleic acids expressing the albumin fusion protein.
Drugs are screened against such transformed cells or supernatants
obtained from culturing such cells, in competitive binding assays.
One may measure, for example, the formulation of complexes between
the agent being tested and an albumin fusion protein of the present
invention.
[1060] Thus, the present invention provides methods of screening
for drugs or any other agents which affect activities mediated by
the albumin fusion proteins of the present invention. These methods
comprise contacting such an agent with an albumin fusion protein of
the present invention or a fragment thereof and assaying for the
presence of a complex between the agent and the albumin fusion
protein or a fragment thereof, by methods well known in the art. In
such a competitive binding assay, the agents to screen are
typically labeled. Following incubation, free agent is separated
from that present in bound form, and the amount of free or
uncomplexed label is a measure of the ability of a particular agent
to hind to the albumin fusion protein of the present invention.
[1061] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to an albumin fusion protein of the present invention, and is
described in great detail in European Patent Application 84/03564,
published on Sep. 13, 1984, which is incorporated herein by
reference herein. Briefly stated, large numbers of different small
peptide test compounds are synthesized on a solid substrate, such
as plastic pins or some other surface. The peptide test compounds
are reacted with an albumin fusion protein of the present invention
and washed. Bound peptides are then detected by methods well known
in the art. Purified albumin fusion protein may be coated directly
unto plates for use in the aforementioned drug screening
techniques. In addition, non-neutralizing antibodies may be used to
capture the peptide and immobilize it on the solid support.
[1062] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding an albumin fusion protein of the present invention
specifically compete with a test compound for binding to the
albumin fusion protein or fragments thereof. In this manner, the
antibodies are used to detect the presence of any peptide which
shares one or more antigenic epitopes with an albumin fusion
protein of the invention.
[1063] Binding Peptides and Other Molecules
[1064] The invention also encompasses screening methods for
identifying polypeptides and nonpolypeptides that bind albumin
fusion proteins of the invention, and the binding molecules
identified thereby. These binding molecules are useful, for
example, as agonists and antagonists of the albumin fusion proteins
of the invention. Such agonists and antagonists can be used, in
accordance with the invention, in the therapeutic embodiments
described in detail, below.
[1065] This method comprises the steps of:
[1066] (a) contacting an albumin fusion protein of the invention
with a plurality of molecules; and
[1067] (b) identifying a molecule that binds the albumin fusion
protein.
[1068] The step of contacting the albumin fusion protein of the
invention with the plurality of molecules may be effected in a
number of ways. For example, one may contemplate immobilizing the
albumin fusion protein on a solid support and bringing a solution
of the plurality of molecules in contact with the immobilized
polypeptides. Such a procedure would be akin to an affinity
chromatographic process, with the affinity matrix being comprised
of the immobilized albumin fusion protein of the invention. The
molecules having a selective affinity for the albumin fusion
protein can then be purified by affinity selection. The nature of
the solid support, process for attachment of the albumin fusion
protein to the solid support, solvent, and conditions of the
affinity isolation or selection are largely conventional and well
known to those of ordinary skill in the art.
[1069] Alternatively, one may also separate a plurality of
polypeptides into substantially separate fractions comprising a
subset of or individual polypeptides. For instance, one can
separate the plurality of polypeptides by gel electrophoresis,
column chromatography, or like method known to those of ordinary
skill for the separation of polypeptides. The individual
polypeptides can also be produced by a transformed host cell in
such a way as to be expressed on or about its outer surface (e.g.,
a recombinant phage). Individual isolates can then be "probed" by
an albumin fusion protein of the invention, optionally in the
presence of an inducer should one be required for expression, to
determine if any selective affinity interaction takes place between
the albumin fusion protein and the individual clone. Prior to
contacting the albumin fusion protein with each fraction comprising
individual polypeptides, the polypeptides could first be
transferred to a solid support for additional convenience. Such a
solid support may simply be a piece of filter membrane, such as one
made of nitrocellulose or nylon. In this manner, positive clones
could be identified from a collection of transformed host cells of
an expression library, which harbor a DNA construct encoding a
polypeptide having a selective affinity for an albumin fusion
protein of the invention. Furthermore, the amino acid sequence of
the polypeptide having a selective affinity ter an albumin fusion
protein of the invention can be determined directly by conventional
means or the coding sequence of the DNA encoding the polypeptide
can frequently be determined more conveniently. The primary
sequence can then be deduced from the corresponding DNA sequence.
If the amino acid sequence is to be determined from the polypeptide
itself, one may use microsequencing techniques. The sequencing
technique may include mass spectroscopy.
[1070] In certain situations, it may be desirable to wash away any
unbound polypeptides from a mixture of an albumin fusion protein of
the invention and the plurality of polypeptides prior to attempting
to determine or to detect the presence of a selective affinity
interaction. Such a wash step may be particularly desirable when
the albumin fusion protein of the invention or the plurality of
polypeptides am bound to a solid support.
[1071] The plurality of molecules provided according to this method
may be provided by way of diversity libraries, such as random or
combinatorial peptide or nonpeptide libraries which can be screened
for molecules that specifically bind an albumin fusion protein of
the invention. Many libraries are known in the art that can be
used, e.g., chemically synthesized libraries, recombinant (e.g.,
phage display libraries), and in vitro translation-based libraries.
Examples of chemically synthesized libraries are described in Fodor
et al., Science 251:767 773 (1991); Houghten as al., Nature
354:84-86 (1991); Lam et al., Nature 354:82-84 (1991); Medynski,
Bio/Technology 12:709-710 (1994); Gallop et al., J. Medicinal
Chemistry 37(9):1233-1251 (1994); Ohlmeyer et al Proc. Natl. Acad.
Sci. USA 90:10922-10926 (1993); Erb et al, Proc. Natl. Acad. Sci.
USA 91:11422-11426 (1994); Houghten et al., Biotechniques 13:412
(1992); Jayawickreme et al., Proc. Natl. Acad. Sci. USA
91:1614-1618 (1994); Salmon et al., Proc. Natl. Acad. Sci. USA
90:11708-11712 (1993); PCT Publication No. WO 93/20242; and Brenner
and Lerner, Proc. Natl. Acad. Sci. USA 89:5381-5383 (1992).
[1072] Examples of phage display libraries are described in Scott
et al., Science 249:386-390 (1990); Devlin et al., Science,
249:404-406 (1990); Christian et al., 1992, J. Mol. Biol.
227:711-718 1992); Lenstra, J. Immunol. Meth. 152:149-157 (1992);
Kay et al., Gene 128:59-65 (1993); and PCT Publication No. WO
94/18318 dated Aug. 18, 1994.
[1073] In vitro translation-based libraries include but ale not
limited to those described in PCT Publication No. WO 91/05058 dated
Apr. 18, 1991; and Mattheakis et al. Proc. Natl. Acad. Sci. USA
91:9022-9026 (1994).
[1074] By way of examples of nonpeptide libraries, a benzodiazepine
library (see e.g., Bunin et al., Proc. Natl. Acad. Sci. USA
91:4708-4712 (1994)) can be adapted for use. Peptoid libraries
(Simon et al., Proc. Natl. Acad. Sci. USA 89:9367-9371 (1992)) can
also be used. Another example of a library that can be used, in
which the amide functionalities in peptides have been permethylated
to generate a chemically transformed combinatorial library, is
described by Ostresh et at (Proc. Natl. Acad. Sci. USA
91:11138-11142 (1994)).
[1075] The variety of non-peptide libraries that are useful in the
present invention is great. For example, Ecker and Crooke
(Bio/Technology 13:351-360 (1995) list benzodiazepines, hydantoins,
piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones,
arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines,
aminimides, and oxazolones as among the chemical species that form
the basis of various libraries.
[1076] Non-peptide libraries can be classified broadly into two
types: decorated monomers and oligomers. Decorated monomer
libraries employ a relatively simple scaffold structure upon which
a variety functional groups is added. Often the scaffold will be a
molecule with a known useful pharmacological activity. Far example,
the scaffold might be the benzodiazepine structure.
[1077] Non-peptide oligomer libraries utilize a large number of
monomers that are assembled together in ways that create new shapes
that depend on the order of the monomers. Among the monomer units
that have been used are carbamates, pyrrolinones, and morpholines.
Peptoids, peptide-like oligomers in which the side chain is
attached to the alpha amino group rather than the alpha carbon,
form the basis of another version of non-peptide oligomer
libraries. The first non-peptide oligomer libraries utilized a
single type of monomer and thus contained a repeating backbone.
Recent libraries have utilized more than one monomer, giving the
libraries added flexibility.
[1078] Screening the libraries can be accomplished by any of a
variety of commonly known methods. See, e.g., the following
references, which disclose screening of peptide libraries: Parmley
et al., Adv. Exp. Med. Biol. 7, 51:215-218 (1989); Scott et al.,
Science 249:386-390 (1990); Fowlkes at al., BioTechniques
13:422-427 (1992); Oldenburg et al., Proc. Natl. Acad. Sci. USA
89:5393-5397 (1992); Yu et al., Cell 76:933-945 (1994); Staudt et
al., Science 241:577-580 (1988); Bock at al., Nature 355:564-566
(1992); Tuerk et al., Proc. Natl. Acad. Sci. USA 89:6988-6992
(1992); Ellington et al., Nature 355:850-852 (1992); U.S. Pat. No.
5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346,
all to Ladner et al.; Rehar et al., Science 264:671-613 (1993); and
PCT Publication No. WO 94/18318.
[1079] In a specific embodiment, screening to identify a molecule
that binds an albumin fusion protein of the invention can be
carried out by contacting the library members with an albumin
fusion protein of the invention immobilized on a solid phase and
harvesting those library members that bind to the albumin fusion
protein. Examples of such screening methods, termed "panning"
techniques are described by way of example in Parmley et al., Gene
73:305-318 (1988); Fowlkes et al., BioTechniques 13:422-427 (1992);
PCT Publication No. WO 94/18318; and in references cited
herein.
[1080] In another embodiment, the two-hybrid system for selecting
interacting proteins in yeast (Fields et al., Nature 340:245-246
(1989); Chien et al., Proc. Natl. Acad. Sci. USA 88:9518-9582
(1991) can be used to identify molecules that specifically bind to
polypeptides of the invention.
[1081] Where the binding molecule is a polypeptide, the polypeptide
can be conveniently selected from any peptide library, including
random peptide libraries, combinatorial peptide libraries, or
biased peptide libraries. The term "biased" is used herein to mean
that the method of generating the library is manipulated so as to
restrict one or more parameters that govern the diversity of the
resulting collection of molecules, in this case peptides.
[1082] Thus, a truly random peptide library would generate a
collection of peptides in which the probability of finding a
particular amino acid at a given position of the peptide is the
same for all 20 amino acids. A bias can be introduced into the
library, however; by specifying, for example, that a lysine occur
every fifth amino acid or that positions 4, 8, and 9 of a
decapeptide library be fixed to include only arginine. Clearly,
many types of biases can be contemplated, and the present invention
is not restricted to any particular bias. Furthermore, the present
invention contemplates specific types of peptide Libraries, such as
phage displayed peptide libraries and those that utilize a DNA
construct comprising a lambda phage vector with a DNA insert.
[1083] As mentioned above, in the case of a binding molecule that
is a polypeptide, the polypeptide may have about 6 to less than
about 60 amino acid residues, preferably about 6 to about 10 amino
acid residues, and most preferably, about 6 to about 22 amino
acids. In another embodiment, a binding polypeptide has in the
range of 15-100 amino acids, or 20-50 amino acids.
[1084] The selected binding polypeptide can be obtained by chemical
synthesis or recombinant expression.
[1085] Other Activities
[1086] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention,
may be employed in treatment for stimulating re-vascularization of
ischemic tissues due to various disease conditions such as
thrombosis, arteriosclerosis, and other cardiovascular conditions.
The albumin fusion proteins of the invention and/or polynucleotides
encoding albumin fusion proteins of the invention may also be
employed to stimulate angiogenesis and limb regeneration, as
discussed above.
[1087] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be employed for treating wounds due to injuries, burns,
post-operative tissue repair, and ulcers since they are mitogenic
to various cells of different origins, such as fibroblast cells and
skeletal muscle cells, and therefore, facilitate the repair or
replacement of damaged or diseased tissue.
[1088] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be employed stimulate neuronal growth and to treat and
prevent neuronal damage which occurs in certain neuronal disorders
or neuro-degenerative conditions such as Alzheimer's disease,
Parkinson's disease, and AIDS-related complex. An albumin fusion
protein of the invention and/or polynucleotide encoding an albumin
fusion protein of the invention may have the ability to stimulate
chondrocyte growth, therefore, they may be employed to enhance bone
and periodontal regeneration and aid in tissue transplants or bone
grafts.
[1089] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may be also be employed to prevent skin aging due to sunburn by
stimulating keratinocyte growth.
[1090] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be employed for preventing hair loss, since FGF family
members activate hair-forming cells and promotes melanocyte growth.
Along the same lines, an albumin fusion protein of the invention
and/or polynucleotide encoding an albumin fusion protein of the
invention may be employed to stimulate growth and differentiation
of hematopoietic cells and bone marrow cells when used in
combination with other cytokines.
[1091] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be employed to maintain organs before transplantation or
for supporting cell culture of primary tissues. An albumin fusion
protein of the invention and/or polynucleotide encoding an albumin
fusion protein of the invention may also be employed for inducing
tissue of mesodermal origin to differentiate in early embryos.
[1092] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also increase or decrease the differentiation or proliferation
of embryonic stem cells, besides, as discussed above, hematopoietic
lineage.
[1093] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be used to modulate mammalian characteristics, such as
body height, weight, hair color, eye color, skin, percentage of
adipose tissue, pigmentation, size, and shape (e.g., cosmetic
surgery). Similarly, an albumin fusion protein of the invention
and/or polynucleotide encoding an albumin fusion protein of the
invention may be used to modulate mammalian metabolism affecting
catabolism, anabolism, processing, utilization, and storage of
energy.
[1094] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may be used to change a mammal's mental state or physical state by
influencing biorhythms, caricadic rhythms, depression (including
depressive disorders), tendency for violence, tolerance for pain,
reproductive capabilities (preferably by Activin or Inhibin-like
activity), hormonal or endocrine levels, appetite, libido, memory,
stress, or other cognitive qualities.
[1095] An albumin fusion protein of the invention and/or
polynucleotide encoding an albumin fusion protein of the invention
may also be used as a food additive or preservative, such as to
increase or decrease storage capabilities, fat content, lipid,
protein, carbohydrate, vitamins, minerals, cofactors or other
nutritional components.
[1096] The above-recited applications have uses in a wide variety
of hosts. Such hosts include, but are not limited to, human,
marine, rabbit, goat, guinea pig, camel, horse, mouse, rat,
hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat,
non-human primate, and human. In specific embodiments, the host is
a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig,
sheep, dog or cat. In preferred embodiments, the host is a mammal.
In most preferred embodiments, the host is a human.
[1097] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
[1098] Without further description, it is believed that one of
ordinary skill in the an can, using the preceding description and
the following illustrative examples, make and utilize the
alterations detected in the present invention and practice the
claimed methods. The following working examples therefore,
specifically point out preferred embodiments of the present
invention, and are not to be construed as limiting in any way the
remainder of the disclosure.
EXAMPLES
Example 1
Generation Of pScNCHSA And pScCHSA
[1099] The vectors pScNHSA (ATCC Deposit No. PTA-3279) and pScCHSA
(ATCC Deposit No. PTA-3276) are derivatives of pPPC0005 (ATCC
Deposit No. PTA-3278) and are used as cloning vectors into which
polynucleotides encoding a therapeutic protein or fragment or
variant thereof is inserted adjacent to and in translation frame
with polynucleotides encoding human serum albumin "HSA". pScCHSA
may be used for generating Therapeutic protein-HSA fusions, while
pScNHSA may be used to generate USA-Therapeutic protein
fusions.
[1100] Generation of pScCHSA: Albumin Fusion with the Albumin
Moiety C-Terminal to the Therapeutic Portion
[1101] A vector to facilitate cloning DNA encoding a Therapeutic
protein N-terminal to DNA encoding the mature albumin protein was
made by altering the nucleic acid sequence that encodes the
chimeric HSA signal peptide in pPPC0005 to include the Xho I and
Cla I restriction sites.
[1102] First, the Xho I and Cla I sites inherent to pPPC0005
(located 3' of the ADH1 terminator sequence) were eliminated by
digesting pPPC0005 with Xho I and Cla I, filling in the sticky ends
with T4 DNA polymerase, and religating the blunt ends to create
pPPC0006.
[1103] Second, the Xho I and Cla I restriction sites were
engineered into the nucleic acid sequence that encodes the signal
peptide of HSA (a chimera of the HSA leader and a kex2 site from
mating factor alpha, "MAF") in pPPC0006 using two rounds of PCR. In
the first round of PCR, amplification with primers shown as SEQ ID
NO:328 and SEQ ID NO:329 was performed. The primer whose sequence
is shown as SEQ ID NO:328 comprises a nucleic acid sequence that
encodes part of the signal peptide sequence of HSA, a kex2 site
from the mating factor alpha leader sequence, and part of the
amino-terminus of the mature form of HSA. Four point mutations were
introduced in the sequence, creating the Xho I and Cla I sites
found at the junction of the chimeric signal peptide and the mature
form of HSA. These for mutations are underlined in the sequence
shown below. In pPPC0005 the nucleotides at these four positions
from 5' so 3' are T, G, T, and G.
5'-GCCTCGAGAAAAGAGATGCACACAAGAGTGAGOTTGCTCATCGATTTAAAGATTTGGG-3'
(SEQ ID NO:328) and
5'-AATCGATGAGCAACCTCACTCTTGTGTOCATCTCTTTTCTCGAGGCTCCTGGAATAAGC-3'
(SEQ ID NO:329). A second round of PCR was then performed with an
upstream flanking primer, 5'-TACAAACTTAAGAGTCCAATTAGC-3' (SEQ ID
NO:330) and a downstream flanking primer
5'-CACTTCTCTAGAGTGGTTTCATATGTCTT-3' (SEQ ID NO:331). The resulting
PCR product was then purified and digested with Afl II and Xba I
and ligated into the same sites in pPPC0006 creating pScCHSA. The
resulting plasmid has Xho I and Cla I sites engineered into the
signal sequence. The presence of the Xho I site creates a single
amino acid change in the end of the signal sequence from LDKR to
LEKR. The D to E change will not be present in the final albumin
fusion protein expression plasmid when a nucleic acid sequence
comprising a polynucleotide encoding the Therapeutic portion of the
albumin fusion protein with a 5' Sal I site (which is compatible
with the Xho I site) and a 3' Cla I site is ligated into the Xho I
and Cla I sites of pScCHSA. Ligation of Sal I to Xho I restores the
original amino acid sequence of the signal peptide sequence. DNA
encoding the Therapeutic portion of the albumin fusion protein may
be inserted after the Kex2 site (Kex2 cleaves after the dibasic
amino acid sequence KR at the end of the signal peptide) and prior
to the Cla I site.
[1104] Generation of pScNHSA: Albumin Fusion with the Albumin
Moiety N-Terminal to the Therapeutic Portion.
[1105] A vector to facilitate cloning DNA encoding a Therapeutic
protein portion C-terminal to DNA encoding the mature albumin
protein, was made by adding three, eight-base-pair restriction
sites to pScCHSA. The Asc I, Fse I, and Pme I restriction sites
were added in between the Bsu36 I and Hind III sites at the end of
the nucleic acid sequence encoding the mature HSA protein. This was
accomplished through the use of two complementary synthetic primers
containing the Asc I, Fse I, and Pme I restriction sites underlined
(SEQ ID NO:332 and SEQ ID NO:333).
TABLE-US-00005 (SEQ ID NO: 332)
5'-AAGCTGCCTTAGGCTTATAATAAGGCGCGCCGGCCGGCCGTT TAAACTAAGCTTAATTCT-3'
and (SEQ ID NO: 334) 5'-AGAATTAAGCTTAGTTTAAACGGCCGGCCGGCGCGCCTTATT
ATAAGCCTAAGGCAGCTT-3'.
These primers s were annealed and digested with Bsu36 I and Hind
III and ligated into the same sites in pScCHSA creating
pScNHSA.
Example 2
General Construct Generation for Yeast Transformation
[1106] The vectors pScNHSA and pScCHSA may be used as cloning
vectors into which polynucleotides encoding a therapeutic protein
or fragment or variant thereof is inserted adjacent to
polynucleotides encoding mature human serum albumin "HSA". pScCHSA
is used for generating Therapeutic protein-HSA fusions, while
pScNHSA may be used to generate HSA-Therapeutic protein
fusions.
[1107] Generation of Albumin Fission Constructs Comprising
HSA-Therapeutic Protein Fusion Products
[1108] DNA encoding a Therapeutic protein (e.g., sequences shown in
SEQ ID NO:X or known in the art) may be PCR amplified using the
primers which facilitate the generation of a fusion construct
(e.g., by adding restriction sites, encoding seamless fusions,
encoding linker sequences, etc.) For example, one skilled in the
art could design a 5' primer that adds polynucleotides encoding the
last four amino acids of the mature form of HSA (and containing the
Bsu36I site) onto the 5' end of DNA encoding a Therapeutic protein,
and a 3' primer that acids a STOP codon and appropriate cloning
sites onto the 3' end of the Therapeutic protein coding sequence.
For instance, the forward primer used to amplify DNA encoding a
Therapeutic protein might have the sequence,
5'-aagctGCCTTAGGCTIA(N).sub.15-3' (SEQ ID NO.sub.334) where the
underlined sequence is a Bsu36I site, the upper case nucleotides
encode the last four amino acids of the mature HSA protein (ALGL),
and (N).sub.15 is identical to the first 15 nucleotides encoding
the Therapeutic protein of interest. Similarly, the reverse primer
used to amplify DNA encoding a Therapeutic protein might have the
sequence,
##STR00001##
where the italicized sequence is a Pme I site, the double
underlined sequence is an Fse I site, the singly underlined
sequence is an Asc I site, the boxed nucleotides are the reverse
complement of two tandem stop codons, and (N).sub.15 is identical
to the reverse complement of the last 15 nucleotides encoding the
Therapeutic protein of interest. Once the PCR product is amplified
it may be cut with Bsu36I and one of (Asc I, Fse I, or Pme I) and
ligated into pScNHSA.
[1109] The presence of the Xho I site in the HSA chimeric leader
sequence creates a single amino acid change in the end of the
chimeric signal sequence, i.e. the HSA-kex2 signal sequence, from
LDKR to LEKR.
[1110] Generation of Albumin Fusion Constructs Comprising Gene-HSA
Fusion Products.
[1111] Similar to the method described above, DNA encoding a
Therapeutic protein may be PCR amplified using the following
primers: A 5' primer that adds polynucleotides containing a Sal 1
site and encoding the last three amino acids of the HSA leader
sequence. DKR, onto the 5' and of DNA encoding a Therapeutic
protein; and a 3' primer that adds polynucleotides encoding the
fast few amino acids of the mature HSA containing a Cla I site onto
the 3' end of DNA encoding a Therapeutic protein. For instance, the
forward primer used to amplify the DNA encoding a Therapeutic
protein might have the sequence, 5'-aggagegtcGACAAAAGA(N).sub.15-3'
(SEQ ID NO:336) where the underlined sequence is a Sal I site, the
upper case nucleotides encode the last three amino acids of the HSA
leader sequence (DKR), and (N).sub.15 is identical to the first 15
nucleotides encoding the Therapeutic protein or interest.
Similarly, the reverse primer used to amplify the DNA encoding a
Therapeutic protein might have the sequence,
TABLE-US-00006 (SEQ ID NO: 337)
5'-CTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATC(N).sub.15-3'
wherein the italicized sequence is a Cla I site, the underlined
nucleotides are the reverse complement of the DNA encoding the
first 9 amino acids of the mature form of HSA (DAHKSEVAH, SEQ ID
NO.sub.391), and (N).sub.15 is identical to the reverse complement
of the last 15 nucleotides encoding the Therapeutic protein of
interest. Once the PCR product is amplified it may be cut with Sal
1 and Cla I and ligated into pScCHSA digested with Xho I and Cla I.
A different sinful or leader sequence may be desired, for example,
invertase "INV" (Swiss-Prot Accession P00124), mating factor alpha
"MAF" (Genbank Accession AA A18405), MPIF (Geneseq AAF82936),
Fibulin B (Swiss-Prot Accession P23142), Clusterin (Swiss-Prot
Accession P10909), Insulin-Like Growth Factor-Binding Protein 4
(Swiss-Prot Accession P22692), and permutations of the HSA leader
sequence can be subcloned into the appropriate vector by means of
standard methods known in the art.
[1112] Generation of Albumin Fusion Construct Compatible for
Expression in Yeast S. cerevisiae.
[1113] The Not I fragment containing the DNA encoding either an
N-terminal or C-terminal albumin fusion protein generated from
pScNHSA or pScCHSA may then be cloned into the Nat I site of pSAC3S
which has a LEU2 selectable marker. The resulting vector is then
used in transformation of a yeast S. cerevisiae expression
system.
Example 3
General Expression in Yeast S. cerevisiae
[1114] An expression vector compatible with yeast expression can be
transformed into yeast S. cerevisiae by lithium acetate
transformation, electroporation, or other methods known in the art
and or as described in part in Sambrook, Fritsch, and Maniatis.
(1989). "Molecular Cloning: A Laboratory Manual, 2.sup.nd edition",
volumes 1-3, and in Ausubel et al. (2000). Massachusetts General
Hospital and Harvard Medical School "Current Protocols in Molecular
Biology", volumes 1-4. The expression vectors are introduced into
S. cerevisiae strains DXY1, D88, or HXP10 by transformation,
individual transformants can be grown, for example, for 3 days at
30.degree. C. in 10 mL YEPD (1% w/v yeast extract, 2% peptone, 2%
with, dextrose), and cells can be collected at stationary phase
after 60 hours of growth. Supernatants are collected by clarifying
cells at 3000 g for 10 minutes.
[1115] pSAC35 (Sleep et al., Biotechnology 8:42 (1990) and see FIG.
4) comprises, in addition to the LEU2 selectable marker, the entire
yeast 2 .mu.m plasmid to provide replication functions, the PRB1
promoter, and the ADH1 termination signal.
Example 4
General Purification of an Albumin Fusion Protein Expressed from an
Albumin Fusion in Yeast S. cerevisiae
[1116] In preferred embodiments, albumin fusion proteins of the
invention comprise the mature form of HSA fused to either the N- or
C-terminus of the mature form of a therapeutic protein or portions
thereof (e.g., the mature form of a therapeutic protein listed in
Table 1, or the mature form of a therapeutic protein shown in Table
2 as SEQ ID NO:Z). In one embodiment of the invention, albumin
fusion proteins of the invention further comprise a signal sequence
which directs the nascent fusion polypeptide in the secretory
pathways of the host used for expression. In a preferred
embodiment, the signal peptide encoded by the signal sequence is
removed, and the mature albumin fusion protein is secreted directly
into the culture medium. Albumin fusion proteins of the invention
preferably comprise heterologous signal sequences (e.g., the
non-native signal sequence of a particular therapeutic protein)
including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin,
Insulin-Like Growth Factor Binding Protein 4, variant HSA leader
sequences including, but not limited to, a chimeric HSA/MAF leader
sequence, or other heterologous signal sequences known in the art.
In preferred embodiments, the fusion proteins of the invention
further comprise an N-terminal methionine residue. Polynucleotides
encoding these polypeptides, including fragments and/or variants,
are also encompassed by the invention.
[1117] Albumin fusion proteins expressed in yeast as described
above can be purified on a small-scale over a Dyax peptide affinity
column as follows. Supernatants from yeast expressing an albumin
fusion protein is dinfiltrated against 3 mM phosphate buffer pH
6.2, mM NaCl and 0.01% Tween 20 to reduce the volume and to remove
the pigments. The solution is then filtered through a 0.22 .mu.m
device. The filtrate is loaded onto a Dyax peptide affinity column.
The column is eluted with 100 mM Tris/HCl, pH 8.2 buffer. The peak
fractions containing protein are collected and analyzed on SDS-PAGE
after concentrating 5-fold.
[1118] For large scale purification, the following method can be
utilized. The supernatant in excess of 2 L is diafiltered and
concentrated to 500 mL in 20 mM Tris/HCl pH 8.0. The concentrated
protein solution is loaded onto a pre-equilibrated 50 mL
DEAE-Sepharose Fast Flow column, the column is washed, and the
protein is eluted with a linear gradient of NaCl from 0 to 0.4 M
NaCl in 20 mM Tris/HCl, pH 8.0. Those fractions containing the
protein are pooled, adjusted to pH 6.8 with 0.5 M sodium phosphate
(NaH.sub.2PO.sub.4). A final concentration of 0.9 M
(NH.sub.4).sub.2SO.sub.4 is added to the protein solution and the
whole solution is loaded onto a pre-equilibrated 50 mL Butyl650S
column. The protein is eluted with a linear gradient of ammonium
sulfate (0.9 to 0 M (NH.sub.4).sub.2SO.sub.4). Those fractions with
the albumin fusion are again pooled, diafiltered against 10 mM
Na.sub.2HPO.sub.4/citric acid buffer pH 5.75, and loaded onto a 50
mL pre-equilibrated SP-Sepharose Fast Flow column. The protein is
eluted with a NaCl linear gradient from 0 to 0.5 M. The fractions
containing the protein of interest are combined, the buffer is
changed to 10 mM Na.sub.2HPO.sub.4/citric acid pH 6.25 with an
Amicon concentrator, the conductivity is <2.5 mS/cm. This
protein solution is loaded onto a 15 mL, pre-equilibrated
Q-Sepharose high performance column, the column is washed, and the
protein is eluted with a NaCl linear gradient from 0 to 0.15 M
NaCl. The purified protein can then be formulated into a specific
buffer composition by buffer exchange.
Example 5
General Construct Generation for Mammalian Cell Transfection
[1119] Generation of Albumin Fusion Construct Compatible for
Expression in Mammalian Cell-Lines.
[1120] Albumin fusion constructs can be generated in expression
vectors for use in mammalian cell culture systems. DNA encoding a
therapeutic protein can be cloned N-terminus or C-terminus to HSA
in a mammalian expression vector by standard methods known in the
art (e.g., PCR amplification, restriction digestion, and ligation).
Once the expression vector has been constructed, transfection into
a mammalian expression system can proceed. Suitable vectors are
known in the art including, but not limited to, for example, the
pC4 vector, and/or vectors available from Lonza Biologics, Inc.
(Portsmouth, N.H.).
[1121] The DNA encoding human serum albumin has been cloned into
the pC4 vector which is suitable for mammalian culture systems,
creating plasmid pC4:HSA (ATCC Deposit # PTA-3277). This vector has
a DiHydroFolate Reductase, "DHFR", gene that will allow for
selection in the presence of methotrexate.
[1122] The pC4:HSA vector is suitable for expression of albumin
fusion proteins in CHO cells. For expression, in other mammalian
cell culture systems, it may be desirable to subclone a fragment
comprising, or alternatively consisting of, DNA which encodes for
an albumin fusion protein into an alternative expression vector.
For example, a fragment comprising, or alternatively consisting, of
DNA which encodes for a mature albumin fusion protein may be
subcloned into another expression vector including, but not limited
to, any of the mammalian expression vectors described herein.
[1123] In a preferred embodiment, DNA encoding an albumin fusion
construct is subcloned into vectors provided by Lonza Biologics,
Inc. (Portsmouth, N.H.) by procedures known in the act for
expression in NS0 cells.
[1124] Generation of Albumin Fission Constructs Comprising
HSA-Therapeutic Protein Fusion Products.
[1125] Using pC4:HSA (ATCC Deposit 4 PTA-3277), albumin fusion
constructs can be generated in which the Therapeutic protein
portion is C terminal to the mature albumin sequence. For example,
one can clone DNA encoding a Therapeutic protein of fragment or
variant thereof between the Bsu 36f and Asc 1 restriction sites of
the vector. When cloning into the Bsu 36I and Asc I, the same
primer design used to clone into the yeast vector system (SEQ ID
NO:334 and 335) may be employed (see Example 2).
[1126] Generation of Albumin Fusion Constructs Comprising Gene-HSA
Fusion Products.
[1127] Using pC4:HSA (ATCC Deposit # PTA-3277), albumin fusion
constructs can be generated in which a Therapeutic protein portion
is cloned N terminal to the mature albumin sequence. For example,
one can clone DNA encoding a Therapeutic protein that has its own
signal sequence between the Bam HI (or Hind III) and Cla I sites of
pC4:HSA. When cloning into either the Bam HI or Hind III site, it
is preferable to include a Kozak sequence (CCGCCACCATG, SEQ ID
NO:392) prior to the translational start codon of the DNA encoding
the Therapeutic protein. If a Therapeutic protein does not have a
signal sequence, DNA encoding that Therapeutic protein may be
cloned in between the Xho I and Clo I sites of pC4:HSA. When using
the Xho I site, the following 5' (SEQ ID NO:338) and 3' (SEQ ID
NO:339) exemplary PCR primers may be used:
TABLE-US-00007 (SEQ ID NO: 338)
5'-CCGCCGCTCGAGGGGTGTGTTTCGTCGA(N).sub.18-3'; and (SEQ ID NO: 339)
5'-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATC(N).sub.18-3'
[1128] In the 5' primer (SEQ ID NO:338), the underlined sequence is
a Xho I site; and the Xho I site and the DNA following the Xho I
site code for the last seven amino acids of the leader sequence of
natural human serum albumin. In SEQ ID NO:338, "(N).sub.15" is DNA
identical to the first 18 nucleotides encoding the Therapeutic
protein of interest. In the 3' primer (SEQ ID NO:339), the
underlined sequence is a Cla I site; and the Cla I site and the DNA
following it are the reverse complement of the DNA encoding the
first 10 amino acids of the mature HSA protein (SEQ ID NO:327). In
SEQ ID NO:339 "(N).sub.15" is the reverse complement of DNA
encoding the last 18 nucleotides encoding the Therapeutic protein
of interest. Using these two primers, one may PCR amplify the
Therapeutic protein of interest, purify the PCR product, digest it
with Xho I and Cla I restriction enzymes and clone it into the Xho
I and Cla I sites in the pC4:HSA vector.
[1129] If an alternative leader sequence is desired, the native
albumin leader sequence can be replaced with the chimeric albumin
leader, i.e., the HSA-kex2 signal peptide, or an alternative leader
by standard methods known in the art. (For example, one skilled in
the art could routinely PCR amplify an alternate leader and
subclone the PCR product into an albumin fusion construct in place
of the albumin leader while maintaining the reading frame).
Example 6
General Expression in Mammalian Cell Lines
[1130] An albumin fusion construct generated in an expression
vector compatible with expression in mammalian cell-lines can be
transfected into appropriate cell-lines by calcium phosphate
precipitation, lipofectamine, electroporation, or other
transfection methods known in the err and/or as described in
Sambrook, Fritsch, and Maniatis. 1989. "Molecular Cloning: A
Laboratory Manual, 2.sup.nd edition" and in Ausubel et al. 2000.
Massachusetts General Hospital and Harvard Medical School "Current
Protocols in Molecular Biology", volumes 1-4. The transfected cells
are then selected for by the presence of a selecting agent
determined by the selectable marker in the expression vector.
[1131] The pC4 expression vector (ATCC Accession No. 209646) is a
derivative of the plasmid pSV2-DHFR (ATCC Accession No. 37146). pC4
contains the strong promoter Long Terminal Repeats "LTR" of the
Rous Sarcoma Virus (Cullen et al., March 1985, Molecular and
Cellular Biology, 438-447) and a fragment of the CytoMeganloVirus
"CMV"-enhancer (Boshart et al., Cell 41: 521-530 (1985)). The
vector also contains the 3' intron, the polyadenylation and
termination signal of the rat preproinsulin gene, and the mouse
DEER gene under control of the SV40 early promoter. Chinese hamster
ovary "CHO" cells or other cell-lines lacking an active DHFR gene
are used for transfection. Transfection of an albumin fusion
construct in pC4 into CHO cells by methods known in the art will
allow for the expression of the albumin fusion protein in CHO
cells, followed by leader sequence cleavage, and secretion into the
supernatant. The albumin fusion protein is then further purified
from the supernatant.
[1132] The pEE12.1 expression vector is provided by Lanza
Biologics, Inc. (Portsmouth, N.H.) and is a derivative of pEE6
(Stephens and Cockett, Nucl. Acids Res. 17: 7110 (1989).sub.3. This
vector comprises a promoter, enhancer and complete 5'-untranslated
region of the Major Immediate Early gene of the human
CytoMegaloVirus, "hCMV-MIE" (International Publication #
WO89/01016), upstream of a sequence of interest, and a Glutamine
Synthetase gene (Murphy et al., Biochem J. 227: 277-279 (1991):
Bebbington et al., Bio/Technology 10:169-175 (1992); U.S. Pat. No.
5,122,464) for purposes of selection of transfected cells in
selective methionine sulphoximine containing medium. Transfection
of albumin fusion constructs made in pEE12.1 into NS0 cells
(International Publication # WO86/05807) by methods known in the
art will allow for the expression of the albumin fusion protein in
NS0 cells, followed by leader sequence cleavage, and secretion into
the supernatant. The albumin fusion protein is then further
purified front the supernatant using techniques described herein or
otherwise known in the art.
[1133] Expression of an albumin fusion protein may be analyzed, for
example, by SDS-PAGE and Western blot, reversed phase HPLC
analysis, or other methods known in the art.
[1134] Stable CHO and NS0 transfected with albumin fusion
constructs are generated by methods known in the art (e.g.,
lipofectamine transfection) and selected, for example, with 100 nM
methotrexate for vectors having the DiHydroFolate Reductase `DHFR`
gene as a selectable marker or through growth in the absence of
glutamine. Expression levels can be examined for example, by
immunoblotting, primarily, with an anti-HSA serum as the primary
antibody, or, secondarily, with serum containing antibodies
directed to the Therapeutic protein portion of a given albumin
fusion protein as the primary antibody.
[1135] Expression levels are examined by immunoblot detection with
ant-HSA serum as the primary antibody. The specific productivity
rates are determined via ELISA in which the capture antibody can be
a monoclonal antibody towards the therapeutic protein portion of
the albumin fusion and the detecting antibody can be the monoclonal
anti-HSA-biotinylated antibody (or vice verse), followed by
horseradish peroxidase/streptavidin binding and analysis according
to the manufacturer's protocol.
Example 7
General Purification of an Albumin Fusion Protein Expressed from an
Albumin Fusion Construct in Mammalian Cell-Lines
[1136] In preferred embodiments, albumin fusion proteins of the
invention comprise the mature form of HSA fused to either the N- or
C-terminus of the mature form of a therapeutic protein or portions
thereof (e.g., the mature form of a therapeutic protein listed in
Table 1, or the mature form of a therapeutic protein shown in Table
2 as SEQ ID NO:Z). In one embodiment of the invention, albumin
fusion proteins of the invention further comprise a signal sequence
which directs the nascent fusion polypeptide in the secretory
pathways of the host used for expression. In a preferred
embodiment, the signal peptide encoded by the signal sequence is
removed, and the mature albumin fusion protein is secreted directly
into the culture medium. Albumin fusion proteins of the invention
preferably comprise heterologous signal sequences (e.g., the
non-native signal sequence of a particular therapeutic protein)
including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin,
Insulin-Like Growth Factor Binding Protein 4, variant HSA leader
sequences including, but not limited to, a chimeric HSA/MAF leader
sequence, or other heterologous signal sequences known in the art.
In preferred embodiments, the fusion proteins of the invention
further comprise an N-terminal methionine residue. Polynucleotides
encoding these polypeptides, including fragments and/or variants,
are also encompassed by the invention.
[1137] Albumin fusion proteins from mammalian cell-line
supernatants are purified according to different protocols
depending on the expression system used.
[1138] Purification from CHO and 293T Cell-Lines.
[1139] Purification of an albumin fusion protein from CHO cell
supernatant or from transiently transfected 293T cell supernatant
may involve initial capture with an anionic HQ resin using a sodium
phosphate buffer and a phosphate gradient elution, followed by
affinity chromatography on a Blue Sepharose FF column using a salt
gradient elution. Blue Sepharose FF removes the main BSA/fetuin
contaminants. Further purification over the Poros PI 50 resin with
a phosphate gradient may remove and lower endotoxin contamination
as well as concentrate the albumin fusion protein.
[1140] Purification from NS0 Cell-Line
[1141] Purification of an albumin-fusion protein from NS0 cell
supernatant may involve Q-Sepharose anion exchange chromatography,
followed by SP-sepharose purification with a step elution, followed
by Phenyl-650M purification with a step elution, and, ultimately,
diafiltration.
[1142] The purified protein may then be formulated by buffer
exchange.
Example 8
Construct Id 2448, Glp1-Hsa, Generation
[1143] Construct ID 2448, pSAC35:GLP1.H98-R127.HSA, encodes for a
GLP1-HSA fusion protein which comprises the HSA chimeric leader
sequence, i.e. the HSA-kex2 signal peptide, the mature GLP1
protein, i.e. H98-R127, fused to the amino-terminus of the mature
form of HSA cloned into the S. cerevisiae yeast expression vector
pSAC35.
[1144] Cloning of GLP1 cDNA for Construct 2448
[1145] The DNA encoding the mature form of GLP1 was codon optimized
so as not to hybridize to the wild-type DNA encoding the mature
form of GLP1. The polynucleotide encoding the codon optimized GLP1
was generated with two overlapping primers GLP1-1 and GLP1-2,
described below, with optimal carton usage for yeast S. cerevisiae,
cut with SalI/ClaI, and ligated into XhoI/ClaI cut pScCHSA. After
sequence confirmation, the NotI fragment encompassing the GLP1
albumin fusion was subcloned into pSAC35. Construct ID #2448
encodes an albumin fusion protein containing the chimeric leader
sequence of HSA, the mature form of GLP1, followed by the mature
HSA protein (SEQ ID NO:327).
[1146] Two overlapping oligonucleotides suitable for synthesis of
the polynucleotide encoding the mature form of GLP1, GLP1-1 and
GLP1-2, are:
TABLE-US-00008 GLP1-1: (SEQ ID NO: 312)
5'-AGGAGCGTCGACAAAAGACACGCTGAAGGTACTTTCACTTCTGATG
TTTCTTCTTACTTGGAAGGTCAAGCTGCTAAGGAATTCATTGCT-3' GLP1-2: (SEQ ID NO:
313) 5'-CTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATCTCTACCCTT
AACCAACCAAGCAATGAATTCCTTAGCAG-3'
GLP1-1 incorporates a SalI cloning site (shown in italics) and the
DNA encoding the first 24 amino acids (shown in bold) of the ORF of
the mature form of GLP1, i.e. His-98 to Ala-121. In GLP1-2, the
italicized sequence is a ClaI site; and the ClaI site and the DNA
following it are the reverse complement of DNA encoding the first
10 amino acids of the nature HSA protein. In GLP1-2, the bolded
sequence is the reverse complement of the 38 nucleotides encoding
the last 12 amino acid residues Ala-116 to Arg-127 of the mature
form of GLP1. Using these two printers, the mature GLP1 protein was
generated by annealing, extension of the annealed primers,
digestion with SalI and ClaI, and subcloning into XhoI/ClaI
digested pScCHSA. The NotI fragment from this clone was then
ligated into the NotI site of pSAC35 to generate construct ID 2448.
Construct ID #2448 encodes an albumin fusion protein containing the
chimeric leader sequence, the mature form of GLP1, i.e. His-98 to
Arg-127, and the mature form of HSA.
[1147] Expression and Purification of Construct ID 2448.
[1148] Expression in Yeast S. Cerevisiae.
[1149] Construct 2448 was transformed into yeast S. cerevisiae by
methods known in the art (see Example 3). Expression levels can be
examined by immunoblot detection with anti-HSA serum as the primary
antibody.
[1150] Purification from Yeast S. cerevisiae Cell Supernatant.
[1151] The cell supernatant containing the secreted GLP1-HSA fusion
protein expressed from construct ID #2448 in yeast S. cerevisiae
was purified as described below (also see Example 4). Specifically,
the yeast supernatant was equilibrated to pH 7.0 and 2 M NaCl,
loaded onto a POROS HP2 resin (phenyl) column, washed with Tris pH
7.0/2 M NaCl and eluted with a linear gradient from 2 M to 0 M NaCl
at pH 7.0. The albumin fusion tinted with a >1 M NaCl and was
.about.90% pure. N-terminal sequencing should generate the
amino-terminus sequence (i.e. HAEGT) of the mature form of
GLP1.
[1152] In Vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2448.
[1153] Method
[1154] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the GLP1 albumin fusion protein
encoded by construct 24-48 can be carried out as described below in
Example 28.
[1155] Result
[1156] At all concentrations including 1 nM, 10 nM, and 100 nM, the
GLP1 albumin fusion encoded by construct 2448 demonstrated enhanced
glucose uptake by the 3T3-L1 adipocytes and, consequently, greater
insulin sensitivity as compared to the GLP1 protein.
[1157] In Vitro Pancreatic Cell-Lines Proliferation Assns. In the
Presence of the Albumin Fusion Protein Encoded by Construct
2448.
[1158] Method
[1159] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARID cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
GLP1 albumin fusion protein encoded by construct 2448 can be
carried out as described below under heading: "Example 29: In vitro
Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1160] The Activity of the Albumin Fusion Protein Encoded by
Construct 2448 can be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1161] The activity of the GLP1 albumin fusion protein encoded by
construct 2443 can be measured using NOD and/or NIDDM mouse models
described below under the headings, "Example 31: Occurrence of
Diabetes in NOD Mice", "Example 32: Histological Examination of NOD
Mice", and "Example 34: In vivo Mouse Model of NIDDM".
Example 9
Construct Id 2455, Hsa-Glp1, Generation
[1162] Construct ID 2455, pSAC35:HSA.GLP1.H98-R127, comprises DNA
encoding for a GLP1 albumin fusion protein which has the HSA
chimeric leader sequence, i.e. the HSA-kex2 signal peptide,
followed by the mantle form of HSA fused to the amino terminus of
the mature form of GLP1, cloned into the S. cerevisiae yeast
expression vector pSAC35.
[1163] Cloning of GLP1 cDNA for construct 2455
[1164] The DNA encoding the mature form of GLP1 was codon optimized
so as not to hybridize to the wild-type DNA encoding the mature
form of GLP1. The polynucleotide encoding the codon optimized GLP1
was generated with two overlapping primers GLP1-3 and GLP1-4,
described below, with optimal codon usage for yeast S. cerevisiae,
that were annealed, extended, cut with Bsu36I/AscI, and ligated
into Bsu36I/AscI cut pScNHSA. After sequence confirmation, the NotI
fragment encompassing the GLP1 albumin fusion was subcloned into
pSAC35. Construct ID #2455 encodes an albumin fusion protein
containing the chimeric leader sequence and mature form of HSA and
the mature form of GLP1, His-98 to Arg-127.
[1165] Two overlapping oligonucleotides suitable for synthesis of
the polynucleotide encoding the mature form of GLP1, GLP1-3 and
GLP1-4, are:
TABLE-US-00009 GLP1-3: (SEQ ID NO: 314)
5'-AAGCTGCCTTAGGCTTACACGCTGAAGGTACTTTCACTTCTGATGT
TTCTTCTTACTTGGAAGGTCAAGCTGCTAAGGAA TTCATTGCT-3' GLP1-4: (SEQ ID NO:
315) 5'-GGCGCGCCTCATCTACCCTTAACCAACCAAGCAATGAATTCCTTAG CAG-3'
GLP1-3 incorporates a Bsu36I cloning site (shown in italics) and
the DNA encoding the first 24 amino acids (shown in bold) of the
ORF of the mature form of GLP1, i.e. His-98 to Ala-121, In GLP1-4,
the italicized sequence is an AscI site and the last 38 nucleotides
in bold are the reverse complement of DNA encoding the last 12
amino acid residues Ala-116 to Arg-127 of the mature form of GLP1
(for general construct cloning see Example 2). Using these two
primers, the mature GLP1 protein was generated by annealing,
extension of the annealed primers, digestion with Bsu36I and AscI,
and subcloning into Bsu36I/AscI digested pScNHSA. The NotI fragment
from this clone was then ligated into the NotI site of pSAC35 to
generate construct ID 2455.
[1166] Expression and Purification of Construct ID 2455.
[1167] Expression Yeast S. cerevisiae.
[1168] Construct 2455 was transformed into yeast N cerevisiae by
methods known in the art (see Example 3). Expression levels can be
examined by immunoblot detection with anti-HSA serum as the primary
antibody.
Purification from yeast S. cerevisiae cell supernatant.
[1169] The cell supernatant containing the secreted HSA-GLP1 fusion
protein expressed from construct ID #2455 in yeast S. cerevisiae
was purified as described below (also see Example 4). Specifically,
the yeast supernatant was equilibrated to pH 7.0 and 2 M NaCl,
loaded onto a FORDS HP2 resin (phenyl) column, washed with Tris pH
7.0/2 M NaCl and eluted with a linear gradient from 2 M to 0 M NaCl
at pH 7.0. The albumin fusion eluted with >1 M NaCl and was
.about.90% pure. N-terminal sequencing should generate the
amino-terminus sequence (i.e. DARKS) of the mature form of HSA.
[1170] In vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2455.
[1171] Method
[1172] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the GLP1 albumin fusion protein
encoded by construct 2455 can be carried out as described below in
Example 28.
[1173] Result
[1174] At all concentrations including 1 nM, 10 nM, and 100 nM, the
GLP1 albumin fusion encoded by construct 2455 demonstrated enhanced
glucose uptake by the 3T3-L1 adipocytes and, consequently, greater
insulin sensitivity as compared to the GLP1 protein.
[1175] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2455.
[1176] Method
[1177] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-tine into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
GLP1 albumin fusion protein encoded by construct 2455 can be
carried out as described below under heading: "Example 29: In vitro
Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1178] The Activity of the Albumin Fusion Protein Encoded by
Construct 2455 can be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1179] The activity of the GLP1 albumin fusion protein encoded by
construct 2455 can be measured using NOD and/or NIDDM mouse models
described below under the headings, "Example 31: Occurrence of
Diabetes in NOD Mice", "Example 32: Histological Examination of NOD
Mice", and "Example 34: In vivo Mouse Model of NIDDM".
Example 141
Construct Id 2456, GLP1-HSA, Generation
[1180] Construct ID 2456, pSAC35:GLP1.H98-R127,HSA, encodes for a
GLP1-HSA fusion protein which comprises the HSA chimeric leader
sequence, the HSA-kex2 signal peptide, the mature GLP1 protein,
i.e. H98-R127, with the exception of Ala-99 mutated to Gly, fused
to the amino-terminus of the mature form of HSA cloned into the S.
cerevisiae yeast expression vector pSAC35. Construct ID #2456 is
designed to produce a GLP-1 albumin fusion protein that will be
resistant to cleavage by Dipeptidyl Peptidase IV, "DIV"
(Hildebrandt et al., Scared J Gastroenterol, 36(10): 1067-72
(2001)), in viva.
[1181] Cloning of GLP1 cDNA for Construct 2956
[1182] The DNA encoding the mature form of GLP1 was codon optimized
so as not to hybridize to the wild-type DNA encoding the mature
form of GLP1. The polynucleotide encoding the codon optimized GLP1
was generated with two overlapping primers GLP1-5 and GLP1-6,
described below, with optimal codon usage for yeast S. cerevisiae,
cut with SalI/ClaI, and ligated into XhoI/ClaI cut pScCHSA. After
sequence confirmation, the NotI fragment encompassing the GLP1
albumin fusion was subcloned into pSAC35, Construct ID #2456
encodes an albumin fusion protein containing the chimeric leader
sequence of HSA, the mature form of GLP1 with the exception of
Ala-99 mutated to Gly, followed by the mature HSA protein
[1183] Two overlapping oligonucleotides suitable for synthesis of
the polynucleotide encoding the mature form of GLP1, GLP1-5 and
GLP1-6, are:
TABLE-US-00010 GLP1-5: (SEQ ID NO: 316)
5'-AGGAGCGTCGACAAAAGACACGGTGAAGGTACTTTCACTTCTGATG
TTTCTTCTTACTTGGAAGGTCAAGCTGCTAAGGAATTCATTGCT-3' GLP1-6: (SEQ ID NO:
317) 5'-CTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATCTCTACCCTT
AACCAACCAAGCAATGAATTCCTTAGCAG-3'
GLP1-5 incorporates a SalI cloning site (shown in italics) and the
DNA encoding the first 24 amino acids (shown in bold) of the ORF of
the mature form of GLP1, i.e. His-98 to Ala-121, with the exception
of Ala-99 mutated to Gly. In GLP1-6, the italicized sequence is a
ClaI site; and the ClaI site and the DNA following it are the
reverse complement of DNA encoding the first 10 amino acids of the
mature HSA protein. In GLP1-6, the bolded sequence is the reverse
complement of the 38 nucleotides encoding the last 12 amino acid
residues Ala-116 to Arg-127 of the mature form of GLP1. Using these
two primers, the mature GLP1 protein was generated by annealing,
extension of the annealed primers, digestion with SalI and ClaI,
and subcloning into XhoI/ClaI digested pScCHSA. The NotI fragment
from this clone was then ligated into the NotI site of pSAC35 to
generate construct ID 2456. Construct ID #2456 encodes an albumin
fusion protein containing the chimeric leader sequence, the mature
form of GLP1, i.e. His-98 to Arg-127, with the exception of Ala-99
mutated to Gly, and the mature form of HSA.
[1184] Expression and Purification of Construct ID 2456.
[1185] Expression yeast S. cerevisiae.
[1186] Construct 2456 was transformed into yeast S. cerevisiae by
methods known in the art (see Example 3). Expression levels can be
examined by immunoblot detection with anti-HSA serum as the primary
antibody.
Purification from Yeast S. cerevisiae Cell Supernatant.
[1187] The cell supernatant containing the secreted GLP1-HSA fusion
protein expressed from construct ID #2456 in yeast S. cerevisiae
was purified as described below (also see Example 4). Specifically,
the yeast supernatant was equilibrated to pH 7.0 and 2 M NaCl,
loaded onto a POROS HP2 resin (phenyl) column, washed with Tris pH
7.012 M NaCl and eluted with a linear gradient from 2 M to 0 M NaCl
at pH 7.0. N-terminal sequencing should generate the amino-terminus
sequence (i.e. HAEGT) of the mature form of GLP1.
[1188] In Vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2456.
[1189] Method
[1190] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the GLP1 albumin fusion protein
encoded by construct 2456 can be carried out as described below in
Example 28.
[1191] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2456
[1192] Method
[1193] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
GLP1 albumin fusion protein encoded by construct 2456 can be
carried out as described below under heading: "Example 29: In vitro
Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1194] The activity of the GLP1 albumin fusion protein encoded by
construct 2456 can be assayed in vivo using diabetic NOD and/or
NIDDM mouse models.
[1195] The activity of the GLP1 albumin fusion protein encoded by
construct 2456 can be measured using NOD and/or NIDDM mouse models
described below under the headings, "Example 31: Occurrence of
Diabetes in NOD Mice", "Example 32: Histological Examination of NOD
Mice", and "Example 34: In vivo Mouse Model of NIDDM".
Example 11
Construct ID 2457, HSA-GLP1, Generation
[1196] Construct ID 2457, pSAC35HSA.GLP1.H98-R127, comprises DNA
encoding for a GLP1 albumin fusion protein which has the HSA
chimeric leader sequence, i.e. the HSA-kex2 signal peptide,
followed by the mature form of HSA fused to the amino terminus of
the mature form of GLP1 with the exception of the Ala-99 to Gly
mutation, cloned into the S. cerevisiae yeast expression vector
pSAC35. Construct ID #2457 is designed so produce a GLP-1 albumin
fusion protein that will be resistant to cleavage by Dipeptidyl
Peptidase IV, "DIV" (Hildebrandt et al., Scand J Gastroenterol,
36(10): 1067-72 (2001)), in vivo.
[1197] Cloning of GLP1 cDNA for Construct 2457
[1198] The DNA encoding the mature form of GLP1 was codon optimized
so as not to hybridize to the wild-type DNA encoding the mature
form of GLP1. The polynucleotide encoding the codon optimized GLP1
was generated with two overlapping primers GLP1-7 and GLP1-8,
described below, with optimal endue usage for yeast S. cerevisiae,
that were annealed, extended, cut with Bsu 36I/Asc I, and ligated
into Ban 36I/Asc I cut pScNHSA. After sequence confirmation, the
NotI fragment encompassing the GLP1 albumin fusion with the Ala-99
to Gly mutation was subcloned into pSAC35. Construct ID #2457
encodes an albumin fusion protein containing the chimeric leader
sequence and mature form of HSA and the mature form of GLP1, His-98
to Arg-127, with the exception of the Ala-99 to Gly mutation.
[1199] Two overlapping oligonucleotides suitable for synthesis of
the polynucleotide encoding the mature form of GLP1, GLP1-7 and
GLP1-8, are:
TABLE-US-00011 GLP1-7: (SEQ ID NO: 318)
5'-AAGCTGCCTTAGGCTTACACGGTGAAGGTACTTTCACTTCTGATGT
TTCTTCTTACTTGGAAGGTCAAGCTGCTAAGGAA TTCATTGCT-3' GLP1-8: (SEQ ID NO:
319) 5'-GGCGCGCCTCATCTACCCTTAACCAACCAAGCAATGAATTCCTTAG CAG-3'
GLP1-7 incorporates a Bsu36I cloning site (shown in italics) and
the DNA encoding the first 24 amino acids (shown in bold) of the
ORF of the mature form of GLP1, i.e. His-98 to Ala-121 with the
exception of the Ala-99 to Gly mutation. In GLP1-8, the italicized
sequence is an AscI site and the last 38 nucleotides in bold are
the reverse complement of DNA encoding the last 12 amino acid
residues of the mature form of GLP1, i.e. Ala-116 to Arg-127 (for
general construct cloning see Example 2). Using these two primers,
the mature GLP1 protein was generated by annealing, extension of
the annealed primers, digestion with Bsu 36I and Asc I, and
subcloning into Bsu 36I/Asc I digested pScNHSA. The NotI fragment
from this clone was then ligated into the NotI site of pSAC35 to
generate construct ID 2457.
[1200] Expression and Purification of Construct ID 2457.
[1201] Expression yeast S. cerevisiae.
[1202] Construct 2457 was transformed into yeast S. cerevisiae by
methods known in the art (see Example 3). Expression levels can be
examined by immunoblot detection with anti-HSA serum as the primary
antibody.
Purification from Yeast S. cerevisiae Cell Supernatant.
[1203] The cell supernatant containing the secreted HSA-GLP1 fusion
protein expressed from construct ID #2457 in yeast S. cerevisiae
was purified as described below (also see Example 4). Specifically,
the yeast supernatant was equilibrated to pH 7.0 and 2 M NaCl,
loaded onto a POROS HP2 resin (phenyl) column, washed with Tris pH
7.0/2 M NaCl and eluted with a linear gradient from 2 M to 0 M NaCl
at pH 7.0. N-terminal sequencing should generate the amino-terminus
sequence (i.e. DA1IKS) of the mature form of HSA.
[1204] In Vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Asian Protein Encoded by Construct
2457.
[1205] Method
[1206] The in vitro assay to measure the glucose uptake in 3T3L1
adipocytes in the presence of the GLP1 albumin fusion protein
encoded by construct 2457 can be carried out as described below in
Example 28.
[1207] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2457.
[1208] Method
[1209] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
GLP1 albumin fusion protein encoded by construct 2457 can be
carried out as described below under heading: "Example 29: In vitro
Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1210] The Activity of the Albumin Fusion Protein Encoded by
Construct 2457 can be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1211] The activity of the GLP1 albumin fusion protein encoded by
construct 2457 can be measured using NOD and/or NIDDM mouse models
described below under the headings, "Example 31: Occurrence of
Diabetes in NOD Mice", "Example 32: Histological Examination of NOD
Mice", and "Example 34: In vivo Mouse Model of NIDDM".
Example 12
Construct ID 2470, Exendin-4-HSA, Generation
[1212] Construct ID 2470, pSAC35.exendin.H48-S86.HSA, encodes for
an EXENDIN4-HSA fusion protein which comprises the HSA chimeric
leader sequence, i.e. the HSA-kex2 signal peptide, the mature
EXENDIN4 protein, i.e. H48-S86, fused to the amino-terminus of the
mature form of HSA cloned into the S. cerevisiae yeast expression
vector pSAC35.
[1213] Cloning of EXENDIN4 cDNA for Construct 2470
[1214] The DNA encoding the mature form of EXENDIN4 was codon
optimized so as not to hybridize to the wild-type DNA encoding the
mature form of EXENDIN4. The polynucleotide encoding the codon
optimized EXENDIN4 was generated with four overlapping primers
EXTC-1, EXTC-2, EXTC-3, and EXTC-4 described below, with optimal
codon usage for yeast S. cerevisiae, cut with SalI/ClaI, and
ligated into XhoI/ClaI cut pScCHSA. After sequence confirmation,
the NotI fragment encompassing the EXENDIN4 albumin fusion was
sub-cloned into pSAC35. Construct ID #2470 encodes an albumin
fusion protein containing the chimeric leader sequence of HSA, the
mature form of EXENDIN4, followed by the mature HSA protein.
[1215] Four overlapping oligonucleotides suitable for synthesis of
the polynucleotide encoding the mature form of EXENDIN4, EXTC-1,
EXTC-2, EXTC-3, and EXTC-4, are:
TABLE-US-00012 EXTC-1: (SEQ ID NO: 850)
5'-TCCAGGAGCGTCGACAAAAGACACGGTGAAGGTACTTTCACTTCTG
ATTTGTCTAAGCAAATGG-3' EXTC-2: (SEQ ID NO: 851)
5'-TCTTCAACCATTCAATGAACAATCTAACAGCTTCTTCTTCCATTTG
CTTAGACAAATCAGAAGT-3' EXTC-3: (SEQ ID NO: 852)
5'-AGATTGTTCATTGAATGGTTGAAGAACGGTGGTCCATCTTCTGGTG
CTCCACCACCATCTGATG-3' EXTC-4: (SEQ ID NO: 853)
5'-AGACTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATCAGATGG
TGGTGGAGCACCAGAAGA-3'
EXTC-1 incorporates a SalI cloning site (shown in italics) and the
DNA encoding the first 14 amino acids (shown in bold) of the ORE of
the mature form of EXENDIN4, i.e. His-48 to Met-61. In EXTC-4, the
italicized sequence is a ClaI site; and the ClaI site and the DNA
following it are the reverse complement of DNA encoding the first
10 amino acids of the mature HSA protein. In EXTC-4, the bolded
sequence is the reverse complement of the 24 nucleotides encoding
the last 8 amino acid residues Ser-79 to Ser-86 of the mature form
of EXENDIN4. Using these two primers along with EXTC-2 and EXTC-3,
the mature EXENDIN4 protein was generated by annealing, extension
of the annealed primers, digestion with SalI and ClaI, and
subcloning into XhoI/ClaI digested pScCHSA. The NotI fragment from
this clone was then ligated into the NotI site of pSAC35 to
generate construct ID 2470. Construct ID #2470 encodes an albumin
fusion protein containing the chimeric leader sequence, the mature
form of EXENDIN4, i.e. His-48 to Ser-36, and the mature form of
HSA.
[1216] Expression and Purification of Construct ID 2470.
[1217] Expression in Yeast S. cerevisiae.
[1218] Construct 2470 was transformed into yeast S. cerevisiae by
methods known in the art (see Example 3). Expression levels can be
examined by immunoblot detection with anti-HSA serum as the primary
antibody.
Purification from Yeast S. cerevisiae Cell Supernatant.
[1219] The cell supernatant containing the secreted EXENDIN4-HSA
fusion protein expressed from construct ID #2470 in yeast S.
cerevisiae was purified as described below (also see Example 4).
Briefly, the yeast supernatant was equilibrated to pH 7.4 and
loaded onto Pharmacia's Blue Sepharose 6 Fast Flow column. The
column was washed in 20 mM Tris pH 7.4/200 mM NaCl. The EXENDIN4
albumin fusion was eluted via step elution with NaCl from 0.2 M to
2M. Proteins were .about.90% pure. N-terminal sequencing should
generate the amino-terminus sequence (i.e. HGEGT) of the mature
form of EXENDIN4.
[1220] In vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2470.
[1221] Method
[1222] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the EXENDIN4 albumin fusion protein
encoded by construct 2470 was carried out as described below in
Example 28.
[1223] Result
[1224] At all concentrations including 1 nM, 10 nM, and 100 nM, the
EXENDIN4 albumin fusion encoded by construct 2470 demonstrated
glucose uptake activity in 3T3-L1 adipocytes.
[1225] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fission Protein Encoded by Construct
2470.
[1226] Method
[1227] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARM cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
EXENDIN4 albumin fusion protein encoded by construct 2470 can be
carried out as described below under heading: "Example 29: In vitro
Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1228] The activity of the Albumin Fusion Protein Encoded by
Construct 2470 can be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1229] The activity of the EXENDIN4 albumin fusion protein encoded
by construct 2470 can be measured using NOD and/or NIDDM mouse
models described below under the headings, "Example 31: Occurrence
of Diabetes in NOD Mice", "Example 32; Histological Examination of
NOD Mice", and "Example 34: In vivo Mouse Model of NIDDM".
Example 13
Construct ID 2469, HSA-Exendin4, Generation
[1230] Construct ID 2469, pSAC35:HSA.EXENDIN4.H48-S86, comprises
DNA encoding for a EXENDIN4 albumin fusion protein which has the
HSA chimeric leader sequence, i.e. the HSA-kex2 signal peptide,
followed by the mature form of HSA fused to the amino terminus of
the mature form of EXENDIN4, cloned into the S. cerevisiae yeast
expression vector pSAC35.
[1231] Cloning of EXENDIN4 cDNA for Construct 2469
[1232] The DNA encoding the mature form of EXENDIN4 was codon
optimized so as not to hybridize to the wild-type DNA encoding the
mature form of EXENDIN4. The polynucleotide encoding the codon
optimized EXENDIN4 was generated with four overlapping primers
EXTN-1, EXTN-2, EXTN-3, and EXTN-4, described below, with optimal
codon usage for yeast S. cerevisiae, that were annealed, extended,
cut with Bsu36I/AscI, and ligated into Bsu36I/AscI cut pScNHSA.
After sequence confirmation, the NotI fragment encompassing the
EXENDIN4 albumin fusion was subcloned into pSAC35. Construct ID
#2469 encodes an albumin fusion protein containing the chimeric
leader sequence and mature form of HSA and the mature form of
EXENDIN4. His-48 to Set-86.
[1233] Four overlapping oligonucleotides suitable for synthesis of
the polynucleotide encoding the mature form of EXENDIN4, EXTN-1,
EXTN-2, EXTN-3, and EXTN-4, are:
TABLE-US-00013 EXTN-1: (SEQ ID NO: 854) 5'
GTCAAGCTGCCTTAGGCTTACACGGTGAAGGTACTTTCACTTCTGA TTTGTCTAAGCAAA- 3'
EXTN-2: (SEQ ID NO: 855)
5'-CCATTCAATGAACAATCTAACAGCTTCTTCTTCCATTTGCTTAGAC AAATCAGAAGTGAA-3'
EXTN-3: (SEQ ID NO: 856)
5'-AGCTGTTAGATTGTTCATTGAATGGTTGAAGAACGGTGGTCCATCT TCTGGTGCTCCACC-3'
EXTN-4: (SEQ ID NO: 857)
5'-ATCGCATATGGCGCGCCCTATTAAGATGGTGGTGGAGCACCAGAAG
ATGGACCACCGTT-3'
[1234] EXTN-1 incorporates a Bsu36I cloning site (shown in italics)
and the DNA encoding the first 13 amino acids (shown in bold) of
the ORE of the mature form of EXENDIN4, i.e. His-48 to Gin-60. In
EXTN-4, the italicized sequence is an AscI site and the last 36
nucleotides in bold are the reverse complement of DNA encoding the
last 12 amino acid residues Asn-75 to Ser-86 of the mature form of
EXENDIN4 (for general construct cloning see Example 2). Using these
two primers along with EXTN-2 and EXTN-3, the mature EXENDIN4
protein was generated by annealing, extension of the annealed
primers, digestion with Bsu36I and AscI, and subcloning into
Bsu36I/AscI digested pScNHSA. The NotI fragment from this clone was
then ligated into the NotI site of pSAC35 to generate construes ID
2469.
[1235] Expression and Purification of Construct ID 2469.
[1236] Expression Yeast S. cerevisiae.
[1237] Construct 2469 was transformed into yeast S. cerevisiae by
methods known in the art (see Example 3). Expression levels can be
examined by immunoblot detection with anti-HSA serum as the primary
antibody.
Purification from Yeast S. cerevisiae Cell Supernatant.
[1238] The cell supernatant containing the secreted HSA-EXENDIN4
fusion protein expressed from construct ID #2469 in yeast S.
cerevisiae was purified as described below (also see Example 4).
Specifically, the yeast supernatant was equilibrated to pH 7.4 and
loaded directly onto Pharmacia's Blue Sepharose 6 Fast Flow column.
The column was washed in 21) mM. Tris pH 7.4+200 mM NaCl and eluted
via step elation with NaCl from 0.2 M to 2 M. Proteins were
.about.90% pure. N-terminal sequencing should generate the
amino-terminus sequence (i.e. DARKS) of the mature form of HSA.
[1239] In vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2469
[1240] Method
[1241] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the EXENDIN4 albumin fusion protein
encoded by construct 2469 was carried out as described below in
Example 28.
[1242] Result
[1243] At all concentrations including 1 nM, 10 nM, and 100 nM, the
EXENDIN4 albumin fusion encoded by construct 2469 demonstrated
glucose uptake activity in 3T3-L1 adipocytes.
[1244] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2469.
[1245] Method
[1246] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
EXENDIN4 albumin fusion protein encoded by construct 2469 can be
carried out as described below under heading: "Example 29: In vitro
Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1247] The Activity of the Albumin Fusion Protein Encoded by
Construct 2469 can be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1248] The activity of the EXENDIN4 albumin fusion protein encoded
by construct 2469 can be measured using NOD and/or NIDDM mouse
models described below under the headings, "Example 31: Occurrence
of Diabetes in NOD Mice", "Example 32: Histological Examination of
NOD Mice", and "Example 34: In vivo Mouse Model of NIDDM".
Example 14
Construct ID 2328 and 2359, HLDOU18-HSA, Generation
[1249] Construct ID 2328, pC4.HLDOU18:K23-R429.HSA encodes for an
HLDOU18-HSA fusion protein which comprises the HSA chimeric leader
sequence and the mature bone-morphogenic protein 9, "HLDOU18",
protein, i.e. K23-R429, fused to the amino-terminus of the mature
form of HSA cloned into the mammalian expression vector pC4.
[1250] Construct ID 2359, pEE12.1.HLDOU18:K23-R429.HSA, encodes for
an HLDOU18-HSA fusion protein which comprises the HSA chimeric
leader sequence and the mature bone-morphogenic protein 9,
"HLDOU18", protein, i.e. K23-R429, fused to the amino-terminus of
the mature form of HSA cloned into the mammalian expression vector
pEE12.1.
[1251] Cloning of HLDOU18 cDNA for Constructs 2328 and 2359
[1252] The DNA encoding HLDOU18 was amplified with primers
HLDOU18-1 and HLDOU18-2, described below, cut with XhoI/ClaI, and
ligated into XhoI/ClaI cut pC4:HSA. Construct ID #2328 encodes an
albumin fusion protein containing the chimeric leader sequence of
HSA and the mature form of HLDOU18, followed by the mature HSA
protein.
[1253] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the mature form of HLDOU18, HLDOU18-1 and
HLDOU18-2, were synthesized.
TABLE-US-00014 HLDOU18-1: (SEQ ID NO: 286)
5'-CCGCCGCTCGAGGGGTGTGTTTCGTCGAAAGCCACTGCAGAGCTGG GGA-3' HLDOU18-2:
(SEQ ID NO: 287) 5'-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATCCCTGCACCC
ACACTCTGC-3'
HLDOU18-1 incorporates a XhoI cloning site (shown in italics), DNA
encoding for the last seven amino acids of the chimeric leader
sequence of HSA, and DNA encoding the first 7 amino acids of the
ORF of the mature form of HLDOU18 (shown in bold). In HLDOU18-2,
the ClaI site (shown in italics) and the DNA following it are the
reverse complement of DNA encoding the first 10 amino acids of the
mature HSA protein. In HLDOU18-2, the bolded sequence is the
reverse complement of the last 18 nucleotides encoding amino acid
residues Ala-424 to Arg-429 of the mature form of HLDOU18. Using
these two primers, the mature form of HLDOU18 protein was PCR
amplified. Annealing and extension temperatures and times must be
empirically determined for each specific primer pair and
template.
[1254] The PCR product was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested
with XhoI and ClaI. After further purification of the XhoI-ClaI
fragment by gel electrophoresis, the product was cloned into
XhoI/ClaI digested pC4:HSA to give construct ID #2328.
[1255] A HindIII/EcoRI fragment encompassing the HLDOU18 albumin
fusion was also subcloned from construct 2328 into the mammalian
expression vector pEE12.1 generating construct ID #2359 to be used
in the NS0 expression system.
[1256] The DNA encoding the mature form of HLDOU18 can also be
codon optimized so as not to hybridize to the wild-type DNA
encoding the marine form of HLDOU18. The polynucleotide encoding
the codon optimized HLDOU18 can be generated with overlapping
synthetic primers that take advantage of optimal codon usage for
mammalian cell-line expression. After PCR amplification and
digestion of the codon optimized DNA encoding the mature form of
HLDOU18 with the desired restriction enzymes, subsequent
construction of appropriate expression vectors comprising the
chimeric leader sequence of HSA fused to this codon optimized DNA
encoding the mature form of HLDOU18 fused to the amino-terminus of
die mature form of HSA can be carried out as previously described
in Example 5 and as described above.
[1257] Further analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing should confirm the presence
of the expected HLDOU18 sequence.
[1258] Expression and Purification of Construct ID 2328 and
2359.
[1259] Expression in Either 293T, CHO or NS0 Cells
[1260] Construct 2328 can be transfected into either 293T cells or
CHO cells by methods known in the an (e.g., lipofectamine
transfection) and selected with 100 nM methotrexate (see Example
5). Construct 2359 can be transfected into NS0 cells also by
methods known in the art.
[1261] Purification from 293T Cell Supernatant.
[1262] The 293T cell supernatant containing the secreted
HLDOU18-albumin fusion protein expressed from construct ID #2328 in
293T cells can be purified as described in Example 6.
[1263] Purification from CHO Cell Supernatant.
[1264] The cell supernatant containing the HLDOU18-albumin fusion
protein expressed from construct ID #2328 in CHO cells can be
purified as described in Example 7.
[1265] Purification from NS0 Cell Supernatant.
[1266] The cell supernatant containing the HLDOU18-albumin fusion
protein expressed from construct ID 42359 in NS0 cells can be
purified as described in Example 7.
[1267] In Vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct 2328
and 2359.
[1268] Method
[1269] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the HLDOU18 albumin fusion protein
encoded by either construct 2328 or construct 2359 can be carried
out as described below in Example 28. Other assays known in the art
used to test HLDOU18's involvement in insulin action can also be
employed including, but not limited to, L6 Rat Myoblast
Proliferation Assay via glycogen synthase kinase-3 (CSK-3) and
H4Ile reporter assays (see Example 35) including the rat Malic
Enzyme Promoter (rMEP)-SEAP, Sterol Regulatory Element Binding
Protein (SREBP)-SEAP, Fatty Acid Synthetase (FAS)-SEAP,
PhosphoEnolPyruvate CarboxyKinase (PEPCK)-SEAP reporters.
[1270] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct 2328
and 2359.
[1271] Method
[1272] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
HLDOU18 albumin fusion protein encoded by either constructs 2328 or
2359 can be carried out as described below under heading: "Example
29: In vitro Assay of [.sup.3H]-Thymidine Incorporation into
Pancreatic Cell-lines".
[1273] The Activity of the Albumin Fusion Protein Encoded by
Construct 2328 or by Construct 2359 can be Assayed In Vivo Using
Diabetic NOD and/or NIDDM Mouse Models.
[1274] The activity of the HLDOU18 albumin fusion protein encoded
by construct 2328 or by construct 2359 can be measured using NOD
and/or NIDDM mouse models described below under the headings,
"Example 31: Occurrence of Diabetes in NOD Mice", "Example 32:
Histological Examination of NOD Mice", and "Example 34: In vivo
Mouse Model of NIDDM".
Example 15
Construct ID 2340 and 2362, HSA-HLDOU18, Generation
[1275] Construct ID 2340, pC4.HSA.HLDOU18:K23-R429, encodes for an
HSA-HLDOU18 fusion protein which comprises the HSA chimeric leader
sequence and the mature form of HSA fused to the amino-terminus of
the mature bone-morphogenic protein 9, "HLDOU18", protein, i.e.
K23-R429, cloned into the mammalian expression vector pC4.
[1276] Construct ID 2362, pEE12.1.HSA.HLDOU18:K23-R429, encodes for
an HSA-HLDOU18 fusion protein which comprises the HSA chimeric
leader sequence and the mature form of HSA fused to the
amino-terminus of the mature bone-morphogenic protein 9, "HLDOU18",
protein, i.e. K23-R429, cloned into the mammalian expression vector
pEE12.1.
[1277] Cloning of HLDOU18 cDNA for Constructs 2340 and 2362
[1278] The DNA encoding the mature form of HLDOU18 was amplified
with primers HLDOU18-3 and HLDOU18-4, described below, cut with
Esu36I/Asp718, and ligated into Bsu36I/Asp718 cut pC4:HSA.
Construct ID #2340 encodes an albumin fusion protein containing the
chimeric leader sequence and the mature form of HSA fused to the
amino terminus of the mature form of HLDOU18 protein.
[1279] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the mature form of HLDOU18, HLDOU18-3 and
HLDOU18-4, were synthesized.
TABLE-US-00015 HLDOU18-3: (SEQ ID NO: 288)
5'-AAGCTGCCTTAGGCTTAAAGCCACTGCAGAGCTGG-3' HLDOU18-4: (SEQ ID NO:
289) 5'-GCGCGGGTACCTTACTACCTGCACCCACACTCTG-3'
HLDOU18-3 incorporates a Bsu36I cloning site (shown in italics) and
encodes the first 6 amino acids (shown in bold) of the ORF of the
mature form of HLDOU18. In HLDOU18-4, the italicized sequence is an
Asp718 site; the Asp718 site and the DNA following it are the
reverse complement of DNA encoding the last 6 amino acids (shown in
bold) of the mature HLDOU18 protein. In HLDOU18-4, the bolded
sequence is the reverse complement of the last 22 nucleotides
encoding amino acid residues Glu-425 to Arg-429 of the mature form
of HLDOU18. Using these two primers, the mature HLDOU18 protein was
PCR amplified. Annealing and extension temperatures and times must
be empirically determined for each specific primer pair and
template.
[1280] The PCR product was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested
with Bsu36I and Asp718. After further purification of the
Bsu36I-Asp718 fragment by gel electrophoresis, the product was
cloned into Bsu36I/Asp718 digested pC4:HSA to give construct ID
#2340.
[1281] A HindIII/EcoRI fragment encompassing the HLDOU18 albumin
fusion was also subcloned from construct 2340 into the mammalian
expression vector pEE12.1 generating construct ID #2362 to be used
in the NS0 expression system.
[1282] The DNA encoding the mature form of HLDOU18 can also be
codon optimized so as not to hybridize to the wild-type DNA
encoding the mature form of HLDOU18. The polynucleotide encoding
the codon optimized HLDOU18 can be generated with overlapping
synthetic primers that take advantage of optimal codon usage for
mammalian cell-line expression. After PCR amplification and
digestion of the codon optimized DNA encoding the mature form of
HLDOU18 with the desired restriction enzymes, subsequent
construction of appropriate expression vectors comprising the
chimeric leader sequence of and the mature form of HSA fused to the
cotton optimized DNA encoding the mature form of HLDOU18 can be
carried out as previously described in Example 5 and as described
above.
[1283] Further analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing should confirm the presence
of the expected mature HSA sequence.
[1284] Expression and Purification of Construct ID 2340 and
2362.
[1285] Expression in either 293T, CHO or NS0 cells.
[1286] Construct 2340 can be transfected into either 293T cells or
CHO cells by methods known in the art (e.g., lipofectamine
transfection) and selected with 100 nM methotrexate (see Example
5). Construct 2362 can be transfected into NS0 cells also by
methods known in the art.
[1287] Purification from 293T Cell Supernatant.
[1288] The 293T cell supernatant containing the secreted HLDOU18
albumin fusion protein expressed from construct ID #2340 in 293T
cells can be purified as described in Example 6.
[1289] Purification from CHO Cell Supernatant.
[1290] The cell supernatant containing the HLDOU18 albumin fusion
protein expressed from construct ID #2340 in CHO cells can be
purified as described in Example 6.
[1291] Purification from NS0 Cell Supernatant.
[1292] The cell supernatant containing the HLDOU18 albumin fusion
protein expressed from construct ID #2362 in NS0 cells can be
purified as described in Example 6.
[1293] In Vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct 2340
and 2362.
[1294] Method
[1295] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the HLDOU18 albumin fusion protein
encoded by either construct 2340 or construct 2362 can be carried
out as described below in Example 28. Other assays known in the art
used to test HLDOU18 albumin fusions' involvement in insulin action
can also be employed including, but not limited to, L6 Rat Myoblast
Proliferation Assay via glycogen synthase kinase-3 (GSK-3) and
H4Ile reporter assays (see Example 35) including the rat Matic
Enzyme Promoter (rMEP)-SEAP, Sterol Regulatory Element Binding
Protein (SREBP)-SEAP, Fatty Acid Synthetase (FAS)-SEAP,
PhosphoEnolPyruvate CarboxyKinase (PEPCK)-SEAP reporters.
[1296] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct 2340
and 2362.
[1297] Method
[1298] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
HLDOU18 albumin fusion protein encoded by either constructs 2340 or
2362 can be carried out as described below under heading: "Example
29: In vitro Assay of [.sup.3H]-Thymidine Incorporation into
Pancreatic Cell-lines".
[1299] The activity of the albumin fusion protein encoded by
construct 2340 or by construct 2362 can be assayed in vivo using
diabetic NOD and/or NIDDM mouse models.
[1300] The activity of the HLDOU18 albumin fusion protein encoded
by construct 2340 or by construct 2362 can be measured using NOD
and/or NIDDM mouse models described below under the headings,
"Example 31: Occurrence of Diabetes in NOD Mice", "Example 32:
Histological Examination of NOD Mice", and "Example 34: in vivo
Mouse Model of NIDDM".
Example 16
Construct ID 2369 and 2370, HLDOU18-HSA, Generation
[1301] Construct ID 2369, pC4.1-HLDOU18:M1-R429.HSA, encodes for an
HLDOU18-HSA fusion protein which comprises the full-length of
bone-morphogenic protein 9, "HLDOU18", protein, i.e. M1-R429, which
includes its native signal sequence, fused to the amino-terminus of
the mature form of HSA cloned into the mammalian expression vector
pC4.
[1302] Construct ID 2370, pEE12.1.HLDOU18:M1-R429.HSA, encodes for
an HLDOU18-HSA fusion protein which comprises the full-length of
bone-morphogentic protein 9, "HLDOU18", protein. i.e. M1-R429,
which includes its native signal sequence, fused to the
amino-terminus of the mature loon of HSA cloned into the mammalian
expression vector pEE12.1.
[1303] Cloning of HLDOU18 cDNA for Constructs 2369 and 2370
[1304] The DNA encoding the full-length HLDOU18 was amplified with
primers HLDOU18-5 and HLDOU18-6, described below, cut with
BamHI/ClaI, and ligated into cut pC4:1-HSA. Construct ID #2369
encodes an albumin fusion protein containing the full-length form
of HLDOU18 which includes its native signal sequence, followed by
the mature HSA protein.
[1305] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the full-length form of HLDOU18, HLDOU18-5
and HLDOU18-6, were synthesized.
TABLE-US-00016 HLDOU18-5: (SEQ ID NO: 300)
5'-AATTAAGGATCCAAGCTTGCCACCATGTGTCCTGGGGCACTGTGG-3' HLDOU18-6: (SEQ
ID NO: 301) 5'-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATCCCTGCACCC
ACACTCTGCCACGCTC-3'
HLDOU18-5 incorporates a BamHI cloning site (shown in italics), the
Kozak sequence shown underlined, and DNA encoding the first 7 amino
acids of the ORE of the full-length form of HLDOU18 (shown in
bold), i.e. M-1 to Trp-7. In HLDOU18-6, the ClaI site (shown in
italics) and the DNA following it are the reverse complement of DNA
encoding the first 10 amino acids of the mature HSA protein. In
HLDOU18-6, the bolded sequence is the reverse complement of the
last 25 nucleotides encoding amino acid residues Ser-422 to Arg-429
of the full-length form of HLDOU18, Using these two primers, the
full-length form of HLDOU18 protein was PCR amplified. Annealing
and extension temperatures and times must be empirically determined
for each specific primer pair and template.
[1306] The PCR pro-duct was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested
with BamHI and ClaI. After further purification of the BamHI-ClaI
fragment by gel electrophoresis, the product was cloned into
BamHI/ClaI digested pC4:HSA to give construct up #2369.
[1307] A HindIII/EcoRI fragment encompassing the HLDOU18 albumin
fusion was also subcloned from construct 2369 into the mammalian
expression vector pEE12.1 generating construct ID #2370 to be used
in the NS0 expression system.
[1308] The DNA encoding the full-length form of HLDOU18 can also be
codon optimized so as not to hybridize to the wild-type DNA
encoding the full-length form of HLDOU18. The polynucleotide
encoding the codon optimized HLDOU18 can be generated with
overlapping synthetic primers that take advantage of optimal codon
usage for mammalian cell-line expression. After PCR amplification
and digestion of the codon optimized DNA encoding the full-length
form of HLDOU18 with the desired restriction enzymes, subsequent
construction of appropriate expression vectors comprising the
native leader sequence and mature form of HLDOU18 encoded by this
codon optimized DNA fused to the amino-terminus of the mature form
of HSA can be carried out as previously described in Example 5 and
as described above.
[1309] Further analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing should confirm the presence
of the expected HLDOU18 sequence.
[1310] Expression and Purification of Construct ID 2369 and
2370.
[1311] Expression in Either 293T, CHO or NS0 Cells.
[1312] Construct 2369 can be transfected into either 293T cells or
CHO cells by methods known in the art (e.g., lipofectamine
transfection) and selected with 100 nM methotrexate (see Example
5). Construct 2370 can be transfected into NS0 cells also by
methods known in the art.
[1313] Purification from 293T Cell Supernatant.
[1314] The 293T cell supernatant containing the secreted HLDOU18
albumin fusion protein expressed from construct ID #2369 in 293T
cells can be purified as described in Example 6.
[1315] Purification from CHO Cell Supernatant.
[1316] The cell supernatant containing the HLDOU18 albumin fusion
protein expressed from construct ID #2369 in CHO cells can be
purified as described in Example 6.
[1317] Purification from NS0 Cell Supernatant.
[1318] The cell supernatant containing the HLDOU18 albumin fusion
protein expressed from construct ID #2370 in NS0 cells can be
purified as described in Example 6.
[1319] In vitro [.sup.3H]-2-Deoxyglucose Uptake Asset in the
Presence of the Albumin Fusion Protein Encoded by Construct 2369
and 2370.
[1320] Method
[1321] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the HLDOU18 albumin fusion protein
encoded by either construct 2369 or construct 2370 can be carried
out as described below in Example 28. Other assays known in the art
used to test HLDOU18 albumin fusions' involvement in insulin action
can also be employed including, but not limited to, L6 Rat Myoblast
Proliferation Assay via glycogen synthase kinase-3 (GSK-3) and Wile
reporter assays (see Example 35) including the rat Malic Enzyme
Promoter (rMEP)-SEAP, Sterol Regulatory Element Binding Protein
(SREBP)-SEAP, Fatty Acid Synthetase (FAS)-SEAP, PhosphoEnolPyruvate
CarboxyKinase (PEPCK)-SEAP reporters.
[1322] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct 2369
and 2370.
[1323] Method
[1324] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
HLDOU18 albumin fusion protein encoded by either construct 2369 or
2370 can be carried out as described below under heading: "Example
29: In vitro Assay of [.sup.3H]-Thymidine Incorporation into
Pancreatic Cell-lines".
[1325] The Activity of the Albumin Fusion Protein Encoded by
Construct 2369 or by Construct 2370 can be Assayed In Vivo Using
Diabetic NOD and/or NIDDM Mouse Models.
[1326] The activity of the HLDOU18 albumin fusion protein encoded
by construct 2369 or by construct 2370 can be measured using NOD
and/or NIDDM mouse models described below under the headings,
"Example 31: Occurrence of Diabetes in NOD Mice", "Example 32:
Histological Examination of NOD Mice", and "Example 34: In vivo
Mouse Model of NIDDM".
Example 17
Construct ID 2350, HLDOU18 S320-R429-HSA, Generation
[1327] Construct ED 2350, pC4.MPIF.HLDOU18:S320-R429.HSA, encodes
for an HLDOU18-HSA fusion protein which comprises the Myeloid
Progenitor Inhibitor Factor (MPIF) signal sequence fused to the
amino-terminus of a fragment of the bone-morphogenic protein 9,
"HLDOU18", protein, i.e. S320-R429, fused to the amino-terminus of
the mature form of HSA cloned into the mammalian expression vector
pC4.
[1328] Cloning of HLDOU18 cDNA for Construct 2350.
[1329] The DNA encoding a fragment of HLDOU18, i.e. Ser-320 to
Arg-429, fused to the MPIF signal sequence was amplified with
primers HLDOU18-7 and HLDOU18-8, described below, cut with
BglII/ClaI, and ligated into BglII/ClaI cut pC4:HSA. Construct ID
#2350 encodes an albumin fusion protein containing the MPIF signal
sequence fused to the amino-terminus of a fragment of HLDOU18, i.e.
Ser-320 to Arg-429, followed by the mature HSA protein.
[1330] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding a fragment of HLDOU18. HLDOU18-7 and
HLDOU18-8, were synthesized.
TABLE-US-00017 HLDOU18-7: (SEQ ID NO: 292)
5'-GCCGGAAGATCTCCGCCACCATGAAGGTCTCCGTGGCTGCCCTCTC
CTGCCTCATGCTTGTTACTGCCCTTGGATCCCAGGCCAGCGCCGGGGCT GGCAGCCAC-3'
HLDOU18-8: (SEQ ID NO: 293)
5'-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATCCCTGCACCC
ACACTCTGCCACGC-3'
HLDOU18-7 incorporates a BglII cloning site (shown in italics), the
DNA encoding the signal peptide of MPIF, and DNA encoding the 6
amino acid residues Ser-320 to Ser-325 of HLDOU18 (shown in bold).
In HLDOU18-8, the ClaI site (shown in italics) and the DNA
following it are the reverse complement of DNA encoding the first
10 amino acids of the mature HSA protein. In HLDOU18-8, the bolded
sequence is the reverse complement of the last 23 nucleotides
encoding amino acid residues Val-423 to Arg-429 of HLDOU18. Using
these two primers, the fragment Ser-320 to Arg-429 of HLDOU18
protein was PCR amplified. Annealing and extension temperatures and
times must be empirically determined for each specific primer pair
and template.
[1331] The PCR product was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested
with BglII and ClaI. After further purification of the BglII-ClaI
fragment by gel electrophoresis, the product was cloned into
BglII/ClaI digested pC4:HSA to give construct ID #2350.
[1332] The DNA encoding the Ser-320 to Arg-429 fragment of HLDOU18
can also be codon optimized so as not to hybridize to the wild-type
DNA encoding that fragment. The polynucleotide encoding the codon
optimized fragment of HLDOU18 can be generated with overlapping
synthetic primers that take advantage of optimal codon usage for
mammalian cell-line expression. After PCR amplification and
digestion of the codon optimized DNA encoding the Ser-320 to
Arg-429 fragment of HLDOU18 with the desired restriction enzymes,
subsequent construction of appropriate expression vectors
comprising the MPIF leader sequence fused to this codon optimized
DNA encoding fragment Ser-320 to Arg-429 of HLDOU18 fused to the
amino-terminus of the mature form of HSA can be carried out as
previously described in Example 5 and as described above.
[1333] Further analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing should confirm the presence
of the expected HLDOU18 sequence.
[1334] Expression and Purification of Construct ID 2350.
[1335] Expression in Either 293T or CHO Cells.
[1336] Construct 2350 can be transfected into either 293T cells or
CHO cells by methods known in the art (e.g., lipofectamine
transfection) and selected with 100 nM methotrexate (see Example
5).
[1337] Purification from 293T Cell Supernatant.
[1338] The 293T cell supernatant containing the secreted HLDOU18
albumin fusion protein expressed from construct ID #2350 in 293T
cells can be purified as described in Example 6.
Purification from CHO Cell Supernatant.
[1339] The cell supernatant containing the HLDOU18 albumin fusion
protein expressed from construct ID #2350 in CHO cells can be
purified as described in Example 6.
[1340] In Vitro PHI-2-Deoxyglucose Uptake Assay in the Presence of
the Albumin Fusion Protein Encoded by Construct 2350.
[1341] Method
[1342] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the HLDOU18 albumin fusion protein
encoded by construct 2350 can be carried out as described below in
Example 28. Other assays known in the art used to test HLDOU18
albumin fusions' involvement in insulin action can also be employed
including, but not limited to, L6 Rat Myoblast Proliferation Assay
via glycogen synthase kinase-3 (GSK-3) and H4Ile reporter assays
(see Example 35) including the rat Malic Enzyme Promoter
(rMEP)-SEAP, Sterol Regulatory Element Binding Protein
(SREBP)-SEAP, Fatty Acid Synthetase (FAS)-SEAP, PhosphoEnolPyruvate
CarboxyKinase (PEPCK)-SEAP reporters.
[1343] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2350.
[1344] Method
[1345] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
HLDOU18 albumin fusion protein encoded by construct 2350 can be
carried out as described below under heading: "Example 29: In vitro
Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1346] The Activity of the Albumin Fusion Protein Encoded by
Construct 2350 can be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1347] The activity of the HLDOU18 albumin fusion protein encoded
by construct 2350 can be measured using NOD and/or NIDDM mouse
models described below under the headings, "Example 31: Occurrence
of Diabetes in NOD Mice", "Example 32: Histological Examination of
NOD Mice", and "Example 34: In vivo Mouse Model of NIDDM".
Example 18
Construct ID 2351, HSA-HLDOU18 S320-R429, Generation
[1348] Construct ID 2351, pC4.HSA.HLDOU18:S320-R429, encodes for an
HSA-HLDOU18 fusion protein which comprises the HSA chimeric leader
sequence and the mature form of HSA fused to the amino-terminus of
a fragment of the bone-morphogenic protein 9, "HLDOU18", protein,
i.e. S320-R429, cloned into the mammalian expression vector
pC4.
[1349] Cloning of HLDOU18 cDNA for Construct 2351.
[1350] The DNA encoding HLDOU18 was amplified with primers
HLDOU18-9 and HLDOU18-10, described below, cut with Bsu36I/Asp718,
and ligated into Bsu36I/Asp718 cut pC4:HSA Construct ID #2351
encodes an albumin fusion protein containing the chimeric leader
sequence and the mature form of HSA, followed by the S320-R429
fragment of HLDOU18.
[1351] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the HLDOU18 fragment, HLDOU18-9 and
HLDOU18-10, were synthesized.
TABLE-US-00018 HLDOU18-9: (SEQ ID NO: 294)
5'-AAGCTGCCTTAGGCTTAAGCGCCGGGGCTGGC-3' HLDOU18-10: (SEQ ID NO: 295)
5'-GCCCGGGGTACCCTACCTGCACCCACACTCTGCC-3'
HLDOU18-9 incorporates a Bsu36I cloning site (shown in italics) and
encodes the 5 amino acid residues Ser-320 to Gly-324 (shown in
bold) of HLDOU18. In HLDOU18-10, the italicized sequence is an
Asp718 site; the Asp718 site and the DNA following it are the
reverse complement of DNA encoding the last 6 amino acids (shown in
bold) of the mature HLDOU18 protein. In HLDOU18-10, the bolded
sequence is the reverse complement of the last 22 nucleotides
encoding amino acid residues Ala-424 to Arg-429 of the mature form
of HLDOU18. Using these two primers, the Ser-320 to Arg-429
fragment of the HLDOU18 protein was PCR amplified. Annealing and
extension temperatures and times most be empirically determined for
each specific primer pair and template.
[1352] The PCR product was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested
with Bsu36I and Asp718. After further purification of the
Bsu36I-Asp718 fragment by gel electrophoresis, the product was
cloned into Esu36I/Asp718 digested pC4:HSA to give construct ID
#2351.
[1353] A HindIII/EcoRI fragment encompassing the HLDOU18 albumin
fusion can also be subcloned from construct 2351 into the mammalian
expression vector pEE12.1 to be used in the NS0 expression
system.
[1354] The DNA encoding the Ser-320 to Arg-429 fragment of HLDOU18
can also be codon optimized so as not to hybridize to the wild-type
DNA encoding that fragment. The polynucleotide encoding the codon
optimized fragment of HLDOU18 can be generated with overlapping
synthetic primers that take advantage of optimal codon usage for
mammalian cell-line expression. After PCR amplification and
digestion of the codon optimized DNA encoding the Ser-320 to
Arg-429 fragment of HLDOU18 with the desired restriction enzymes,
subsequent construction of appropriate expression vectors
comprising the chimeric leader sequence and mature form of HSA
fused to this codon optimized DNA encoding fragment Ser-320 to
Arg-429 of HLDOU18 can be carried out as previously described in
Example 5 and as described above.
[1355] Further analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing should confirm the presence
of the expected mature HSA sequence.
[1356] Expression and Purification of Construct ID 2351.
[1357] Expression in Either 293T or CHO Cells.
[1358] Construct 2351 can be transfected into either 293T cells or
CHO cells by methods known in the art (e.g., lipofectamine
transfection) and selected with 100 nM methotrexate (see Example
5).
Purification from 293T Cell Supernatant.
[1359] The 293T cell supernatant containing the secreted HLDOU18
albumin fusion protein expressed from construct ID #2351 in 293T
cells can be purified as described in Example 6.
Purification from G110 Cell Supernatant.
[1360] The cell supernatant containing the HLDOU18 albumin fusion
protein expressed from construct ID #2351 in CHO cells can be
purified as described in Example 6.
[1361] In Vitro [.sup.3H]-Deoxyglucose Uptake Assay in the Presence
of the Albumin Fusion Protein Encoded by Construct 2351.
[1362] Method
[1363] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the HLDOU18 albumin fusion protein
encoded by construct 2351 can be earned out as described below in
Example 28. Other assays known in the art used to test HLDOU18
albumin fusions' involvement in insulin action can also be employed
including, but not limited to, L6 Rat Myoblast Proliferation Assay
via glycogen synthase kinase-3 (GSK-3) and H4Ile reporter assays
(see Example 35) including the rat Malic Enzyme Promoter
(rMEP)-SEAP, Sterol Regulatory Element Binding Protein
(SREBP)-SEAP, Fatty Acid Synthetase (FAS)-SEAP, PhosphoEnolPyruvate
CarboxyKinase (PEPCK)-SEAP reporters.
[1364] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2351.
[1365] Method
[1366] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
HLDOU18 albumin fusion protein encoded by either constructs 2351
can be carried out as described below under heading: "Example 29:
In vitro Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1367] The activity of the Albumin Fusion Protein Encoded be
Construct 2351 can be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1368] The activity of the HLDOU18 albumin fusion protein encoded
by construct 2351 can be measured using NOD and/or NIDDM mouse
models described below under the headings, "Example 31: Occurrence
of Diabetes in NOD Mice", "Example 32: Histological Examination of
NOD Mice", and "Example 34: In vivo Mouse Model of NIDDM".
Example 19
Construct ID 2053, IFNb-HSA, Generation
[1369] Construct ID 2053, pEE12.1:1 IFNb-HSA, comprises DNA
encoding an IFNb albumin fusion protein which has the full-length
IFNb protein including the native IFNb leader sequence fused to the
amino-terminus of the mature form of HSA in the NS0 expression
vector pEE12.1.
[1370] Cloning of IFNb cDNA
[1371] The polynucleotide encoding IFNb was PCR amplified using
primers IFNb-1 and IFNb-2, described below, cut with Bam HI/Cla I,
and ligated into Bam HI/Cla I cut pC4:HSA, resulting in construct
2011. The Eco RI/Eco RI fragment from Construct ID It 2011 was
subcloned into the Eco RI site of pEE12, I generating construct ID
#2053 which comprises DNA encoding an albumin fusion protein
containing the leader sequence and the mature form of IFNb,
followed by the mature HSA protein.
[1372] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the full-length of IFNb, IFNb-1 and IFNb-2,
were synthesized:
TABLE-US-00019 IFNb-1: (SEQ ID NO: 256)
5'-GCGCGGATCCGAATTCCGCCGCCATGACCAACAAGTGTCTCCTCCA
AATTGCTCTCCTGTTGTGCTTCTCCACTACAGCTCTTTCCATGAGCTACA ACTTGCTTGG-3'
IFNb-2: (SEQ ID NO: 257)
5'-GCGCGCATCGATGAGCAACCTCACTCTTGTGTGCATCGTTTCGGA GGTAACCTCT-3'
[1373] The IFNb-1 primer incorporates a Bam HI cloning site (shown
underlined), an Eco RI cloning site, and a Kozak sequence (shown in
italics), followed by 80 nucleotides encoding the first 27 amino
acids of the full-length form of IFNb. In IFNb-2, the Cla I site
(shown underlined) and the DNA following it are the reverse
complement of DNA encoding the first 10 amino acids of the mature
HSA protein (SEQ ID NO:327) and the last 18 nucleotides are the
reverse complement of DNA encoding the last 6 amino acid residues
of IFNb (see Example 2). A PCR amplimer was generated using these
primers, purified, digested with Bam HI and Cla I restriction
enzymes, and cloned into the Bam HI and Cla I sites of the pC4:HSA
vector. After the sequence was confirmed, an Eco RI fragment
containing the IFNb albumin fusion protein expression cassette was
subcloned into Eco RI digested pEE12.1.
[1374] Further, analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing can confirm the presence of
the expected IFNb sequence (see below).
[1375] IFNb albumin fusion proteins of the invention preferably
comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to
either the N- or C-terminus of the mature form of IFNb, i.e.,
Met-22 to Asn-187. In one embodiment of the invention, IFNb albumin
fusion proteins of the invention further comprise a signal sequence
which directs the nascent fusion polypeptide in the secretory
pathways of the host used for expression. In a further preferred
embodiment, the signal peptide encoded by the signal sequence is
removed, and the mature IFNb albumin fusion protein is secreted
directly into the culture medium. IFNb albumin fusion proteins of
the invention may comprise heterologous signal sequences including,
but not limited to, MAF, INV, Ig, Fibulin B, Clusterin,
Insulin-Like Growth Factor Binding Protein 4, variant HSA leader
sequences including, but not limited to, a chimeric HSA/MAF leader
sequence, or other heterologous signal sequences known in the art.
In a preferred embodiment, IFNb albumin fusion proteins of the
invention comprise the native IFNb. In further preferred
embodiments, the IFNb albumin fusion proteins of the invention
further comprise an N-terminal methionine residue. Polynucleotides
encoding these polypeptides, including fragments and/or variants,
are also encompassed by the invention.
[1376] Expression and Purification of Construct ID 2053.
[1377] Expression in Marine Myeloma NS0 Cell-Lines.
[1378] Construct ID #2053, pEE12.1:IFNb-HSA, was electroporated
into NS0 cells by methods known in the art (see Example 6).
[1379] Purification from NS0 Cell Supernatant.
[1380] Purification of IFNb-HSA from NS0 cell supernatant may
follow the methods described in Example 7 which involve Q-Sepharose
anion exchange chromatography at pH 7.4 using a NaCl gradient from
0 to 1 M in 20 mM Tris-HCl, followed by Poros PI 50 anion exchange
chromatography at pH 6.5 with a sodium citrate gradient from 5 to
40 mM, and diafiltrating for 6 DV into 10 mM citrate, pH 65 and 140
mM NaCl, the final buffer composition. N-terminal sequencing should
yield the sequence MSYNLL which is the amino terminus of the mature
form of IFNb. The protein has an approximate MW of 88.5 kDa.
[1381] For larger scale purification, e.g., 50 L of NS0 cell
supernatant can be concentrated into .about.8 to 10 L. The
concentrated sample can then be passed over the Q-Sepharose anion
exchange column (10.times.19 cm, 1.5 L) at pH 7.5 using a step
elution consisting of 50 mM NaOAc, pH 6.0 and 150 mM NaCl. The
eluted sample can then be virally inactivated with 0.75% Triton-X
100 for 60 min at room temperature. SDR-Reverse Phase
chromatography (10 cm.times.10 cm, 0.8 L) can then be employed at
pH 6.0 with 50 mM NaOAc and 150 mM NaCl, or alternatively, the
sample can be passed over an SP sepharose column at pH 4.8 using a
step elution of 50 mM NaOAc, pH 6.0, and 150 mM NaCl. DV 50
filtration would follow to remove any viral content. Phenyl-650M
chromatography (20 cm.times.12 cm, 3.8 L) at pH 6.0 using a step
elution consisting of 350 mM (NH.sub.4).sub.2SO.sub.4 and 50 mM
NaOAc, or alternatively consisting of 50 mM NaOAc pH 6.0, can
follow. Diafiltration for 6-8 DV will allow for buffer exchange
into the desired final formulation buffer of either 10 mM
Na.sub.2HPO.sub.4+58 mM sucrose+120 mM NaCl, pH 7.2 or 10 mM
citrate, pH 6.5, and 140 mM NaCl or 25 mM Na.sub.2HPO.sub.4, 100 mM
NaCl, pH 7.2.
[1382] The Activity of INFb can be Assayed Using an In Vitro
ISRE-SEAP Assay.
[1383] All type I Interferon proteins signal through a common
receptor complex and a similar Jak/STAT signaling pathway that
culminates in the activation of Interferon, "IFN", responsive genes
through the Interferon Sequence Responsive Element, "ISRE". A
convenient assay for type I IFN activity is a promoter-reporter
based assay system that contains multiple copies of the ISRE
element fused to a downstream reporter gene. A stable HEK293
cell-line can be generated and contains a stably integrated copy of
an ISRE-SEAP reporter gene that is extremely sensitive to type I
IFNs and displays linearity over 5 logs of concentration,
Method of Screening of IFNb-HSA NS0 Stable Clones.
[1384] Construct 2053 was electroporated into NS0 cells as
described in Example 6. The NS0 cells transfected with construct ID
#2053 were screened for activity by testing conditioned growth
media in the ISRE-SEAP assay. The ISRE-SEAP/293F reporter cells
were plated at 3.times.10.sup.4 cell/well in 96-well, poly-D-lysine
coated, plates, one day prior to treatment. Reporter cells were
treated with various dilutions (including but not limited to 1:500
and 1:5000) of conditioned supernatant or purified preparations of
IFNb albumin fusion protein encoded by construct ID 2053 or rhIFNb
as a control. The reporter cells were then incubated for 24 hours
prior to removing 40 CL for use in the SEAP Reporter Gene
Chemiluminescent Assay (Roche catalog #1779842). Recombinant human
Interferon beta, "rhIFNb" (Biogen), was used as a positive
control.
[1385] Result
[1386] The purified preparation of NS0 expressed IFNb-HSA had a
greater EC50 of 9.3.times.10.sup.-9 g/mL than rhIFNb (Biogen) which
had an EC50 of 1.8.times.10.sup.-10 g/mL (see FIG. 6).
[1387] In Vivo Induction of OAS by an Interferon.
[1388] Method
[1389] The OAS enzyme, 2'-5'-OligoAdenylate Synthetase, is
activated at the transcriptional level by interferon in response to
antiviral infection. The effect of interferon constructs can be
measured by obtaining blood samples from treated monkeys and
analyzing these samples for transcriptional activation of two OAS
mRNA, p41 and p69. A volume of 0.5 mL of whole blood can be
obtained from 4 animals per group at 7 different time points, day
0, day 1, day 2, day 4, day 8, day 10, and day 14 per animal. The
various groups may include injection of vehicle control,
intravenous and/or subcutaneous injection of either 30 .mu.g/kg
and/or 300 .mu.g/kg IFN albumin fusion protein on day 1, and
subcutaneous injection of 40 .mu.g/kg of Interferon alpha
(Schering-Plough) as a positive control on days 1, 3, and 5. The
levels of the p41 and the p69 mRNA transcripts can be determined by
real-time quantitative PCR (Taqman) using probes specific for
p41-OAS and p69-OAS. OAS mRNA levels can be quantitated relative to
18S ribosomal RNA endogenous control.
[1390] In Vivo Induction of OAS by Interferon Beta Albumin Fusion
Encoded by Construct ID 2053.
[1391] Method
[1392] The activity of the HSA-IFNb fusion protein encoded by
construct 2053 can be assayed in the or vivo OAS assay as
previously described above under subsection heading, "In viva
induction of OAS by an Interferon".
Example 20
Indications for IFNb Albumin Fusion Proteins
[1393] IFN beta albumin fusion proteins (including, but not limited
to, those encoded by construct 2053) can be used to treat, prevent,
ameliorate and/or detect multiple sclerosis. Other indications
include, but are not limited to, melanoma, solid tumors, cancer,
bacterial infections, chemoprotection, thrombocytopenia, HIV
infections, prostate cancer, cancer, hematological malignancies,
hematological disorders, preleukemia, glioma, hepatitis B,
hepatitis C, human papillomavirus, pulmonary fibrosis, age-related
macular degeneration, brain cancer, glioblastoma multiforme, liver
cancer, malignant melanoma, colorectal cancer, Crohn's disease,
neurological disorders, non-small cell lung cancer, rheumatoid
arthritis, and ulcerative colitis.
Example 21
Construct ID 2250, HSA-Insulin (GYG), Generation
[1394] Construct ID 2250, pSAC35,HSA.INSULIN(GYG).F1-N62, encodes
for an HSA-INSULIN (GYG) fusion protein which comprises the HSA
chimeric leader sequence and the mature form of HSA fused to the
amino-terminus of the synthetic single-chain long-acting insulin
analog (INSULIN (GY.sup.32G)) with a Tyr at position 32, cloned
into the yeast S. cerevisiae expression vector pSAC35.
[1395] Cloning of INSULIN (GYG) cDNA for Construct 2250.
[1396] The DNA encoding the synthetic single-chain form of INSULIN
(GYG) was PCR generated using four overlapping primers. The
sequence corresponding to the C-peptide in the middle region of the
proinsulin cDNA was replaced by the C-domain of Insulin Growth
Factor 1, "IGF-1" (GY.sup.32GSSSRRAPQT) (SEQ ID NO:832), to avoid
the need for proinsulin processing and to ensure proper folding of
the single-chain protein. The sequence was codon optimized for
expression in yeast S. cerevisiae. The PCR fragment was digested
and subcloned into Bsu 36I/Asc I digested pScNHSA. A Nor I fragment
was then subcloned into the pSAC35 plasmid. Construct ID #2250
encodes for the chimeric leader sequence and the mature fonts of
HSA fused to the amino-terminus of the synthetic single-chain form
of INSULIN (GYG).
[1397] The 5' and 3' primers of the four overlapping
oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the synthetic single-chain form of INSULIN
(GYG), INSULIN (GYG)-1 and INSULIN (GYG)-2, were synthesized:
TABLE-US-00020 INSULIN (GYG)-1: (SEQ ID NO: 268)
5'-GTCAAGCTGCCTTAGGCTTATTCGTTAACCAACACTTGTGTGGTT
CTCACTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAA-3' INSULIN (GYG)-2: (SEQ ID
NO: 269) 5'-ATCGCATATGGCGCGCCCTATTAGTTACAGTAGTTTTCCAATTG
GTACAAAGAACAAATAGAAGTACAA-3'
INSULIN (GYG)-1 incorporates a Bsu 36I cloning site (shown in
italics) and encodes the first 21 amino acids (shown in bold) of
the ORE of the synthetic single-chain form of INSULIN (GYG). In
INSULIN (GYG)-2, the italicized sequence is an Asc I site. In
INSULIN (GYG)-2, the bolded sequence is the reverse complement of
the last 49 nucleotides encoding amino acid residues Cys-49 to
Asn-63 of the synthetic single-chain form of INSULIN (GYG). With
these two primers, the synthetic single-chain form of INSULIN (GYG)
was PCR amplified. Annealing and extension temperatures and times
must be empirically determined for each specific primer pair and
template.
[1398] The PCR product was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested
with Bsu36I and AscI. After further purification of the Bsu36I-AscI
fragment by gel electrophoresis, the product was cloned into
Bsu36I/AscI digested pScNHSA. A Not I fragment was further
subcloned into pSAC35 to give construct ID #2250.
[1399] Further analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing should confirm the presence
of the expected mature HSA sequence (see below).
[1400] INSULIN albumin fusion proteins of the invention preferably
comprise the mature form of HSA, i.e., Asp-25 to Len-609, fused to
either the N- or C-terminus of the synthetic single-chain analog of
INSULIN, i.e., Phe-1 so Asn-62; the sequence corresponding to the
C-peptide in the middle region of the proinsulin cDNA was replaced
by the C-domain of Insulin Growth Factor 1, "IGF-1"
(GY.sup.32GSSSRRAPQT) (SEQ ID NO:832). In one embodiment of the
invention, INSULIN albumin fusion proteins of the invention further
comprise a signal sequence which directs the nascent fusion
polypeptide in the secretory pathways of the host used for
expression. In a further preferred embodiment, the signal peptide
encoded by the signal sequence is removed, and the mature INSULIN
albumin fusion protein is secreted directly into the culture
medium. INSULIN albumin fusion proteins of the invention may
comprise heterologous signal sequences including, but not limited
to, MAF, INV, Ig, Fibulin B, Chisterin, Insulin-Like Growth Factor
Binding Protein 4, variant HSA leader sequences including, but not
limited to, a chimeric HSA/MAF leader sequence, or other
heterologous signal sequences known in the art. In a preferred
embodiment, INSULIN albumin fusion proteins of the invention
comprise the native INSULIN. In further preferred embodiments, the
INSULIN albumin fusion proteins of the invention further comprise
an N-terminal methionine residue. Polynucleotides encoding these
polypeptides, including fragments and/or variants, are also
encompassed by the invention.
[1401] Expression and Purification of Construct ID 2250.
[1402] Expression in Yeast S. cerevisiae.
[1403] Construct 2250 can be transformed into yeast S. cerevisiae
by methods known in the art (see Example 3). Expression levels can
be examined by immunoblot detection with anti-HSA serum as the
primary antibody.
Purification from Yeast S. cerevisiae Cell Supernatant.
[1404] The cell supernatant containing the secreted INSULIN (GYG)
albumin fusion protein expressed from construct ID #2250 in yeast
S. cerevisiae can be purified as described in Example 4. N-terminal
sequencing of the albumin fusion protein should result in the
sequence DAHKS which corresponds to the amino terminus of the
mature form of HSA.
[1405] In Vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2250.
[1406] Method
[1407] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the INSULIN (GYG) albumin fusion
protein encoded by construct 2250 was carried out as described
below in Example 28. Other assays known in the art that may be used
to test INSULIN (GYG) albumin fusion proteins' include, but are not
limited to, L6 Rat Myoblast Proliferation Assay via glycogen
synthase kinase-3 (GSK-3) and H4Ile reporter assays (see Example
35) including the rat Malic Enzyme Promoter (rMEP)-SEAP, Sterol
Regulatory Element Binding Protein (SREBP)-SEAP, Fatty Acid
Synthetase (FAS)-SEAP, and PhosphoEnolPyruvate CarboxyKinase
(PEPCK)-SEAP reporters.
[1408] Result
[1409] The supernatant derived from transformed yeast S. cerevisiae
expressing insulin albumin fusion encoded by construct 2250
demonstrated glucose uptake/transport activity in 3T3-L1 adipocytes
(see FIG. 2).
[1410] In Vitro Pancreatic Cell-Lines Proliferation Assess in the
Presence if the Albumin Fusion Protein Encoded by Construct
2250.
[1411] Method
[1412] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
INSULIN (GYG) albumin fusion protein encoded by construct 2250 can
be carried out as described below under heading: "Example 29: In
vitro Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1413] The activity of the Albumin Fusion Protein Encoded by
Construct 2250 can be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1414] The activity of the INSULIN (GYG) albumin fusion protein
encoded by construct 2250 can be measured using NOD and/or NIDDM
mouse models described below under the headings, "Example 31:
Occurrence of Diabetes in NOD Mice", "Example 32: Histological
Examination of NOD Mice", and "Example 34: In vivo Mouse Model of
NIDDM".
Example 22
Construct ID 2255, Insulin (GYG)-HSA, Generation
[1415] Construct ID 2255, pSAC35.INSULIN(GYG).F1-N62.HSA, encodes
for an INSULIN (GYG)-HSA fusion protein which comprises the HSA
chimeric leader sequence of HSA fused to the amino-terminus of the
synthetic single-chain long-acting insulin analog (INSULIN
(GY.sup.32G)) with a Tyr in position 32, which is, in turn, fused
to the mature form of HSA, cloned into the yeast S. cerevisiae
expression vector pSAC35.
[1416] Cloning of INSULIN IGYG) cDNA for Construct 2255.
[1417] The DNA encoding the synthetic single-chain form of INSULIN
(GYG) was PCR generated using four overlapping primers. The
sequence corresponding to the C-peptide in the middle region of the
proinsulin cDNA was replaced by the C-domain of Insulin Growth
Factor 1. "IGF-1" (GY.sup.32GSSSRRAPQT) (SEQ ID NO: 832), to avoid
the need for proinsulin processing and to ensure proper folding of
the single-chain protein. The sequence was codon optimized for
expression in yeast S. cerevisiae. The PCR fragment was digested
with Sal I/Cla I and subcloned into Xho I/Cla I digested pScCHSA. A
Not I fragment was then subcloned into the pSAC35 plasmid.
Construct ID #2255 encodes for the chimeric leader sequence of HSA
fused to the amino-terminus of the synthetic single-chain form of
INSULIN (GYG) followed by the mature form of HSA.
[1418] The 5' and 3' primers of the four overlapping
oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the synthetic single-chain form of INSULIN
(GYG), INSULIN (GYG)-3 and INSULIN (GYG)-4, were synthesized:
TABLE-US-00021 INSULIN (GYG)-3: (SEQ ID NO: 271)
5'-TCCAGGAGCGTCGACAAAAGATTCGTTAACCAACACTTGTGTGG
TTCTCACTTGGTTGAAGCTTTGTACTTGGTTTCTGCTGAA-3' INSULIN (GYG)-4: (SEQ
ID NO: 272) 5'-AGACTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATCGTTA
CAGTAGTTTTCCAATTGGTACAAAGAACAAATAGAAGTACAA-3'
INSULIN (GYG)-3 incorporates a Sal I cloning site (shown in
italics) and the DNA encoding the first 21 amino acids (shown in
hold) of the ORF of the synthetic single-chain form of
INSULIN(GYG). In INSULIN (GYG)-4, the italicized sequence is a Cla
I site; and the Cla I site and the DNA following it are the reverse
complement of DNA encoding the first 10 amino acids of the mature
HSA protein. The bolded sequence is the reverse complement of the
46 nucleotides encoding the last 15 amino acid residues Cys-49 to
Asn-63 of the synthetic single-chain form of INSULIN (GYG). With
these two primers, the synthetic single-chain INSULIN (GYG) protein
was generated by annealing, extension of the annealed primers,
digestion with Sal I and Cla I, and subcloning into Xho I/Cla I
digested pScCHSA. The Not I fragment from this clone was then
ligated into the Not 1 site of pSAC35 to generate construct ID
2255. Construct ID #2255 encodes an albumin fusion protein
containing the chimeric leader sequence, the synthetic single-chain
form of INSULIN (GYG), and the mature form of HSA.
[1419] Further analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing should confirm the presence
of the expected INSULIN (GYG) sequence (see below).
[1420] INSULIN albumin fusion proteins of the invention preferably
comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to
either the N- or C-terminus of the synthetic single-chain analog of
INSULIN, Phe-1 to Asn-62; the sequence corresponding to the
C-peptide in the middle region of the proinsulin cDNA was replaced
by the C-domain of Insulin Growth Factor 1, "IGF-1"
(GY.sup.32GSSSRRAPQT) (SEQ ID NO:832). In one embodiment of the
invention, INSULIN albumin fusion proteins of the invention further
comprise a signal sequence which directs the nascent fusion
polypeptide in the secretory pathways of the host used for
expression. In a further preferred embodiment, the signal peptide
encoded by the signal sequence is removed, and the mature INSULIN
albumin fusion protein is secreted directly into the culture
medium. INSULIN albumin fusion proteins of the invention may
comprise heterologous signal sequences including, but not limited
to, MAF, INV, Ig, B, Clusterin, Insulin-Like Growth Factor Binding
Protein 4, variant HSA leader sequences including, but not limited
to, a chimeric HSA/MAF leader sequence, or other heterologous
signal sequences known in the art. In a preferred embodiment,
INSULIN albumin fusion proteins of the invention comprise the
native INSULIN. In further preferred embodiments, the INSULIN
albumin fusion proteins of the invention further comprise an
N-terminal methionine residue. Polynucleotides encoding these
polypeptides, including fragments and/or variants, are also
encompassed by the invention.
[1421] Expression and Purification of Construct ID 2255.
[1422] Expression in Yeast S. cerevisiae.
[1423] Construct 2255 can be transformed into yeast S. cerevisiae
by methods known in the art (see Example 3) Expression levels can
be examined by immunoblot detection with anti-HSA serum as the
primary antibody.
Purification from Yeast S. cerevisiae Cell Supernatant.
[1424] The cell supernatant containing the secreted INSULIN (GYG)
albumin fusion protein expressed from construct ID #2255 in yeast
S. cerevisiae can be purified as described in Example 4. N-terminal
sequencing of the expressed and purified albumin fusion protein
should generate FVNQH which corresponds to the amino terminus of
the synthetic single-chain long-acting insulin analog (INSULIN
(GY.sup.32G)).
[1425] In Vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Album n Fusion Protein Encoded by Construct
2255.
[1426] Method
[1427] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the INSULIN (GYG) albumin fusion
protein encoded by construct 2255 can be carried out as described
below in Example 28. Other assays known in the art that may be used
to test INSULIN (GYG) albumin fusion proteins' include, but are not
limited to, L6 Rat Myoblast Proliferation Assay via glycogen
synthase kinase-3 (GSK-3) and H4Ile reporter assays (see Example
35) including the rat Malic Enzyme Promoter (rMEP)-SEAP, Sterol
Regulatory Element Binding Protein (SREBP)-SEAP, Fatty Acid
Synthetase (FAS)-SEAP, and PhosphoEnolPyravate CarboxyKinase
(PEPCK)-SEAP reporters.
[1428] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded be Construct
2255.
[1429] Method
[1430] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
INSULIN (GYG) albumin fusion protein encoded by construct 2255 can
be carried out as described below under heading: "Example 29: In
vitro Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1431] The Activity of the Albumin Fusion Protein Encoded by
Construct 2255 can be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1432] The activity of the INSULIN (GYG) albumin fusion protein
encoded by construct 2255 can be measured using NOD and/or NIDDM
mouse models described below under the headings, "Example 31:
Occurrence of Diabetes in NOD Mice", "Example 327 Histological
Examination of NOD Mice", and "Example 34: In vivo Moose Model of
NIDDM".
Example 23
Construct ID 2276, HSA-Insulin (GGG), Generation
[1433] Construct ID 2276, pSAC35.HSA.INSULIN(GGG).F1-N58, encodes
for an HSA-INSULIN (GGG) fusion protein which comprises the HSA
chimeric leader sequence and the mature form of HSA fused to the
amino-terminus of the synthetic single-chain long-acting insulin
analog (INSULIN (GG.sup.32G)) with a Gly at position 32, cloned
into the yeast S. cerevisiae expression vector pSAC35.
[1434] Cloning of INSULIN (GGG) cDNA for Construct 2276.
[1435] The DNA encoding the synthetic single-chain form of INSULIN
(GGG) was PCR generated using four overlapping primers. The
sequence corresponding to the C-peptide in the middle region of the
proinsulin cDNA was replaced by the synthetic linker
"GG.sup.32GPGKR" (SEQ ID NO:858) to avoid the need for proinsulin
processing and to ensure proper folding of the single-chain
protein. The sequence was codon optimized for expression in yeast
S. cerevisiae. The PCR fragment was digested and subcloned into Bsu
36I/Asc I digested pScNHSA. A Not I fragment was then subcloned
into the pSAC35 plasmid. Construct ID #2276 encodes for the
chimeric leader sequence and the mature form of HSA fused to the
amino-terminus of the synthetic single-chain form of INSULIN
(GGG).
[1436] The 5' and 3' primers of the four overlapping
oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the synthetic single-chain form of INSULIN
(GOO), INSULIN (GGG)-1 and INSULIN (GGG)-2, were synthesized:
TABLE-US-00022 INSULIN (GGG)-5: (SEQ ID NO: 268)
5'-GTCAAGCTGCCTTAGGCTTATTCGTTAACCAACACTTGTGTGGT
TCTCACTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAA-3' INSULIN (GGG)-6: (SEQ ID
NO: 269) 5'-ATCGCATATGGCGCGCCCTATTAGTTACAGTAGTTTTCCAATTG
GTACAAAGAACAAATAGAAGTACAA-3'
INSULIN (GGG)-5 incorporates a Bsu 36I cloning site (shown in
italics) and encodes the first 21 amino acids (shown in bold) of
the ORF of the synthetic single-chain form of INSULIN (GGG). In
INSULIN (GGG)-6, the italicized sequence is an Ase I site. In
INSULIN (GGG)-6, the bolded sequence is the reverse complement of
the last 49 nucleotides encoding amino acid residues Cys-44 to
Asn-58 of the synthetic single-chain form of INSULIN (GGG). With
these two primers, the synthetic single-chain form of INSULIN (GGG)
was PCR amplified. Annealing and extension temperatures and times
must be empirically determined for each specific primer pair and
template.
[1437] The PCR product was purified (for example, using Wizard VCR
Preps DNA Purification System (Promega Corp)) and then digested
with Bsu36I and AscI. After further purification of the Bsu35I-AscI
fragment by gel electrophoresis, the product was cloned into
Bsu36I/AscI digested pScNHSA. A Not 1 fragment was further
subcloned into pSAC35 to give construct ID #2276.
[1438] Further, analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing should confirm the presence
of the expected mature HSA sequence (see below).
[1439] INSULIN albumin fusion proteins of the invention preferably
comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to
either the N- or C-terminus of the synthetic single-chain analog of
INSULIN, i.e., Phe-1 to Asn-58; the sequence corresponding to the
C-peptide in the middle region of the proinsulin cDNA was replaced
by the synthetic linker "GG.sup.32GPGKR" (SEQ ID NO:858). In one
embodiment of the invention, INSULIN albumin fusion proteins of the
invention further comprise a signal sequence which directs the
nascent fusion polypeptide in the secretory pathways of the host
used for expression. In a further preferred embodiment, the signal
peptide encoded by the signal sequence is removed, and the mature
INSULIN albumin fusion protein is secreted directly into the
culture medium. INSULIN albumin fusion proteins of the invention
may comprise heterologous signal sequences including, but not
limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth
Factor Binding Protein 4, variant HSA leader sequences including,
but not limited to, a chimeric HSA/MAF leader sequence, or other
heterologous signal sequences known in the art. In a preferred
embodiment, INSULIN albumin fusion proteins of the invention
comprise the native INSULIN. In further preferred embodiments, the
INSULIN albumin fusion proteins of the invention further comprise
an N-terminal methionine residue. Polynucleotides encoding these
polypeptides, including fragments and/or variants, are also
encompassed by the invention.
[1440] Expression and Purification of Construct ID 2276.
[1441] Expression in yeast S. cerevisiae.
[1442] Construct 2276 cart be uarisfinnied into yeast S. cerevisiae
by methods known in the art. (see Example 3). Expression levels can
be examined by immunoblot detection with anti-HSA serum as the
primary antibody.
Purification from Yeast S. cerevisiae Cell Supernatant.
[1443] The cell supernatant containing the secreted INSULIN (GGG)
albumin fusion protein expressed from construct ID #2276 in yeast
S. cerevisiae can be purified as described in Example 4. N-terminal
sequencing should yield DARKS which corresponds to the amino
terminus of the mature form of HSA.
[1444] In Vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2276.
[1445] Method
[1446] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the INSULIN (GGG) albumin fusion
protein encoded by construct 2276 was carried out as described
below in Example 28. Other assays known in the art that may be used
to test INSULIN (GGG) albumin fusion proteins' include, but are not
limited to, L6 Rat Myoblast Proliferation Assay via glycogen
synthase kinase-3 (GSK-3) and H4Ile reporter assays (see Example
35) including the rat Malic Enzyme Promoter (rMEP)-SEAP, Sterol
Regulatory Element Binding Protein (SREBP)-SEAP, Fatty Acid
Synthetase (FAS)-SEAP, and PhosphoEnolPyruvate CarboxyKinase
(PEPCK)-SEAP reporters.
[1447] Result
[1448] The supernatant derived from transformed yeast S. cerevisiae
expressing insulin albumin fusion encoded by construct 2276
demonstrated glucose uptake/transport activity in 3T3-L1 adipocytes
(see FIG. 2).
[1449] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2276.
[1450] Method
[1451] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
INSULIN (GGG) albumin fusion protein encoded by construct 2276 can
be carried out as described below under heading: "Example 29: In
vitro Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1452] The Activity of the Albumin Fusion Protein Encoded by
Construct 2276 can be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1453] The activity of the INSULIN (GGG) albumin fusion protein
encoded by construct 2276 can be measured using NOD and/or NIDDM
mouse models described below under the headings, "Example 31:
Occurrence of Diabetes in NOD Mice", "Example 32: Histological
Examination of NOD Mice", and "Example 34: In vivo Mouse Model of
NIDDM".
Example 24
Construct ID 2278, Insulin (GGG)-HSA, Generation
[1454] Construct ID 2278, pSAC35.INSULIN(GGG).HSA, encodes for an
INSULIN (GGG)-HSA fusion protein which comprises the HSA chimeric
leader sequence of HSA fused to the amino-terminus of the synthetic
single-chain long-acting insulin analog (INSULIN (GG.sup.2G)) with
a Gly in position 32, which is, in turn, fused to the mature form
of HSA, cloned into the yeast S. cerevisiae expression vector
pSAC35.
[1455] Cloning of INSULIN (GGG) cDNA for Construct 2278.
[1456] The DNA encoding the synthetic single-chain form of INSULIN
(GGG) was PCR generated using four overlapping primers. The
sequence corresponding to the C-peptide in the middle region of the
proinsulin cDNA was replaced by the synthetic linker
"GG.sup.32GPGKR" (SEQ ID NO:858) to avoid the need for proinsulin
processing and to ensure proper folding of the single-chain
protein, The sequence was codon optimized for expression in yeast
S. cerevisiae. The PCR fragment was digested with Sal I/Cla I and
subcloned into Xho I/Cla I digested pScCHSA. A Not I fragment was
then subcloned into the pSAC35 plasmid. Construct ID #2278 encodes
for the chimeric leader sequence of HSA fused to the amino-terminus
of the synthetic single-chain form of INSULIN (GGG) followed by the
mature form of HSA.
[1457] The 5' and 3' primers of the four overlapping
oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the synthetic single-chain form of INSULIN
(GGG), INSULIN (GGG)-7 and INSULIN (GGG)-8, were synthesized:
TABLE-US-00023 INSULIN (GGG)-7: (SEQ ID NO: 275)
5'-TCCAGGAGCGTCGACAAAAGATTCGTTAACCAACACTTGTGTGG
TTCTCACTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAA-3' INSULIN (GGG)-8: (SEQ
ID NO: 276) 5'-AGACTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATCGTTA
CAGTAGTTTTCCAATTGGTACAAAGAACAAATAGAAGTACAA-3'
INSULIN (GGG)-7 incorporates a Sal I cloning site (shown in
italics) and the DNA encoding the first 21 amino acids (shown in
bold) of the ORF of the synthetic single-chain form of INSULIN
(GGG). In INSULIN (GGG)-8, the italicized sequence is a Cla I site;
and the Cla I site and the DNA following it are the reverse
complement of DNA encoding the first 10 amino acids of the mature
HSA protein. The bolded sequence is the reverse complement of the
46 nucleotides encoding the last 15 amino acid residues Cys-44 to
Asn-58 of the synthetic single-chain form of INSULIN (GGG). With
these two primers, the synthetic single-chain INSULIN (GGG) protein
was generated by annealing, extension of the annealed primers,
digestion with Sal I and Cla I, and subcloning into Xho I/Cla I
digested pScCHSA. The Not I fragment from this clone was then
ligated into the Not I site of pSAC35 to generate construct ID
2278. Construct ID #2278 encodes an albumin fusion protein
containing the chimeric leader sequence, the synthetic single-chain
form of INSULIN (COG), and the mature form of HSA.
[1458] Further, analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing should confirm the presence
of the expected INSULIN (GGG) sequence (see below).
[1459] INSULIN albumin fusion proteins of the invention preferably
comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to
either the N- or C-terminus of the synthetic single-chain analog of
INSULIN, i.e. Phe-1 to Asn-58; the sequence corresponding to the
C-peptide in the middle region of the proinsulin cDNA was replaced
by the synthetic linker "GG.sup.32GPGKR" (SEQ ID NO:858). In one
embodiment of the invention. INSULIN albumin fusion proteins of the
invention further comprise a signal sequence which directs the
nascent fusion polypeptide in the secretory pathways of the host
used for expression. In a further preferred embodiment, the signal
peptide encoded by the signal sequence is removed, and the mature
INSULIN albumin fusion protein is secreted directly into the
culture medium. INSULIN albumin fusion proteins of the invention
may comprise heterologous signal sequences including, but not
limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin Like Growth
Factor Binding Protein 4, variant HSA leader sequences including,
but not limited to a chimeric HSA/MAE leader sequence, or other
Heterologous signal sequences known in the art. In a preferred
embodiment, INSULIN albumin fusion proteins of the invention
comprise the native INSULIN. In further preferred embodiments, the
INSULIN albumin fusion proteins of the invention further comprise
an N-terminal methionine residue. Polynucleotides encoding these
polypeptides, including fragments and/or variants, are also
encompassed by the invention.
[1460] Expression and Purification of Construct ID 2278,
[1461] Expression in Yeast S. cerevisiae.
[1462] Construct 2278 can be transformed into yeast S. cerevisiae
by methods known in the art (see Example 3). Expression levels can
be examined by immunoblot detection with anti-HSA serum as the
primary antibody.
Purification from Yeast S. cerevisiae Cell Supernatant.
[1463] The cell supernatant containing the secreted INSULIN (GGG)
albumin fusion protein expressed from construct ID #2278 in yeast
S. cerevisiae can be purified as described in Example 4. N-terminal
sequencing of the expressed and purified albumin fusion protein
should generate FVNQH which corresponds to the amino terminus of
the synthetic single-chain long-acting insulin analog (INSULIN
(GG.sup.32G)).
[1464] In Vitro [.sup.3H]-2-Deoxyglucose Uptake Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2278.
[1465] Method
[1466] The in vitro assay to measure the glucose uptake in 3T3-L1
adipocytes in the presence of the INSULIN (GGG) albumin fusion
protein encoded by construct 2278 can be carried out as described
below in Example 28. Other assays known in the art that may be used
to test INSULIN (GGG) albumin fusion proteins' include, but are not
limited to, L6 Rat Myoblast Proliferation Assay via glycogen
synthase kinase-3 (GSK-3) and H4Ile reporter assays (see Example
35) including the rat Malic Enzyme Promoter (rMEP)-SEAP, Sterol
Regulatory Element Binding Protein (SREBP)-SEAP, Fatty Acid
Synthetase (FAS)-SEAP, and PhosphoEnolPyruvate CarboxyKinase
(PEPCK)-SEAP reporters.
[1467] In Vitro Pancreatic Cell-Lines Proliferation Assay in the
Presence of the Albumin Fusion Protein Encoded by Construct
2278.
[1468] Method
[1469] The in vitro assay to measure the differentiation and
proliferation of ductal epithelium pancreatic ARIP cell-line into
insulin-producing beta cells and/or to measure the proliferation of
the insulin-producing RIN-M beta cell-line in the presence of the
INSULIN (GGG) albumin fusion protein encoded by construct 2278 can
be carried out as described below under beading; "Example 29; In
vitro Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-lines".
[1470] The Activity of the Albumin Fusion Protein Encoded by
Construct 2278 Con be Assayed In Vivo Using Diabetic NOD and/or
NIDDM Mouse Models.
[1471] The activity of the INSULIN (GGG) albumin fusion protein
encoded by construct 2278 can be measured using NOD and/or NIDDM
mouse models described below under the headings, "Example 31:
Occurrence of Diabetes in NOD Mice", "Example 32: Histological
Examination of NOD Mice", and "Example 34; In viva Mouse Model of
NIDDM".
Example 25
Indications for Insulin Albumin Fusion Proteins
[1472] Results from in vitro assays described above indicate that
insulin albumin fusion proteins are useful for the treatment,
prevention, and/or diagnosis of hyperglycemia, insulin resistance,
insulin deficiency, hyperlipidemia, hyperketonemia, and diabetes
mellitus, Type 1 and Type 2 diabetes,
Example 26
Isolation of a Selected ellNA Clone from the Deposited Sample
[1473] Many of the albumin fusion constructs of the invention have
been deposited with the ATCC as shown in Table 3. The albumin
fusion constructs may comprise any one of the following expression
vectors, the yeast S. cerevisiae expression vector pSAC35, the
mammalian expression vector pC4, or the mammalian expression vector
pEE12.1.
[1474] pSAC35 (Sleep et al., Biotechnology 8:42 (1990)), pC4 (ATCC
Accession No. 209646; Cullen et al., Molecular and Cellular
Biology, 438-447 (1985); Boshart et al., Cell 41: 521-530 (1985)),
and pEE12.1 (Lanza Biologics, Inc.; Stephens and Cockett, Nucl.
Acids Res. 17: 7110 (1989); International Publication #WO89/01036;
Murphy et al., Biochem J. 227: 277-279 (1991); Bebbington et al.,
Bio/Technology 10:169-175 (1992); U.S. Pat. No. 5,122,464;
International Publication #WO86/05807) vectors comprise an
ampicillin resistance gene for growth in bacterial cells. These
vectors and/or all albumin fusion construct comprising them can be
transformed into an E. coli strain such as Stratagene Blue
(Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La
Jolla, Calif., 92037) using techniques described in the art such as
Hanahan, spread onto Luria-Broth agar plates containing 100
.mu.g/mL ampicillin, and grown overnight at 37.degree. C.
[1475] The deposited material in the sample assigned the ATCC
Deposit Number cited in Table 3 for any given albumin fusion
construct also may contain one or more additional albumin fusion
constructs, each encoding different albumin fusion proteins. Thus,
deposits sharing the same ATCC Deposit Number contain at least an
albumin fusion construct identified in the corresponding row of
Table 3.
[1476] Two approaches can be used to isolate a particular albumin
fusion construct from the deposited sample of plasmid DNAs cited
for that albumin fusion construct in Table 3.
[1477] Method 1: Screening
[1478] First, an albumin fusion construct may be directly isolated
by screening the sample of deposited plasmid DNAs using a
polynucleotide probe corresponding to SEQ ID NO:X using methods
known in the art. For example, a specific polynucleotide with 30-40
nucleotides may be synthesized using an Applied Biosystems DNA
synthesizer according to the sequence reported. The oligonucleotide
can be labeled, for instance, with .sup.32P-.gamma.-ATP using T4
polynucleotide kinase and purified according to routine methods.
(E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). The albumin
fusion construct from a given ATCC deposit is transformed into a
suitable host, as indicated above (such as XL-1 Blue (Stratagene))
using techniques known to those of skill in the art, such as those
provided by the vector supplier or in related publications or
patents cited above. The transformants are plated on 1.5% agar
plates (containing the appropriate selection agent, e.g.,
ampicillin) to a density of about 150 transformants (colonies) per
plate. These plates are screened using Nylon membranes according to
routine methods for bacterial colony screening (e.g., Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989),
Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other
techniques known to those of skill in the art.
[1479] Method 2: PCR
[1480] Alternatively, DNA encoding a given albumin fusion protein
may be amplified from a sample of a deposited albumin fusion
construct with SEQ ID NO:X, for example, by using two primers of
17-20 nucleotides that hybridize to the deposited albumin fusion
construct 5' and 3' to the DNA encoding a given albumin fusion
protein. The polymerase chain reaction is carried out under routine
conditions, for instance, in 25 .mu.l of reaction mixture with 0.5
ug of the above cDNA template. A convenient reaction mixture is
1.5-5 mM MgCl.sub.2, 0.01% (w/v) gelatin, 20 .mu.M each of dATP,
dCTP, dGTP, dTTP, 25 .mu.mol of each primer and 0.25 Unit of Taq
polymerase. Thirty five cycles of PCR (denaturation at 94.degree.
C. for 1 min; annealing at 55.degree. C. for 1 min; elongation at
72.degree. C. for 1 min) are performed with a Perkin-Elmer Cetus
automated thermal cycler. The amplified product is analyzed by
agarose gel electrophoresis and the DNA band with expected
molecular weight is excised and purified. The PCR product is
verified to be the selected sequence by subcloning and sequencing
the DNA product.
[1481] Several methods are available for the identification of the
5' or 3' non-coding portions of a gene which may not be present in
the deposited clone. These methods include but are not limited to,
filter probing, clone enrichment using specific probes, and
protocols similar or identical to 5' and 3' "RACE" protocols which
are known in the art. For instance, a method similar to 5' RACE is
available for generating the missing 5' end of a desired
full-length transcript. (Fromont-Racine et al., Nucleic Acids Res.,
21(7):1683-1684 (1993)).
[1482] Briefly, a specific RNA oligonucleotide is ligated to the 5'
ends of a population of RNA presumably containing full-length gene
RNA transcripts. A primer set containing a primer specific to the
ligated RNA oligonucleotide and a primer specific to a known
sequence of the gene of interest is used to PCR amplify the 5'
portion of the desired full-length gene. This amplified product may
then be sequenced and used to generate the full length gene.
[1483] This above method starts with total RNA isolated from the
desired source, although poly-A+ RNA can be used. The RNA
preparation can then be treated with phosphatase if necessary to
eliminate 5' phosphate groups on degraded or damaged RNA which may
interfere with the later RNA ligase step. The phosphatase should
then be inactivated and the RNA treated with tobacco acid
pyrophosphatase in order to remove the cap structure present at the
5' ends of messenger RNAs. This reaction leaves a 5' phosphate
group at the 5' end of the cap cleaved RNA which can then be
ligated to an RNA oligonucleotide using T4 RNA ligase.
[1484] This modified RNA preparation is used as a template for
first strand cDNA synthesis using a gene specific oligonucleotide.
The first strand synthesis reaction is used as a template for PCR
amplification of the desired 5' end using a primer specific to the
ligated RNA oligonucleotide and a primer specific to the known
sequence of the gene of interest. The resultant product is then
sequenced and analyzed to confirm that the 5' end sequence belongs
to the desired gene.
Example 27
Expression of an Albumin Fusion Protein in Mammalian Cells
[1485] The albumin fusion proteins of the present invention can be
expressed in a mammalian cell. A typical mammalian expression
vector contains a promoter element, which mediates the initiation
of transcription of mRNA, a protein coding sequence, and signals
required for the termination of transcription and polyadenylation
of the transcript. Additional elements include enhancers, Kozak
sequences and intervening sequences flanked by donor and acceptor
sites for RNA splicing. Highly efficient transcription is achieved
with the early and late promoters from SV40, the tong terminal
repeats (LTRs) from Retro-viruses, e.g., RSV, HTLVI, HIVI and the
early promoter of the cytomegalovirus (CMV). However, cellular
elements can also be used (e.g., the human actin promoter).
[1486] Suitable expression vectors for use in practicing the
present invention include, for example, vectors such as, pSVL and
pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr
(ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport
3.0. Mammalian host cells that could be used include, but are not
limited to, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and
C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells
and Chinese hamster ovary (CHO) cells.
[1487] Alternatively, the albumin fusion protein can be expressed
in stable cell lines containing the polynucleotide encoding the
albumin fusion protein integrated into a chromosome. The
co-transfection with a selectable marker such as DHFR, gpt,
neomycin, or hygromycin allows the identification and isolation of
the transfected cells.
[1488] The transfected polynucleotide encoding the fusion protein
can also be amplified to express large amounts of the encoded
fusion protein. The DHFR (dihydrofolate reductase) marker is useful
in developing cell lines that carry several hundred or even several
thousand copies of the gene of interest. (See, e.g., Alt et al., J.
Biol. Chem. 253:1357-1370 (1978); Hamlin et al., Biochem. et
Biophys. Acta, 1097:107-143 (1990); Page et al., Biotechnology
9:64-68 (1991)). Another useful selection marker is the enzyme
glutamine synthase (GS) (Murphy et al., Biochem J. 227:277 279
(1991); Bebbington at al., Bio/Technology 10:169-175 (1992). Using
these markers, the mammalian cells are grown in selective medium
and the cells with the highest resistance are selected. These cell
lines contain the amplified gene(s) integrated into a chromosome.
Chinese hamster ovary (CHO) and NSO cells are often used for the
production of proteins.
[1489] Derivatives of the plasmid pSV2-dhfr (ATCC Accession No.
37146), the expression vectors pC4 (ATCC Accession No. 209646) and
pC6 (ATCC Accession No. 209647) contain the strong promoter (LTR)
of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular
Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer
(Boshart at al., Cell 41:521-530 (1985)). Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites BamHI, XbaI and
Asp718, facilitate the cloning of the gene of interest. The vectors
also contain the 3' intron, the polyadenylation and termination
signal of the rat preproinsulin gene, and the mouse DHFR gene under
control of the SV40 early promoter.
[1490] Specifically, the plasmid pC6, for example, is digested with
appropriate restriction enzymes and then dephosphorylated using
calf intestinal phosphates by procedures known in the art. The
vector is then isolated from a 1% agarose gel.
[1491] A polynucleotide encoding an albumin fusion protein of the
present invention is generated using techniques known in the art
and this polynucleotide is amplified using PCR technology known in
the art. If a naturally occurring signal sequence is used to
produce the fusion protein of the present invention, the vector
does not need a second signal peptide. Alternatively, if a
naturally occurring signal sequence is not used, the vector can be
modified to include a heterologous signal sequence. (See, e.g.,
International Publication No. WO 96/34891.)
[1492] The amplified fragment encoding the fusion protein of the
invention is isolated from a 1% agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Calif.). The
fragment then is digested with appropriate restriction enzymes and
again purified on a 1% agarose gel.
[1493] The amplified fragment encoding the albumin fusion protein
of the invention is then digested with the same restriction enzyme
and purified on a 1% agarose gel. The isolated fragment and the
dephosphorylated vector are then ligated with T4 DNA ligase. E.
coli HB101 or XL-1 Blue cells are then transformed and bacteria are
identified that contain the fragment inserted into plasmid pC6
using, for instance, restriction enzyme analysis.
[1494] Chinese hamster ovary cells lacking an active DHFR gene is
used for transfection. Five .mu.g of the expression plasmid pC6 or
pC4 is cotransfected with 0.5 .mu.g of the plasmid pSVneo using
lipofectin (Feigner et al., supra). The plasmid pSV2-neo contains a
dominant selectable marker, the neo gene from Tn5 encoding an
enzyme that confers resistance to a group of antibiotics including
G418, The cells are seeded in alpha minus MEM supplemented with 1
mg/ml H418. After 2 days, the cells are trypsinized and seeded in
hybridoma cloning plates (Greiner, Germany) in alpha minus MEM
supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml
G418. After about 10-14 days single clones are trypsinized and then
seeded in 6-well petri dishes or 10 ml flasks using different
concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800
nM). Clones growing at the highest concentrations of methotrexate
are then transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 .mu.M, 2 .mu.M, 5 .mu.M, 10 mM,
20 mM). The same procedure is repeated until clones are obtained
which grow at a concentration of 100-200 .mu.M. Expression of the
desired fusion protein is analyzed, for instance, by SDS-PAGE and
Western blot or by reversed phase HPLC analysis.
Example 28
[.sup.3H]-2-Deoxyglucose Uptake Assay
[1495] Adipose, skeletal muscle, and liver are insulin-sensitive
tissues. Insulin can stimulate glucose uptake/transport into these
tissues. In the case of adipose and skeletal muscle, insulin
initiates the signal transduction that eventually leads to the
translocation of the glucose transporter 4 molecule, GLUT4, from a
specialized intracellular compartment to the cell surface. Once on
the cell surface, GLUT4 allows for glucose uptake/transport.
[.sup.3H]-2-Deoxyglucose Uptake
[1496] A number of adipose and muscle related cell-lines can be
used to test for glucose uptake/transport activity in the absence
or presence of a combination of any one or more of the therapeutic
drugs listed for the treatment of diabetes menials. In particular,
the 3T3-L1 murine fibroblast cells and the L6 murine skeletal
muscle cells can be differentiated into 3T3-L1 adipocytes and into
myotubes, respectively, no serve as appropriate in vitro models for
the [.sup.3H]-2-deoxyglucose uptake assay (Urso at al., J Biol
Chem, 274(43): 30864-73 (1999); Wang at al., J Mol Endocrinol,
19(3): 2414 (1997); Haspel et al., J Membr Biol, 169 (1): 45-53
(1999); Tsakiridis et al., Endocrinology, 136(10): 4315-22 (1995)).
Briefly, 2.times.10.sup.5 cells/100 .mu.l, of adipocytes or
differentiated L6 cells are transferred to 96-well Tissue-Culture,
"TC", treated, i.e., coated with 50 .mu.g/mL of poly-L-lysine,
plates in post-differentiation medium and are incubated overnight
at 37.degree. C. in 5% CO.sub.2. The cells are first washed once
with serum free low glucose DMEM medium and are then starved with
10 .mu.L/well of the same medium and with 100 .mu.L/well of either
buffer or of a combination of any one or more of the therapeutic
drugs listed for the treatment of diabetes mellitus, for example,
increasing concentrations of 1 nM, 10 nM, and 100 nM of the
therapeutics of the subject invention (e.g., specific fusions
disclosed as SEQ ID NO:Y and fragments and variants thereof) for 16
hours at 37.degree. C. in the absence or presence of 1 nM insulin.
The plates are washed three times with 100 .mu.L/well of HEPES
buffered saline. Insulin is added at 1 nM in HEPES buffered saline
for 30 min at 37.degree. C. in the presence of 10 .mu.M labeled
[.sup.3H]-2-deoxyglucose (Amersham, #TRK672) and 10 .mu.M unlabeled
2-deoxyglucose (SIGMA, D-3179). As control, the same conditions are
carried out except in the absence of insulin. A final concentration
of 10 .mu.M cytochalasin B (SIGMA, C6762) is added at 100
.mu.l/well in a separate well to measure the non-specific uptake.
The cells are washed three times with HEPES buffered saline.
Labeled, i.e., 10 .mu.M of [.sup.3H]-2-deoxyglucose, and unlabeled,
i.e., 10 .mu.M of 2-deoxyglucose, are added for 10 minutes at room
temperature. The cells are washed three times with cold Phosphate
Buffered Sal ine, "PBS". The cells are lysed upon the addition of
150 .mu.L/well of 0.2 N NaOH and subsequent incubation with shaking
for 20 minutes at room temperature. Samples are then transferred to
a scintillation vial to which is added 5 mL of scintillation fluid.
The vials are counted in a Beta-Scintillation counter. Uptake in
duplicate conditions, the difference being the absence or presence
of insulin, is determined with the following equation: [(Insulin
counts per minute "cpm"-Non-Specific cpm)/(No Insulin
cpm-Non-Specific cpm)]. Average responses fall within the limits of
about 5-fold and 3-fold that of controls for adipocytes and
myotubes, respectively.
[1497] Differentiation of Cells
[1498] The cells are allowed to become fully confluent in a T-75
cm.sup.2 flask. The medium is removed and replaced with 25 mL of
pre-differentiation medium for 48 hours. The cells are incubated at
37.degree. C., in 5% CO.sub.2, 85% humidity. After 48 hours, the
pre-differentiation medium is removed and replaced with 25 mL
differentiation medium for 48 hours. The cells are again incubated
at 37.degree. C., in 5% CO.sub.2, 85% humidity. After 48 hours, the
medium is removed and replaced with 30 mL post-differentiation
medium. Post-differentiation medium is maintained for 14-20 days or
until complete differentiation is achieved. The medium is changed
every 2-3 days. Human adipocytes can be purchased from Zen-Bio, INC
(# SA-1096).
Example 29
In Vitro Assay of [.sup.3H]-Thymidine Incorporation into Pancreatic
Cell-Lines
[1499] It has recently been shown that GLP-1 induces
differentiation of the rat pancreatic ductal epithelial cell-line
ARIP in a time- and dose-dependent manna which is associated with
an increase in Islet Duodenal Homeobox-1 (IDX-1) and insulin mRNA
levels (Hui et al., Diabetes, 50(4): 785-96 (2001)). The IDX-1 in
turn increases mRNA levels of the GLP-1 receptor.
[1500] Cells Types Tested
[1501] RIN-M cells: These cells are available from the American
Type Tissue Culture Collection (ATCC Cell Line Number CRL-2057).
The RIN-M cell line was derived from a radiation induced
transplantable rat islet cell tumor. The line was established from
a nude mouse xenograft of the tumor. The cells produce and secrete
islet polypeptide hormones, and produce L-dopa decarboxylase (a
marker for cells having amine precursor uptake and decarboxylation,
or APUD, activity).
[1502] ARIP cells: These are pancreatic exocrine cells of
epithelial morphology available from the American Type Tissue
Culture Collection (ATCC Cell Line Number CRL-1674). See also,
references: Jessop, N. W. and Hay, R. I., "Characteristics of two
rat pancreatic exocrine cell lines derived from transplantable
tumors," In Vitro 16: 212, (1980): Cookell, M. et al.,
"Identification of a cell-specific DNA-binding activity that
interacts with a transcriptional activator of genes expressed in
the acinar pancreas," Mol. Cell. Biol. 9: 2464-2476, (1989): Roux,
E., et al. "The cell-specific transcription factor PTFI contains
two different subunits that interact with the DNA" Genes Dev. 3:
1613-1624, (1989); and, Hui, H., et al., "Glucagon-like peptide 1
induces differentiation of islet duodenal homeobox-1-positive
pancreatic ductal cells into insulin-secreting cells," Diabetes 50:
785-796 (2001).
[1503] Preparation of Cells
[1504] The RIN-M cell-line is grown in RPMI 1640 medium (Hyclone,
#SH300027.01) with 10% fetal bovine serum (HyClone, #SH30088.03)
and is subcultured every 6 to 8 days at a ratio of 1:3 to 1:6. The
medium is changed every 3 to 4 days.
[1505] The ARIP (ATCC #CRL-1674) cell-line is grown in Ham's F12K
medium (ATCC, #30-2004) with 2 mM L-glutamine adjusted to contain
1.5 g/L sodium bicarbonate and 10% fetal bovine serum. The ARIP
cell-line is subcultured at a ratio of 1:3 to 1:6 twice per week.
The medium is changed every 3 to 4 days.
Assay Protocol
[1506] The cells are seeded at 4000 cells/well in 96-well plates
and cultured for 48 to 72 hours to 50% confluence. The cells are
switched to serum-free media at 100 .mu.L/well. After incubation
for 48-72 hours, serum and/or the therapeutics of the subject
invention (e.g., specific fusions disclosed as SEQ ID NO:Y and
fragments and variants thereof) are added to the well. Incubation
persists for an additional 36 hours. [.sup.3H]-Thymidine (5-20
Ci/mmol) (Amersham Pharmacia, #TRK120) is diluted to 1
microCuries/5 microliters. After the 36 hour incubation, 5
microliters is added per well for a further 24 hours. The reaction
is terminated by washing the cells gently with cold
Phosphate-Buffered Sal ine: "PBS", once. The cells are then fixed
with 100 microliters of 10% ice cold TCA for 15 min at 4.degree. C.
The PBS is removed and 200 microliters of 0.2 N NaOH is added. The
plates are incubated for 1 hour at room temperature with shaking.
The solution is transferred to a scintillation vial and 5 mL of
scintillation fluid compatible with aqueous solutions is added and
mixed vigorously. The vials are counted in a beta scintillation
counter. As negative control, only buffer is used. As a positive
control fetal calf serum is used.
Example 30
Assaying for Glycosuria
[1507] Glycosuria (i.e., excess sugar in the urine), can be readily
assayed to provide an index of the disease state of diabetes
mellitus. Excess urine in a patient sample as compared with a
normal patient sample is symptomatic of IDDM and NIDDM. Efficacy of
treatment of such a patient having IDDM and NIDDM is indicated by a
resulting decrease in the amount of excess glucose in the urine. In
a preferred embodiment for IDDM and NIDDM monitoring, urine samples
from patients are assayed for the presence of glucose using
techniques known in the art, Glycosuria in humans is defined by a
urinary glucose concentration exceeding 100 mg per 100 ml. Excess
sugar levels in those patients exhibiting glycosuria can be
measured even more precisely by obtaining blood samples and
assaying serum glucose.
Example 31
Occurrence of Diabetes in Nod Mice
[1508] Female NOD (non-obese diabetic) mice are characterized by
displaying IDDM with a course which is similar to that found in
humans, although the disease is mom pronounced in female than male
NOD mice. Hereinafter, unless otherwise stated, the term "NOD
mouse" refers to a female NOD mouse. NOD mice have a progressive
destruction of beta cells which is caused by a chronic autoimmune
disease. Thus, NOD mice begin life with euglycemia, or normal blood
glucose levels. By about 15 to 16 weeks of age, however, NOD mice
start becoming hyperglycemic, indicating the destruction of the
majority of their pancreatic beta cells and the corresponding
inability of the pancreas to produce sufficient insulin. Thus, both
the cause and the progression of the disease are similar to human
IDDM patients.
[1509] In vivo assays of efficacy of the immunization regimens can
be assessed in female NOD/LtJ mice (commercially available from The
Jackson Laboratory, Bar Harbor, Me.). In the literature, it's
reported that 80% of female mice develop diabetes by 24 weeks of
age and onset of insulitis begins between 6-8 weeks age. NOD mice
are inbred and highly responsive to a variety of immunoregulatory
strategies. Adult NOD mice (6-8 weeks of age) have an average mass
of 20-25 g.
[1510] These mice can be either untreated (control), treated with
the therapeutics of the subject invention (e.g., specific fusions
disclosed as SEQ ID NO:Y and fragments and variants thereof), alone
or in combination with other therapeutic compounds stated above.
The effect of these various treatments on the progression of
diabetes can be measured as follows:
[1511] At 14 weeks of age, the female NOD mice can be phenotyped
according to glucose tolerance. Glucose tolerance can be measured
with the intraperitoneal glucose tolerance test (IPGTT). Briefly,
blood is drawn from the paraorbital plexus at 0 minutes and 60
minutes after the intraperitoneal injection of glucose (1 g/kg body
weight). Normal tolerance is defined as plasma glucose at 0 minutes
of less than 144 mg %, or at 60 minutes of less than 160 mg %.
Blood glucose levels are determined with a Glucometer Elite
apparatus.
[1512] Based upon this phenotypic analysis, animals can be
allocated to the different experimental groups. In particular,
animals with more elevated blood glucose levels can be assigned to
the impaired glucose tolerance group. The mice can be fed ad
libitum and can be supplied with acidified water (pH 2.3).
[1513] The glucose tolerant and intolerant mice can be further
subdivided into control, albumin fusion proteins of the subject
invention, and albumin fusion proteins/therapeutic compounds
combination groups. Mice in the control group can receive an
interperitoneal injection of vehicle daily, six times per week.
Mice in the albumin fusion group can receive an interperitoneal
injection of the therapeutics of the subject invention (e.g.,
specific fusions disclosed as SEQ ID NO:Y and fragments and
variants thereof) in vehicle daily, six times per week. Mice in the
albumin fusion proteins/therapeutic compounds combination group can
receive both albumin fusion proteins and combinations of
therapeutic compounds as described above.
[1514] The level of urine glucose in the NOD mice can be determined
on a 61-weekly basis using Labstix (Bayer Diagnostics, Hampshire,
England). Weight and fluid intake can also be determined on a
hi-weekly basis. The onset of diabetes is defined after the
appearance of glucosuria on two consecutive determinations. After
10 weeks of treatment, an additional IPGTT can be performed and
animals can be sacrificed the following day.
[1515] Over the 10 week course of treatment, control animals in
both the glucose tolerant and glucose intolerant groups develop
diabetes at a rate of 60% and 86%, respectively (see U.S. Pat. No.
5,866,546, Gross et al.). Thus, high rates of diabetes occur even
in NOD mice which are initially glucose tolerant if no intervention
is made.
[1516] Results can be confirmed by the measurement of blood glucose
levels in NOD mice, before and aftertreatment. Blood glucose levels
are measured as described above in both glucose tolerant and
intolerant mice in all groups described.
[1517] In an alternative embodiment, the therapeutics of the
subject invention (e.g., specific fusions disclosed as SEQ ID NO:Y
and fragments and variants thereof) can be quantified using
spectrometric analysis and appropriate protein quantities can be
resuspended prior to injection in 50 .mm.l phosphate buffered
saline (PBS) per dose. Two injections, one week apart, can be
administered subcutaneously under the dorsal skin of each mouse.
Monitoring can be performed on two separate occasions prior to
immunization and can be performed weekly throughout the treatment
and continued thereafter. Urine can be tested for glucose every
week (Keto-Diastix.RTM.; Miles Inc., Kankakee, Ill.) and glycosuric
mice can be checked for serum glucose (Exaclech.RTM., tylediSense,
Inc., Waltham, Mass.). Diabetes is diagnosed when fasting nlycernia
is greater than 2.5 g/L.
Example 32
Histological Examination of Nod Mice
[1518] Histological examination of tissue samples from NOD mice can
demonstrate the ability of the compositions of the present
invention, and/or a combination of the compositions of the present
invention with other therapeutic agents for diabetes, to increase
the relative concentration of beta cells in the pancreas. The
experimental method is as follows:
[1519] The mice from Example 31 can be sacrificed at the end of the
treatment period and tissue samples can be taken from the pancreas.
The samples can be fixed in 10% formalin in 0.9% saline and
embedded in wax. Two sets of 5 serial 5 .mu.m sections can be cut
for immunolabeling at a cutting interval of 150 .mu.m. Sections can
be immunolabelled for insulin (guinea pig anti-insulin antisera
dilution 1:1000, ICN Thames U.K.) and glucagon (rabbit
anti-pancreatic glucagon antisera dilution 1:2000) and detected
with peroxidase conjugated anti-guinea pig (Dako, High Wycombe,
U.K.) or peroxidase conjugated anti-rabbit antisera (dilution 1:50,
Dako).
[1520] The composition of the present invention may or may not have
as strong an effect on the visible mass of beta cells as it does on
the clinical manifestations of diabetes in glucose tolerant and
glucose intolerant animals.
Example 33
Pancreatic Beta-Cell Transplantation Combination Therapy
[1521] Transplantation is a common form of treatment of autoimmune
disease, especially when the target self tissue has been severely
damaged. For example, and not by way of limitation, pancreas
transplantation and islet cell transplantation are common treatment
options for IDDM (See, e.g., Stewart et al., Journal of Clinical
Endocrinology & Metabolism 86 (3): 984-988 (2001); Brunicardi,
Transplant. Proc. 28: 2138-40 (1996); Kendall & Robertson,
Diabetes Metab. 22: 157-163 (1996); Hamann et al Kobe J. Med. Sci.
42: 93-104 (1996); Larsen & Stratta, Diabetes Metab. 22:
139-146 (1996); and Kinkhabwala, et al, Am. J. Surg. 171: 516-520
(1996)). As with any transplantation method, transplantation
therapies for autoimmune disease patients include treatments to
minimize the risk of host rejection of the transplanted tissue.
However, autoimmune disease involves the additional, independent
risk that the pre-existing host autoimmune response which damaged
the original self tissue will exert the same damaging effect on the
transplanted tissue. Accordingly, the present invention encompasses
methods and compositions for the treatment of autoimmune pancreatic
disease using the albumin fusion proteins of the subject invention
in combination with immunomodulators/immunosuppressants in
individuals undergoing transplantation therapy of the autoimmune
disease.
[1522] In accordance with the invention, the albumin fusion-based
compositions and formulations described above, are administered to
prevent and treat damage to the transplanted organ, tissue, or
cells resulting from the host individual's autoimmune response
initially directed against the original self tissue. Administration
may be carried out both prior and subsequent to transplantation in
2 to 4 doses each one week apart.
[1523] The following immunomodulators/immunosuppressants including,
but not limited to, AI-401, CDP-571 (anti-TNF monoclonal antibody),
CG-1088, Diamyd (diabetes vaccine), ICM3 (anti-ICAM-3 monoclonal
antibody), linomide (Roquinimex), NBI-6024 (altered peptide
ligand). TM-27, VX-740 (HMR-3480), caspase 8 protease inhibitors,
thalidomide, hOKT3gamma1 (Ala-ala) (anti-CD3 monoclonal antibody).
Oral Interferon-Alpha, oral lactobacillus, and LymphoStat-B.TM. can
be used together with the albumin fusion therapeutics of the
subject invention in islet cell or pancreas transplantation.
Example 34
In Vivo Mouse Model Of NIDDM
[1524] Male C57BL/6J mice from Jackson Laboratory (Bar Harbor, Me.)
can be obtained at 3 weeks of age and fed on conventional chow or
diets enriched in either fat (35.5% wt/wt; Bioserv.Frenchtown,
N.J.) or fructose (60% wt/wt; Harlan Teklad, Madison, Wis.). The
regular chow is composed of 4.5% wt/wt fat, 23% wt/wt protein,
31.9% wt/wt starch, 3.7% wt/wt fructose, and 5.3% wt/wt fiber. The
high-fat (lard) diet is composed of 35.5% wt/wt fat, 20% wt/wt
protein, 36.4% wt/wt starch, 0.0% wt/wt fructose, and 0.1% wt/wt
fiber. The high-fructose diet is composed of 5% wt/wt fat, 20%
wt/wt protein, 0.0% wt/wt starch, 60% wt/wt fructose, and 9.4%
wt/wt fiber. The mice may be housed no more than five per cage at
22.degree.+/-3.degree. C. temperature- and 50%+/-20%
humidity-controlled room with a 12-hour light (6 am to 6 pm)/dark
cycle (Luo et al., Metabolism 47(6): 663-8 (1998), "Nongenetic
mouse models of non-insulin-dependent diabetes mellitus"; Larsen et
al., Diabetes 50(11): 2530-9 (2001), "Systemic administration of
the long-acting GLP-1 derivative NN2211 induces lasting and
reversible weight loss in both normal and obese rats"). After
exposure to the respective diets for 3 weeks, mice can be injected
intraperitoneally with either streptozotocin, "STZ" (Sigma, St.
Louis, Mo.), at 100 mg/kg body weight or vehicle (0.05 mol/L citric
acid, pH 4.5) and kept on the same diet for the next 4 weeks. Under
nonfasting conditions, blood is obtained 1, 2, and 4 weeks post-STZ
by nipping the distal part of the tail. Samples are used to measure
nonfasting plasma glucose and insulin concentrations. Body weight
and food intake are recorded weekly.
[1525] To directly determine the effect of the high-fat diet on the
ability of insulin to stimulate glucose disposal, the experiments
can be initiated on three groups of mice, fat-fed, chow-fed
injected with vehicle, and fat-fed injected with STZ at the end of
the 7-week period described above. Mice can be fasted for 4 hours
before the experiments. In the first series of experiments, mice
can be anesthetized with methoxyflurane (Pitman-Moor, Mundelein,
Ill.) inhalation. Regular insulin (Sigma) can be injected
intravenously ([IV] 0.1 U/kg body weight) through a tail vein, and
blood can be collected 3, 6, 9, 12, and 15 minutes after the
injection from a different tail vein, Plasma glucose concentrations
can be determined on these samples, and the half-life (t1/2) of
glucose disappearance front plasma can be calculated using
WinNonlin (Scientific Consulting, Apex, N.C.), a
pharmacokinetics/pharmacodynamics software program.
[1526] In the second series of experiments, mice can be
anesthetized with intraperitoneal sodium pentobarbital (Sigma). The
abdominal cavity is opened, and the main abdominal vein is exposed
and catheterized with a 24-gauge IV catheter (Johnson-Johnson
Medical, Arlington, Tex.). The catheter is secured to muscle tissue
adjacent to the abdominal vein, cut on the bottom of the syringe
connection, and hooked to a prefilled PESO plastic tube, which in
turn is connected to a syringe with infusion solution. The
abdominal cavity is then sutured closed. With this approach, there
would be no blockage of backflow of the blood from the lower part
of the body. Mice can be infused continuously with glucose (24.1
mg/kg/min) and insulin (10 mU/kg/min) at an infusion volume of 10
.mu.L/min. Retro-orbital blood samples (70 .mu.L each) can be taken
90, 105, 120, and 135 minutes after the start of infusion for
measurement of plasma glucose and insulin concentrations. The mean
of these four samples is used to estimate steady-state plasma
glucose (SSPG) and insulin (SSPI) concentrations for each
animal.
[1527] Finally, experiments to evaluate the ability of the albumin
fusion proteins, the therapeutic compositions of the instant
application, either alone or in combination with any one or more of
the therapeutic drugs listed for the treatment of diabetes
mellitus, to decrease plasma glucose can be performed in the
following two groups of "NIDDM" mice models that are STZ-injected:
(1) fat-fed C57BL/J6J, and (2) fructose-fed C57BL/6J. Plasma
glucose concentrations of the mice for these studies may range from
255 to 555 mg/dL. Mice are randomly assigned to treatment with
either vehicle, albumin fusion therapeutics of the present
invention either alone or in combination with any one or more of
the therapeutic drugs listed for the treatment of diabetes
mellitus, A total of three doses can be administered. Tail vein
blood samples can be taken for measurement of the plasma glucose
concentration before the first dose and 3 hours after the final
dose.
[1528] Plasma glucose concentrations can be determined using the
Glucose Diagnostic Kit from Sigma (Sigma No. 315), an enzyme
colorimetric assay. Plasma insulin levels can be determined using
the Rat Insulin RIP, Kit from Linen Research (#RI-13K; St. Charles,
Mo.).
Example 35
In Vitro H4ILE-SEAP Reporter Assays Establishing Involvement in
Insulin Action
[1529] The Various H4Ile Reporters
[1530] H4Ile/rMEP-SEAP: The maim enzyme promoter isolated from rat
(rMEP) contains a PPAR-gamma element which is in the insulin
pathway. This reporter construct is stably transfected into the
liver H4Ile cell-line.
[1531] H4Ile/SREBP-SEAP: The sterol regulatory element binding
protein (SREBP-1c) is a transcription factor which acts on the
promoters of a number of insulin-responsive genes, for example,
fatty acid synthetase (FAS), and which regulates expression of key
genes in fatty acid metabolism in fibroblasts, adipocytes, and
hepatocytes. SREBP-1c, also known as the adipocyte determination
and differentiation factor 1 (ADD-1), is considered as the primary
mediator of insulin effects on gene expression in adipose cells.
It's activity is modulated by the levels of insulin, sterols, and
glucose. This reporter construct is stably transfected into the
liver H4Ile cell-line.
[1532] H4Ile/FAS-SEAP: The fatty acid synthetase reporter
constructs contain a minimal SREBP-responsive FAS promoter. This
reporter construct is stably transfected into the liver H4Ile
cell-line.
[1533] H4Ile/PEPCK-SEAP: The phosphoenolpyruvate carboxykinase
(PEPCK) promoter is the primary site of hormonal regulation of
PEPCK gene transcription modulating PEPCK activity. PEPCK catalyzes
a committed and rate-limiting step in hepatic gluconeogenesis and
must therefore be carefully controlled to maintain blood glucose
levels within normal limits. This reporter construct is stably
transfected into the liver H4Ile cell-line.
[1534] These reporter constructs can also be stably transfected
into 3T3-L1 fibroblasts and L6 myoblasts. These stable cell-lines
are then differentiated into 3T3 L1 adipocytes and 1.6 myotubes as
previously described in Example 28. The differentiated cell lines
can then be used in the SEAP assay described below.
Growth oral Assay Medium
[1535] The growth medium comprises 10% Fetal Bovine Serum (FBS),
10% Calf Serum, 1% NEAA, 1.times. penicillin/streptomycin, and 0.75
mg/mL G418 (for H4Ile/rFAS-SEAP and H4Ile/SREBP-SEAP) or 0.50 mg/mL
G418 (for H4Ile/rMEP-SEAP). For H4Ile/PEPCK-SEAP, the growth medium
consists of 10% FOS, 1% penicillin/streptomycin, 15 mM HEPES
buffered saline, and 0.50 mg/mL G418.
[1536] The assay medium consists of tow glucose DMEM medium (Life
Technologies), 1% NEAA, 1.times. penicillin/streptomycin for the
H4Ile/rFAS-SEAP, H4Ile/SREBP-SEAP, H4Ile/rMEP-SEAP reporters. The
assay medium for H4Ile/PEPCK-SEAP reporter consists of 0.1% FBS, 1%
penicillin/streptomycin, and 15 mM HEPES buffered saline.
[1537] Method
[1538] The 96-well plates are seeded at 75,000 cells/well in 100
.mu.L/well of growth medium until cells in log growth phase become
adherent. Cells are starved for 48 hours by replacing growth medium
with assay medium, 200 .mu.L/well. (For H4Ile/PEPCK-SEAP cells,
assay medium containing 0.5 .mu.M dexamethasone is added at 100
.mu.L/well and incubated for approximately 20 hours). The assay
medium is replaced thereafter with 1110 .mu.L/well of fresh assay
medium, and a 50 .mu.L aliquot of cell supernatant obtained from
transfected expressing the therapeutics of the subject invention
(e.g., specific fusions disclosed as SEQ ID NO:Y and fragments and
variants thereof) is added to the well. Supernatants from empty
vector transfected cell-lines are used as negative control.
Addition of 10 nM and/or 100 nM insulin to the wells is used as
positive control. After 48 hours of incubation, the conditioned
media are harvested and SEAP activity measured (Phospha-Light
System protocol, Trains 31BP2500). Briefly, samples are diluted 1:4
in dilution buffer and incubated at 65.degree. C. for 30 minutes to
inactivate the endogenous non-placental form of SEAP. An aliquot of
50 .mu.L of the diluted samples is mixed with 50 .mu.L of SEAP
Assay Buffer which contains a mixture of inhibitors active against
the non-placental SEAP isoenzymes and is incubated for another 5
minutes. An aliquot of 50 .mu.L of CSPD chemiluminescent substrate
which is diluted 1:20 in Emerald luminescence enhancer is added to
the mixture and incubated for 15-20 minutes. Plates are read in a
Dynex plate luminometer.
Example 36
Preparation of HA-Hormone Fusion Protein (Such as Insulin,
GLP-1)
[1539] The cDNA for the hormone of interest such as insulin can be
isolated by a variety of means including but not exclusively, from
cDNA libraries, by RT PCR and by FOR using a series of overlapping
synthetic oligonucleotide primers, all using standard methods. The
nucleotide sequences for all of these proteins are known and
available, for instance, in public databases such as GenBank. The
cDNA can be tailored at the 5' and 3' ends to generate restriction
sites, such that oligonucleotide linkers can be used, for cloning
of the cDNA into a vector containing the cDNA for HA. This can be
at the Nor C-terminus with or without the use of a spacer sequence.
The hormone cDNA is cloned into a vector such as pPPC0005 (FIG. 3),
pScCHSA, pScNHSA, or pC4:HSA from which the complete expression
cassette is then excised and inserted into the plasmid pSAC35 to
allow the expression of the albumin fusion protein in yeast. The
albumin fusion protein secreted from the yeast can then be
collected and purified from the media and tested for its biological
activity. For expression in mammalian cell lines a similar
procedure is adopted except that the expression cassette used
employs a mammalian promoter, leader sequence and terminator (See
Example 1). This expression cassette is then excised and inserted
into a plasmid suitable for the transfection of mammalian cell
lines.
Example 37
Preparation of HA-Soluble Receptor or HA-Binding Protein Fusion
Protein Such as HA-TNF Receptor
[1540] The cDNA for the soluble receptor or binding protein of
interest such as TNF receptor can be isolated by a variety of means
including but not exclusively, from cDNA libraries, by RT-PCR and
by PCR using a series of overlapping synthetic oligonucleotide
primers, all using standard methods. The nucleotide sequences for
all of these proteins are known and available, for instance, in
GenBank. The cDNA can be tailored at the 5' and 3' ends to generate
restriction sites, such that oligonucleotide linkers can be used,
for cloning of the cDNA into a vector containing the cDNA for HA.
This can be at the N or C-terminus with or without the use of a
spacer sequence. The receptor cDNA is cloned into a vector such as
pPPC0005 (FIG. 3), pScCHSA, pScNHSA, or pC4:HSA from which the
complete expression cassette is then excised and inserted into the
plasmid pSAC35 to allow the expression of the albumin fusion
protein in yeast. The albumin fusion protein secreted from the
yeast can then be collected and purified from the media and tested
for its biological activity. For expression in mammalian cell lines
a similar procedure is adopted except that the expression cassette
used employs a mammalian promoter, leader sequence and terminator
(See Example D. This expression cassette is then excised and
inserted into a plasmid suitable for the transfection of mammalian
cell lines.
Example 38
Preparation of HA-Growth Factors Such as HA-IGF-1 Fusion
Protein
[1541] The cDNA for the growth factor of interest such as IGF-1 can
be isolated by a variety of means including but not exclusively,
from cDNA libraries, by RT-PCR and by PCR using a series of
overlapping synthetic oligonucleotide primers, all using standard
methods (see GenBank Acc. No. NP.sub.--000609). The cDNA can be
tailored at the 5' and 3' ends to generate restriction sites, such
that oligonucleotide linkers can be used, for cloning of the cDNA
into a vector containing the cDNA for HA. This can be at the N or
C-terminus with or without the use of a spacer sequence. The growth
factor cDNA is cloned into a vector such as pPPC0005 (FIG. 3),
pScCHSA, pScNHSA, or pC4:HSA from which the complete expression
cassette is then excised and inserted into the plasmid pSAC35 to
allow the expression of the albumin fusion protein in yeast. The
albumin fusion protein secreted from the yeast can then be
collected and purified from the media and tested for its biological
activity. For expression in mammalian cell lines a similar
procedure is adopted except that the expression cassette used
employs a mammalian promoter, leader sequence and terminator (See
Example 1). This expression cassette is then excised and inserted
into a plasmid suitable for the transfection of mammalian cell
lines.
Example 39
Preparation Of HA-Single Chain Antibody Fusion Proteins
[1542] Single chain antibodies are produced by several methods
including but not limited to: selection from phage libraries,
cloning of the variable region of a specific antibody by cloning
the cDNA of the antibody and using the flanking constant regions as
the primer to clone the variable region, or by synthesizing an
oligonucleotide corresponding to the variable region of any
specific antibody. The cDNA can be tailored at the 5' and 3' ends
to generate restriction sites, such that oligonucleotide linkers
can be used, for cloning of the cDNA into a vector containing the
cDNA for HA. This can be at the N or C-terminus with or without the
use of a spacer sequence. The cell cDNA is cloned into a vector
such as pPEC0005 (FIG. 3), pScCHSA, pScNHSA, or pC4:HSA from which
the complete expression cassette is then excised and inserted into
the plasmid pSAC35 to allow the expression of the albumin fusion
protein in yeast.
[1543] In fusion molecules of the invention, the V.sub.H and
V.sub.L can be linked by one of the following means or a
combination thereof; a peptide linker between the C-terminus of the
V.sub.H and the N-terminus of the V.sub.L; a Kex2p protease
cleavage site between the V.sub.H and V.sub.L such that the two are
cleaved apart upon secretion and then self associate; and cysteine
residues positioned such that the V.sub.H and V.sub.L can form a
disulphide bond between them to link them together. An alternative
option would be to place the V.sub.H at the N-terminus of HA or an
HA domain fragment and the V.sub.L at the C-terminus of the HA or
HA domain fragment.
[1544] The albumin fusion protein secreted from the yeast can then
be collected and purified from the media and tested for its
activity. For expression in mammalian cell lines a similar
procedure is adopted except that the expression cassette used
employs a mammalian promoter, leader sequence and terminator (See
Example 1). This expression cassette is then excised and inserted
into a plasmid suitable for the transfection of mammalian cell
lines. The antibody produced in this manner can be purified from
media and tested for its binding to its antigen using standard
immunochemical methods.
Example 40
Preparation of HA-Cell Adhesion Molecule Fusion Proteins
[1545] The cDNA for the cell adhesion molecule of interest can be
isolated by a variety of means including but not exclusively, from
cDNA libraries, by RT-PCR and by PCR using a series of overlapping
synthetic oligonucleotide primers, all using standard methods. The
nucleotide sequences for the known cell adhesion molecules are
known and available, for instance, in GenBank. The cDNA can be
tailored at the 5' and 3' ends to generate restriction sites, such
that oligonucleotide linkers can be used, for cloning of the cDNA
into a vector containing the cDNA for HA. This can be at the Nor
C-terminus with or without the use of a spacer sequence. The cell
adhesion molecule cDNA is cloned into a vector such as pPPC0005
(FIG. 3), pScCHSA, pScNHSA, or pC4:HSA from which the complete
expression cassette is then excised and inserted into the plasmid
pSAC35 to allow the expression of the albumin fusion protein in
yeast. The albumin fusion protein secreted from the yeast can then
be collected and purified from the media and tested for its
biological activity. For expression in mammalian cell lines a
similar procedure is adopted except that the expression cassette
used employs a mammalian promoter, leader sequence and terminator
(See Example 1). This expression cassette is then excised and
inserted into a plasmid suitable for the transfection of mammalian
cell hues.
Example 41
Preparation of Inhibitory Factors and Peptides as HA Fusion
Proteins (Such as HA-Antiviral, HA-Antibiotic. HA-Enzyme Inhibitor
And HA-Anti-Allergic Proteins)
[1546] The cDNA for the peptide of interest such as an antibiotic
peptide can be isolated by a variety of means including but not
exclusively, from cDNA libraries, by RT-PCR and by PCR using a
series of overlapping synthetic oligonucleotide primers, all using
standard methods. The cDNA can be tailored at the 5' and 3' ends to
generate restriction sites, such that oligonucleotide linkers can
be used, for cloning of the cDNA into a vector containing the cDNA
for HA. This can be at the N or C-terminus with or without the use
of a spacer sequence. The peptide cDNA is cloned into a vector such
as pPPC0005 (FIG. 3), pScCHSA, pScNHSA, or pC4:HSA from which the
complete expression cassette is then excised and inserted into the
plasmid pSAC35 to allow the expression of the albumin fusion
protein in yeast. The albumin fusion protein secreted from the
yeast can then be collected and purified from the media and tested
for its biological activity. For expression in mammalian cell lines
a similar procedure is adopted except that the expression cassette
used employs a mammalian promoter, leader sequence and terminator
(See Example 1). This expression cassette is then excised and
inserted into a plasmid suitable for the transfection of mammalian
cell lines.
Example 42
Preparation of Targeted HA Fusion Proteins
[1547] The cDNA for the protein of interest can be isolated from
cDNA library or can be made synthetically using several overlapping
oligonucleotides using standard molecular biology methods. The
appropriate nucleotides can be engineered in the cDNA to form
convenient restriction sites and also allow the attachment of the
protein cDNA to albumin cDNA similar to the method described for
hGH. Also a targeting protein or peptide cDNA such as single chain
antibody or peptides, such as nuclear localization signals, that
can direct proteins inside the cells can be fused to the other end
of albumin. The protein of interest and the targeting peptide is
cloned into a vector such as pPPC0005 (FIG. 3), pScCHSA, pScNHSA
pC4:HSA which allows the fusion with albumin cDNA. In this manner
both N- and C-terminal end of albumin are fused to other proteins.
The fused cDNA is then excised from pPPC0005 and is inserted into a
plasmid such as pSAC35 to allow the expression of the albumin
fusion protein in yeast. All the above procedures can be performed
using standard methods in molecular biology. The albumin fusion
protein secreted from yeast can be collected and purified from the
media and tested for its biological activity and its targeting
activity using appropriate biochemical and biological tests.
Example 43
Preparation Of HA-Enzymes Fusions
[1548] The cDNA for the enzyme of interest can be isolated by a
variety of means including but not exclusively, from cDNA
libraries, by RT-PCR and by PCR using a series of overlapping
synthetic oligonucleotide primers, all using standard methods. The
cDNA can be tailored at the 5' and 3' ends to generate restriction
sites, such that oligonucleotide linkers can be used, for cloning
of the cDNA into a vector containing the cDNA for HA. This can be
at the Nor C-terminus with or without the use of a spacer sequence.
The enzyme cDNA is cloned into a vector such as pPPC0005 (FIG. 3),
pScCHSA, pScNHSA, or pC4:HSA from which the complete expression
cassette is then excised and inserted into the plasmid pSAC35 to
allow the expression of the albumin fusion protein in yeast. The
albumin fusion protein secreted from the yeast can then be
collected and purified from the media and tested for its biological
activity. For expression in mammalian cell lines a similar
procedure is adopted except that the expression cassette used
employs a mammalian promoter, leader sequence and terminator (See
Example 1). This expression cassette is then excised and inserted
into a plasmid suitable for the transfection of mammalian cell
lines.
Example 44
Bacterial Expression of an Albumin Fusion Protein
[1549] A polynucleotide encoding an albumin fusion protein of the
present invention comprising a bacterial signal sequence is
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' ends of the DNA sequence, to synthesize insertion fragments.
The primers used to amplify the polynucleotide encoding insert
should preferably contain restriction sites, such as BamHI and
XbaI, at the 5' end of the primers in order to clone the amplified
product into the expression vector. For example, BamHI and XbaI
correspond to the restriction enzyme sites on the bacterial
expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This
plasmid vector encodes antibiotic resistance (Amp.sup.r), a
bacterial origin of replication (on), an IPTG-regulatable
promoter/operator (P/O), a ribosome binding site (RBS), a
6-histidine tag (6-His), and restriction enzyme cloning sites.
[1550] The pQE-9 vector is digested with BamHI and XbaI and the
amplified fragment is ligated into the pQE-9 vector maintaining the
reading frame initiated at the bacterial RBS. The ligation mixture
is then used to transform the E. coli strain M15/rep4 (Qiagen,
Inc.) which contains multiple copies of the plasmid pREP4, which
expresses the lad repressor and also confers kanamycin resistance
(Kan.sup.r). Transformants are identified by their ability to grow
on LB plates and ampicillin/kanamycin resistant colonies are
selected. Plasmid DNA is isolated and confirmed by restriction
analysis.
[1551] Clones containing the desired constructs are grown overnight
(O/N) in liquid culture in LB media supplemented with both Amp (100
ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a
large culture at a ratio of 1:100 to 1:250. The cells are grown to
an optical density 600 (O.D..sup.600) of between 0.4 and 0.6. IPTG
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final
concentration of 1 mM. IPTG induces by inactivating the lad
repressor, clearing the P/0 leading to increased gene
expression.
[1552] Cells are grown for an extra 3 to 4 hours. Cells are then
harvested by centrifugation (20 mins at 6000.times.8). The cell
pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl
or preferably in 8 M urea and concentrations greater than 0.14 M
2-mercaptoethanol by stirring for 3-4 hours at 4.degree. C. (sec,
e.g., Burton et al., Eur. J. Biochem. 179:379-387 (1989)). The cell
debris is removed by centrifugation, and the supernatant containing
the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid
("Ni-NTA") affinity resin column (available from QIAGEN, Inc.,
supra). Proteins with a 6.times.His tag bind to the Ni-NTA resin
with high affinity and can be purified in a simple one-step
procedure (for details see: The QIAexpressionist (1995) QIAGEN,
Inc, supra).
[1553] Briefly, the supernatant is loaded onto the column in 6 M
guanidine-HCl, pH E. The column is first washed with 10 volumes of
6 M guanidine HCl, pH 8, then washed with 10 volumes of 6 M
guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M
guanidine.HCl, pH 5.
[1554] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column.
Exemplary conditions are as follows: renature using a linear 6M-1M
urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4,
containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins are eluted by the addition of 250 mM imidazole.
Imidazole is removed by a final dialyzing step against PBS or 50 mM
sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein
is stored at 4.degree. C. or frozen at -80.degree. C.
[1555] In addition to the above expression vector, the present
invention further includes an expression vector, called pHE4a (ATCC
Accession Number 209645, deposited on Feb. 25, 1998) which contains
phage operator and promoter elements operatively linked to a
polynucleotide encoding an albumin fusion protein of the present
invention, called pHE4a. (ATCC Accession Number 209645, deposited
on Feb. 25, 1998.) This vector contains: 1) a
neomycinphosphotransferase gene as a selection marker, 2) an E.
coli origin of replication, 3) a T5 phage promoter sequence, 4) two
lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the
lactose operon repressor gene (lacIq). The origin of replication
(oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter
and operator sequences are made synthetically.
[1556] DNA can be inserted into the pHE4a by restricting the vector
with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted
product on a gel, and isolating the larger fragment (the sniffer
fragment should be about 310 base pairs). The DNA insert is
generated according to PCR protocols described herein or otherwise
known in the art, using PCR primers having restriction sites for
NdeI (5' primer) and XhaI, BamHI, XhoI, or Asp718 (3' primer). The
PCR insert is gel purified and restricted with compatible enzymes.
The insert and vector are ligated according to standard
protocols.
[1557] The engineered vector may be substituted in the above
protocol to express protein in a bacterial system.
Example 45
Multifusion Fusions
[1558] Albumin fusion proteins of the invention containing multiple
Therapeutic protein portions fused at the N- and C-termini of
albumin may include, but are not limited to, GLP1-HSA-GLP-1,
GLP1-HSA-Exendin 4, Exendin 4-HSA-Exendin 4, Exendin 4-HSA-GLP1,
GLP1-HSA-Insulin, Exendin 4-HSA-Insulin, Insulin-HSA-Insulin,
Insulin-HSA-GLP1, Insulin-HSA-Exendin 4, GLP1-HSA-Resistin, Exendin
4-HSA-Resistin, Insulin-HSA-Resistin, Resistin-HSA-Resistin,
Resistin-HSA-GLP1, Resistin-HSA-Exendin 4, Resistin-HSA-Insulin,
GLP1-HSA-Leptin, Exendin 4-HSA-Leptin, Insulin-HSA-Leptin.
Resistin-HSA-Leptin, Leptin-HSA-Leptin, Leptin-HSA-GLP1,
Leptin-HSA-Exendin 4, Leptin-HSA-Insulin, Leptin-HSA-Resistin,
GLP1-HSA-GLP1, IGF1-HSA-IGF1, Exendin 4-HSA-IGF1, insulin-HSA-IGF1,
Resistin-HSA-IGF1, Leptin-HSA-IGF1, IGF1-HSA-GLP1, IGF1-HSA-Exendin
4, IGF1-HSA-Insulin, IGF1-HSA-Resistin, IGF1-HSA-Leptin,
HCEIP80-HSA-HCEIP80, IGF1-HSA-HCEIP80, GLP1-HSA-HCEIP80, Exendin
4-HSA-HCEIP80, Insulin-HSA-HCEIP80, Resistin-HSA-HCEIP80,
Leptin-HSA-HCEIP80, HCEIP80-HSA-IGF1, HCEIP80-HSA-GLP1,
HCEIP80-HSA-Exendin 4, HCEIP80-HSA-Insulin, HCEIP80-HSA-Resistin,
HCEIP80-HSA-Leptin, HCEIP80-HSA-HLDOU18, IGF1-HSA-HLDOU18,
GLP1-HSA-HLDOU18, Exendin 4-HSA-HLDOU18, Insulin-HSA-HLDOU18,
Resistin-HSA-HLDOU18, Leptin-HSA-HLDOU18, HLDOU18-HSA-HCEIP80,
HLDOU18-HSA-IGF1, HLDOU18-HSA-GLP1, HLDOU18-HSA-Exendin 4,
HLDOU18-HSA-Insulin, HLDOU18-HSA-Resistin, HLDOU18-HSA-Leptin, and
HLDOU18-HSA-HLDOU18, HCEIP80-HSA-RegIV, IGF1-HSA-RegIV,
GLP1-HSA-RegIV, Exendin 4-HSA-RegIV, Insulin-HSA-RegIV,
Resistin-HSA-RegIV, Leptin-HSA-RegIV, HLDOU18-HSA-RegIV,
RegIV-HSA-HCEIP80, RegIV-HSA-IGF1, RegIV-HSA-GLP1,
RegIV-HSA-Exendin 4, RegIV-HSA-Insulin, RegIV-HSA-Resistin,
RegIV-HSA-Leptin, RegIV-HSA-HLDOU18, RegIV-HSA-RegIV,
HCEIP80-HSA-HDRMI82, IGF1-HSA-HDRMI82, GLP1-HSA-HDRMI82, Exendin
4-HSA-HDRMI82, Insulin-HSA-HDRMI82, Resistin-HSA-HDRMI82,
Leptin-HSA-HDRMI82, HLDOU18-HSA-HDRMI82, RegIV-HSA-HDRMI82,
HDRMI82-HSA-HCEIP80. HDRMI82-HSA-IGF1, HDRMI82-HSA-GLP1,
HDRMI82-HSA-Exendin 4, HDRMI82-HSA-Insulin, HDRMI82-HSA-Resistin,
HDRMI82-HSA-Leptin, HDRMI82-HSA-HLDOU18, HDRMI82-HSA-RegIV,
HDRMI82-HSA-HDRMI82, HCEIP80-HSA-IFNa, IGF1-HSA-IFNa,
GLP1-HSA-IFNa, Exendin 4-HSA-IFNa, Insulin-HSA-IFNa,
Resistin-HSA-IFNa, Leptin-HSA-IFNa, HLDOU18-HSA-IFNa,
RegIV-HSA-IFNa, HDRMI82-HSA-IFNa, IFNa-HSA-HCEIP80, IFNa-HSA-IGF1,
IFNa-HSA-GLP1, IFNa-HSA-Exendin 4, IFNa-HSA-Insulin,
IFNs-HSA-Resistin, IFNa-HSA-Leptin, IFNa-HSA-HLDOU18,
IFNa-HSA-RegIV, IFNa-HSA-HDRMI82, and IFNa-HSA-IFNa.
[1559] The albumin fusion proteins (e.g., containing a Therapeutic
protein (or fragment or variant thereof) fused to albumin (or a
fragment or variant thereof)) may additionally be fused to other
proteins to generate "multifusion proteins". These multifusion
proteins can be used for a variety of applications. For example,
fusion of the albumin fusion proteins of the invention to His-tag,
HA-tag, protein A, IgG domains, and maltose binding protein
facilitates purification. (See e.g., EP A 394,827; Traunecker et
al., Nature 331:84-86 (1988)). Nuclear localization signals fused
to the polypeptides of the present invention can target the protein
to a specific subcellular localization, while covalent heterodimer
or homodimers can increase or decrease the activity of an albumin
fusion protein. Furthermore, the fusion of additional protein
sequences to the albumin fusion proteins of the invention may
further increase the solubility and/or stability of the fusion
protein. The fusion proteins described above can be made using or
routinely modifying techniques known in the art and/or by modifying
the following protocol, which outlines the fusion of a polypeptide
to an IgG molecule.
[1560] Briefly, the human Fc portion of the IgG molecule can be PCR
amplified, using primers that span the 5' and 3' ends of the
sequence described below. These primers also should have convenient
restriction enzyme sites that will facilitate cloning into an
expression vector, preferably a mammalian or yeast expression
vector.
[1561] For example, if pC4 (ATCC Accession No. 209646) is used, the
human Fc portion can be ligated into the BamHI cloning site. Note
that the 3' BamHI site should be destroyed. Next, the vector
containing the human Fc portion is re-restricted with BamHI,
linearizing the vector, and a polynucleotide encoding an albumin
fusion protein of the present invention (generated and isolated
using techniques known in the art), is ligated into this BamHI
site. Note that the polynucleotide encoding the fusion protein of
the invention is cloned without a stop codon, otherwise a Fc
containing fusion protein will not be produced.
[1562] If the naturally occurring signal sequence is used to
produce the albumin fusion protein of the present invention, pC4
does not need a second signal peptide. Alternatively, if the
naturally occurring signal sequence is not used, the vector can be
modified to include a heterologous signal sequence. (See, e.g.,
International Publication No. WO 96/34691.)
Human IgG Fc Region:
TABLE-US-00024 [1563] (SEQ ID NO: 347)
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACAT
GCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGG
AGGAGCAGTACAACACTCACGTACCGTGTGGTCACCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATCGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACACGTGTACACCCTGCCCCCATCCCGGGAT
GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
TATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGCAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC
TTCCTCTACACTCAAGCTCACCGTGCACAAGAGCAGGTGGCAGCAGGG
GAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCC
GCGACTCTAGAGGAT
Example 46
Production of an Antibody from an Albumin Fusion Protein
[1564] a) Hybridoma Technology
[1565] Antibodies that bind the albumin fusion proteins of the
present invention and portions of the albumin fusion proteins of
the present invention (e.g., the Therapeutic protein portion or
albumin portion of the fusion protein) can be prepared by a variety
of methods. (See, Current Protocols, Chapter 2.) As one example of
such methods, a preparation of an albumin fusion protein of the
invention or a portion of an albumin fusion protein of the
invention is prepared and purified to render it substantially free
of natural contaminants. Such a preparation is then introduced into
an animal in order to produce polyclonal antisera of greater
specific activity.
[1566] Monoclonal antibodies specific for an albumin fusion protein
of the invention, or a portion of an albumin fusion protein of the
invention, are prepared using hybridoma technology (Kohler et al.,
Nature 256:495 (1975); Kohler et al., Ear. J, Immunol. 6:511
(1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et
al. in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier,
N.Y., pp. 563-681 (1981)). In general, an animal (preferably a
mouse) is immunized with an albumin fusion protein of the
invention, or a portion of an albumin fusion protein of the
invention. The splenocytes of such mice are extracted and fused
with a suitable myeloma cell line. Any suitable myeloma cell line
may be employed in accordance with the present invention; however,
it is preferable to employ the parent myeloma cell line (SP2O),
available from the ATCC. After fusion, the resulting hybridoma
cells are selectively maintained in HAT medium, and then cloned by
limiting dilution as described by Wands et al. (Gastroenterology
80:225-232 (1981)). The hybridoma cells obtained through such a
selection are then assayed to identify clones which secrete
antibodies capable of binding an albumin fusion protein of the
invention, or a portion of an albumin fusion protein of the
invention.
[1567] Alternatively, additional antibodies capable of binding to
an albumin fusion protein of the invention, or a portion of an
albumin fusion protein of the invention can be produced in a
two-step procedure using anti-idiotypic antibodies, Such a method
makes use of the fact that antibodies are themselves antigens, and
therefore, it is possible to obtain an antibody which binds to a
second antibody. In accordance with this method, protein specific
antibodies are used to immunize an animal, preferably a mouse. The
splenocytes of such an animal are then used to produce hybridoma
cells, and the hybridoma cells are screened to identify clones
which produce an antibody whose ability to bind to the an albumin
fusion protein of the invention (or portion of an albumin fusion
protein of the invention)-specific antibody can be blocked by the
fusion protein of the invention, or a portion of an albumin fusion
protein of the invention. Such antibodies comprise anti-idiotypic
antibodies to the fusion protein of the invention (or portion of an
albumin fusion protein of the invention)-specific antibody and are
used to immunize an animal to induce formation of further fusion
protein of the invention (or portion of an albumin fusion protein
of the invention)-specific antibodies.
[1568] For in vivo use of antibodies in humans, an antibody is
"humanized". Such antibodies can be produced using genetic
constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric and
humanized antibodies are known in the art and are discussed herein.
(See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al, EP 173494;
Neuberger et al., WO 8601533; Robinson et al., International
Publication No. WO 8702671; Boulianne et al, Nature 312:643 (1984);
Neuberger et al., Nature 314:268 (1985)).
[1569] b) Isolation of Antibody Fragments Directed Against an
Albumin Fusion Protein of the Invention, or a Portion of an Albumin
Fusion Protein of the Invention from a Library of scFvs
[1570] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against an albumin fusion protein of the invention, or
a portion of an albumin fusion protein of the invention, to which
the donor may or may not have been exposed (see e.g., U.S. Pat. No.
5,835,793 incorporated herein by reference in its entirety).
[1571] c) Rescue of the Library.
[1572] A library of scFvs is constructed from the ENA of human PBLs
as described in International Publication No. WO 92/01047. To
rescue phage displaying antibody fragments, approximately 10.sup.9
E. coli harboring the phagemid are used to inoculate 50 ml of
2.times.TY containing 1% glucose and 100 .mu.g/ml of ampicillin
(2.times.TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five
ml of this culture is used to inoculate 50 ml of
2.times.TY-AMP-GLU, 2.times.108 TU of delta gene 3 helper (M13
delta gene III, see International Publication No. WO 92/01047) are
added and the culture incubated at 37.degree. C. fat 45 minutes
without shaking and then at 37.degree. C. for 45 minutes with
shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and
the pellet resuspended in 2 liters of 2.times.TY containing 100
.mu.g/ml ampicillin and 50 ug/ml kanamycin and grown overnight.
Phage are prepared as described in International Publication No. WO
92/01047.
[1573] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene HI protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100 .mu.g,
ampicillin/ml and 25 .mu.g kanamycin/ml (2.times.TY-AMP-KAN) and
grown overnight, shaking at 37.degree. C. Phage particles are
purified and concentrated from the culture medium by two
PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS
and passed through a 0.45 .mu.m filter (Minisart NML; Sartorius) to
give a final concentration of approximately 10.sup.13 transducing
units/ml (ampicillin-resistant clones).
[1574] Panning of the Library.
[1575] Immunotubes (Nunc) are coated overnight in PBS with 4 ml of
either 100 .mu.g/ml or 10 .mu.g/ml of an albumin fusion protein of
the invention, or a portion of an albumin fusion protein of the
invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at
37.degree. C. and then washed 3 times in PBS. Approximately
10.sup.13 TU of phage is applied to the tube and incubated for 30
minutes at room temperature tumbling on an over and under turntable
and then left to stand for another 1.5 hours. Tubes are washed 10
times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are
diluted by adding 1 ml of 100 mM triethylamine and rotating 15
minutes on an under and over turntable after which the solution is
immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage
are then used to infect 10 ml of mid-log E. coli TGI by incubating
eluted phage with bacteria for 30 minutes at 37.degree. C. The E.
coli are then plated on TYE plates containing 1% glucose and 100
.mu.g/ml ampicillin. The resulting bacterial library is then
rescued with delta gene 3 helper phage as described above to
prepare phage for a subsequent round of selection. This process is
then repeated for a total of 4 rounds of affinity purification with
tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20
times with PBS for rounds 3 and 4.
[1576] Characterization of Binders.
[1577] Eluted phage from the 3rd and 4th rounds of selection are
used to infect E. coli HB 2151 and soluble scFv is produced (Marks,
et al., 1991) front single colonies for assay. ELISAs are performed
with microtitre plates coated with either 10 pg/ml of an albumin
fusion protein of the invention, or a portion of an albumin fusion
protein of the invention, in 50 mM bicarbonate pH 9.6. Clones
positive in ELISA are further characterized by PCR fingerprinting
(see, e.g., International Publication No. WO 92/01047) and then by
sequencing. These ELISA positive clones may also be further
characterized by techniques known in the art, such as, for example,
epitope mapping, binding affinity, receptor signal transduction,
ability to block or competitively inhibit antibody/antigen binding,
and competitive agonistic or antagonistic activity.
Example 47
Method Of Treatment Using Gene Therapy-Ex Vivo
[1578] One method of gene therapy transplants fibroblasts, which
are capable of expressing an albumin fusion protein of the present
invention, onto a patient. Generally, fibroblasts are obtained from
a subject by skin biopsy. The resulting tissue is placed in
tissue-culture medium and separated into small pieces. Small chunks
of the tissue are placed on a wet surface of a tissue culture
flask, approximately ten pieces are placed in each flask. The flask
is turned upside down, closed tight and left at room temperature
over night. After 24 hours at room temperature, the flask is
inverted and the chunks of tissue remain fixed to the bottom of the
flask and fresh media (e.g., Ham's F12 media, with 10% FBS,
penicillin and streptomycin) is added. The flasks are then
incubated at 37 degree C. for approximately one week.
[1579] At this time, fresh media is added and subsequently changed
every several days. After an additional two weeks in culture, a
monolayer of fibroblasts emerge. The monolayer is trypsinized and
scaled into larger flasks.
[1580] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)),
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[1581] Polynucleotides encoding an albumin fusion protein of the
invention can be generated using techniques known in the art
amplified using PCR primers which correspond to the 5' and 3' end
sequences and optionally having appropriate restriction sites and
initiation/stop codons, if necessary. Preferably, the 5' primer
contains an EcoRI site and the 3' primer includes a HindIII site.
Equal quantities of the Moloney murine sarcoma virus linear
backbone and the amplified EcoRI and HindIII fragment are added
together, in the presence of T4 DNA ligase. The resulting mixture
is maintained under conditions appropriate for ligation of the two
fragments. The ligation mixture is then used to transform bacteria
HB101, which are then plated onto agar containing kanamycin for the
purpose of confirming that the vector has the gene of interest
properly inserted.
[1582] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the gene is then added to
the media and the packaging cells transduced with the vector. The
packaging cells now produce infectious viral particles containing
the gene (the packaging cells are now referred to as producer
cells).
[1583] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his. Once the
fibroblasts have been efficiently infected, the fibroblasts are
analyzed to determine whether the albumin fusion protein is
produced.
[1584] The engineered fibroblasts are then transplanted onto the
host, Whet alone or after having been grown to confluence on
eytodex 3 microcarrier beads.
Example 48
Method of Treatment Using Gene Therapy--In Vivo
[1585] Another aspect of the present invention is using in vivo
gene therapy methods to treat disorders, diseases and conditions.
The gene therapy method relates to the introduction of naked
nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences
encoding an albumin fusion protein of the invention into an animal.
Polynucleotides encoding albumin fusion proteins of the present
invention may be operatively linked to (i.e., associated with) a
promoter or any other genetic elements necessary for the expression
of the polypeptide by the target tissue. Such gene therapy and
delivery techniques and methods are known in the art, see, for
example, WO90/11092, WO98/11779; U.S. Pat. No. 5,693,622,
5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479
(1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff.
Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene
Ther. 3(5):405-411 (1996); Tsurami et al., Circulation
94(12):3281-3290 (1996) (incorporated herein by reference).
[1586] The polynucleotide constructs may be delivered by any method
that delivers injectable materials to the cells of an animal, such
as, injection into the interstitial space of tissues (heart,
muscle, skin, lung, liver, intestine and the like). The
polynucleotide constructs can be delivered in a pharmaceutically
acceptable liquid or aqueous carrier.
[1587] The term "naked" polynucleotide, DNA or RNA, refers to
sequences that are free from any delivery vehicle that acts to
assist, promote, or facilitate entry into the cell, including viral
sequences, viral particles, liposome formulations, lipofectin or
precipitating agents and the like. However, polynucleotides
encoding albumin fusion proteins of the present invention may also
be delivered in liposome formulations (such as those taught in
Feigner P. L. et al. (1995) Ann. NY Acad. Sci, 772:126-139 and
Abdallah B. et al, (1995) Biol. Cell 85(1):1-7) which can be
prepared by methods well known to those skilled in the art.
[1588] The polynucleotide vector constructs used in the gene
therapy method are preferably constructs that will not integrate
into the host genome nor will they contain sequences that allow for
replication. Any strong promoter known to those skilled in the art
can be used for driving the expression of DNA. Unlike other gene
therapy techniques, one major advantage of introducing naked
nucleic acid sequences into target cells is the transitory nature
of the polynucleotide synthesis in the cells. Studies have shown
that non-replicating DNA sequences can be introduced into cells to
provide production of the desired polypeptide for periods of up to
six months.
[1589] The polynucleotide construct can be delivered to the
interstitial space of tissues within an animal, including muscle,
skin, brain, lung, liver, spleen, bone matron, thymus, heart,
lymph, blood, bone, cartilage, pancreas, kidney, gall bladder,
stomach, intestine, testis, ovary, uterus, rectum, nervous system,
eye, gland, and connective tissue. Interstitial space of the
tissues comprises the intercellular fluid, mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers
in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that same matrix within connective tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space
occupied by the plasma of the circulation and the lymph fluid of
the lymphatic channels. Delivery to the interstitial space of
muscle tissue is preferred for the reasons discussed below. They
may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells are particularly competent in their ability to take up
and express polynucleotides.
[1590] For the naked polynucleotide injection, an effective dosage
amount of DNA or RNA will be in the range of from about 0.05 g/kg
body weight to about 50 ml/kg body weight. Preferably the dosage
will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration. The
preferred route of administration is by the parenteral route of
injection into the interstitial space of tissues. However, other
parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
polynucleotide constructs can be delivered to arteries during
angioplasty by the catheter used in the procedure.
[1591] The dose response effects of injected polynucleotide in
muscle in also is determined as follows. Suitable template DNA for
production of mRNA coding for polypeptide of the present invention
is prepared in accordance with a standard recombinant DNA
methodology. The template DNA, which may be either circular or
linear, is either used as naked DNA or complexed with liposomes.
The quadriceps muscles of mice are then injected with various
amounts of the template DNA.
[1592] Five to six week old female and male Balb/C mice are
anesthetized by intraperitoneal injection with 0.3 ml of 2.5%
Avertin, A 1.5 cm incision is made on the anterior thigh, and the
quadriceps muscle is directly visualized. The template DNA is
injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge
needle over one minute, approximately 0.5 cm from the distal
insertion site of the muscle into the knee and about 0.2 cm deep. A
suture is placed over the injection site for future localization,
and the skin is closed with stainless steel clips.
[1593] After an appropriate incubation time (e.g., 7 days) muscle
extracts are prepared by excising the entire quadriceps. Every
fifth 15 um cross-section of the individual quadriceps muscles is
histochemically stained for protein expression. A time course for
fusion protein expression may be done in a similar fashion except
that quadriceps from different mice are harvested at different
times. Persistence of DNA in muscle following injection may be
determined by Southern blot analysis after preparing total cellular
DNA and HIRT supernatants from injected and control mice. The
results of the above experimentation in mice can be used to
extrapolate proper dosages and other treatment parameters in humans
and other animals using naked DNA.
Example 49
Transgenic Animals
[1594] The albumin fusion proteins of the invention can also be
expressed in transgenic animals. Animals of any species, including,
but not limited to, mice, rats, rabbits, hamsters, guinea pigs,
pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g.,
baboons, monkeys, and chimpanzees may be used to generate
transgenic animals. In a specific embodiment, techniques described
herein or otherwise known in the art, are used to express fusion
proteins of the invention in humans, as part of a gene therapy
protocol.
[1595] Any technique known in the art may be used to introduce the
polynucleotides encoding the albumin fusion proteins of the
invention into animals to produce the founder lines of transgenic
animals. Such techniques include, but are not limited to,
pronuclear microinjection (Paterson et al., Appl. Microbial.
Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY)
11:1263.1270 (1993); Wright et al., Biotechnology (NY) 9:830-834
(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989));
retrovirus mediated gene transfer into germ lines (Van der Putten
et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),
blastocysts or embryos; gene targeting in embryonic stem cells
(Thompson et al. Cell 56:313-321 (1989)); electroporation of cells
or embryos (Lo, Mol. Cell. Biol. 3:1803-1814 (1983)); introduction
of the polynucleotides of the invention using a gene gun (see,
e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic
acid constructs into embryonic pleuripotent stem cells and
transferring the stem cells back into the blastocyst; and
sperm-mediated gene transfer (Lavinano et al., Cell 57:717-723
(1989); etc. For a review of such techniques, see Gordon,
"Transgenic Animals." Intl. Rev. Cytol. 115:171-229 (1989), which
is incorporated by reference herein in its entirety.
[1596] Any technique known in the art may be used to produce
transgenic clones containing polynucleotides encoding albumin
fusion proteins of the invention, for example, nuclear transfer
into enucleated oocytes of nuclei from cultured embryonic, fetal,
or adult cells induced to quiescence (Campell et al., Nature
380:64-66 (1996); Wilmot et al., Nature 385:810-813 (1997)).
[1597] The present invention provides for transgenic animals that
carry the polynucleotides encoding the albumin fusion proteins of
the invention in all their cells, as well as animals which carry
these polynucleotides in some, but not all their cells, i.e.,
mosaic animals or chimeric. The transgene may be integrated as a
single transgene or as multiple copies such as in concatamers,
e.g., head-to-head tandems or head-to-tail tandems. The transgene
may also be selectively introduced into and activated in a
particular cell type by following, for example, the teaching of
Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236
(1992)). The regulatory sequences required for such a cell-type
specific activation will depend upon the particular cell type of
interest, and will be apparent to those of skill in the art. When
it is desired that the polynucleotide encoding the fusion protein
of the invention be integrated into the chromosomal site of the
endogenous gene corresponding to the Therapeutic protein portion or
albumin portion of the fusion protein of the invention, gene
targeting is preferred. Briefly, when such a technique is to be
utilized, vectors containing some nucleotide sequences homologous
to the endogenous gene are designed for the purpose of integrating,
via homologous recombination with chromosomal sequences, into and
disrupting the function of the nucleotide sequence of the
endogenous gene. The transgene may also be selectively introduced
into a particular cell type, thus inactivating the endogenous gene
in only that cell type, by following, for example, the teaching of
Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory
sequences required for such a cell-type specific inactivation will
depend upon the particular cell type of interest, and will be
apparent to those of skill in the art.
[1598] Once transgenic animals have been generated, the expression
of the recombinant gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to verify that
integration of the polynucleotide encoding the fusion protein of
the invention has taken place. The level of mRNA expression of the
polynucleotide encoding the fusion protein of the invention in the
tissues of the transgenic animals may also be assessed using
techniques which include, but are not limited to, Northern blot
analysis of tissue samples obtained from the animal, in situ
hybridization analysis, and reverse transcriptase-PCR (rt-PCR).
Samples of fusion protein-expressing tissue may also be evaluated
immunocytochemically or immunohistochemically using antibodies
specific for the fusion protein.
[1599] Once the founder animals are produced, they may be bred,
inbred, outbred, or crossbred to produce colonies of the particular
animal. Examples of such breeding strategies include, but are not
limited to: outbreeding of founder animals with more than one
integration site in order to establish separate lines; inbreeding
of separate lines in order to produce compound transgenics that
express the transgene at higher levels because of the effects of
additive expression of each transgene; crossing of heterozygous
transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate
the need for screening of animals by DNA analysis; crossing of
separate homozygous lines to produce compound heterozygous or
homozygous lines; and breeding to place the transgene (i.e.,
polynucleotide encoding an albumin fusion protein of the invention)
on a distinct background that is appropriate for an experimental
model of interest.
[1600] Transgenic animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating
the biological function of fusion proteins of the invention and the
Therapeutic protein and/or albumin component of the fusion protein
of the invention, studying conditions and/or disorders associated
with aberrant expression, and in screening for compounds effective
in treating (e.g., ameliorating) such conditions and/or
disorders.
Example 50
Assays Detecting Stimulation or Inhibition of B cell Proliferation
and Differentiation
[1601] Generation of functional humoral immune responses requires
both soluble and cognate signaling between B-lineage cells and
their microenvironment. Signals may impart a positive stimulus that
allows a B-lineage cell to continue its programmed development, or
a negative stimulus that instructs the cell to arrest its current
developmental pathway. To date, numerous stimulatory and inhibitory
signals have been found to influence B cell responsiveness
including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and
TL-15. Interestingly, these signals are by themselves weak
effectors but can, in combination with various co-stimulatory
proteins, induce activation, proliferation, differentiation,
homing, tolerance and death among B cell populations.
[1602] One of the best studied classes of B-cell co-stimulatory
proteins is the TNF-superfamily. Within this family CD40, CD27, and
CD30 along with their respective ligands CD 154, CD70, and CD 153
have been found to regulate a variety of immune responses. Assays
which allow for the detection and/or observation of the
proliferation and differentiation of these B-cell populations and
their precursors are valuable tools in determining the effects
various proteins may have on these B-cell populations in terms of
proliferation and differentiation. Listed below are two assays
designed to allow for the detection of the differentiation,
proliferation, or inhibition of B-cell populations and their
precursors.
[1603] In Vitro Assay--Albumin fusion proteins of the invention
(including fusion proteins containing fragments or variants of
Therapeutic proteins and/or albumin or fragments or variants of
albumin) can be assessed for its ability to induce activation,
proliferation, differentiation or inhibition and/or death in B-cell
populations and their precursors. The activity of an albumin fusion
protein of the invention on purified human tonsillar B cells,
measured qualitatively over the dose range from 0.1 to 10,000
ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay
in which purified tonsillar B cells are cultured in the presence of
either formalin-fixed Staphylococcus aureus Cowan I (SAC) or
immobilized anti-human IgM antibody as the priming agent. Second
signals such as IL-2 and IL-15 synergize with SAC and IgM
crosslinking to elicit B cell proliferation as measured by
tritiated-thymidine incorporation. Novel synergizing agents can be
readily identified using this assay. The assay involves isolating
human tonsillar 1-3 cells by magnetic bead (MACS) depletion of
CD3-positive cells. The resulting cell population is greater than
95% B cells as assessed by expression of CD45 R(B220).
[1604] Various dilutions of each sample are placed into individual
wells of a 96-well plate to which are added 10.sup.5 B-cells
suspended in culture medium (RPMI 1640 containing 10% FBS,
5.times.10.sup.-5M 2ME, 100 U/ml penicillin, 10 ug/ml streptomycin,
and 10.sup.-5 dilution of SAC) in a total volume of 150 ul.
Proliferation or inhibition is quantitated by a 20 h pulse (1
uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor
addition. The positive and negative controls are IL2 and medium
respectively.
[1605] In vivo Assay--BALB/c mice are injected (i.p.) twice per day
with buffer only, or 2 mg/Kg of an albumin fusion protein of the
invention (including fusion proteins containing fragments or
variants of Therapeutic proteins and/or albumin or fragments or
variants of albumin). Mice receive this treatment for 4 consecutive
days, at which time they are sacrificed and various tissues and
serum collected for analyses. Comparison of H&E sections from
normal spleens and spleens treated with the albumin fusion protein
of the invention identify the results of the activity of the fusion
protein on spleen cells, such as the diffusion of peri-arterial
lymphatic sheaths, and/or significant increases in the nucleated
cellularity of the red pulp regions, which may indicate the
activation of the differentiation and proliferation of B-cell
populations. Immunohistochemical studies using a B cell marker,
anti-CD45R(B220), are used to determine whether any physiological
changes to splenic cells, such as splenic disorganization, are due
to increased B-cell representation within loosely defined B-cell
zones that infiltrate established T-cell regions.
[1606] Flow cytometric analyses of the spleens from mice treated
with the albumin fusion protein is used to indicate whether the
albumin fusion protein specifically increases the proportion of
ThB+, CD45R(B220)dull B cells over that which is observed in
control mice.
[1607] Likewise, a predicted consequence of increased mature B-cell
representation in vivo is a relative increase in serum Ig titers,
Accordingly, serum IgM and IgA levels are compared between buffer
and fusion protein treated mice.
[1608] The studies described in this example tested activity of
fusion proteins of the invention. However, one skilled in the art
could easily modify the exemplified studies to test the activity of
fusion proteins and polynucleotides of the invention (e.g., gene
therapy).
Example 51
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
[1609] Diabetic db+/db+ Mouse Model.
[1610] To demonstrate that an albumin fusion protein of the
invention accelerates the healing process, the genetically diabetic
mouse model of wound healing is used. The full thickness wound
healing model in the db+/db+ mouse is a well characterized,
clinically relevant and reproducible model of impaired wound
healing. Healing of the diabetic wound is dependent on formation of
granulation fissile and re-epithelialization rather than
contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992);
Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).
[1611] The diabetic animals have many of the characteristic
features observed in Type II diabetes mellitus. Homozygous
(db+/db+) mice are obese in comparison to their normal heterozygous
(db+/db+) littermates. Mutant diabetic (db+/db+) mice have a single
autosomal recessive mutation on chromosome 4 (db+) (Coleman et al.
Proc. Natl. Aced, Sci. USA 77:283-293 (1982)). Animals show
polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+)
have elevated blood glucose, increased or normal insulin levels,
and suppressed cell-mediated immunity (Mandel et al., J. Immunol.
120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.
51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55
(1935)). Peripheral neuropathy, myocardial complications, and
microvascular lesions, basement membrane thickening and glomerular
filtration abnormalities have been described in these animals
(Norido, F. et al., Exp. Neuroi. 83(2):221-232 (1984); Robertson et
al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab invest.
40(41:460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6
(1982)). These homozygous diabetic mice develop hyperglycemia that
is resistant to insulin analogous to human type H diabetes (Mandel
et al., J. Immunol. 120:1375-1377 (1978)).
[1612] The characteristics observed in these animals suggests that
healing in this model may be similar to the healing observed in
human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246
(1990)).
[1613] Genetically diabetic female C57BL/KsJ (db+/db+) mice and
their non-diabetic (db+/+m) heterozygous littermates are used in
this study (Jackson Laboratories). The animals use purchased at 6
weeks of age and are 8 weeks old at the beginning of the study.
Animals are individually housed and received food and water ad
libitum. All manipulations are performed using aseptic techniques.
The experiments are conducted according to the rules and guidelines
of Human Genome Sciences, Inc. Institutional Animal Care and Use
Committee and the Guidelines for the Care and Use of Laboratory
Animals.
[1614] Wounding protocol is performed according to previously
reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med.
172:245-251 (1990)). Briefly, on the day of wounding, animals are
anesthetized with an intraperitoneal injection of Avertin (0.01
mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in
deionized water. The dorsal region of the animal is shaved and the
skin washed with 70% ethanol solution and iodine. The surgical area
is dried with sterile gauze prior to wounding. An 8 mm
full-thickness wound is then created using a Keyes tissue punch.
Immediately following wounding, the surrounding skin is gently
stretched to eliminate wound expansion. The wounds are left open
for the duration of the experiment. Application of the treatment is
given topically for 5 consecutive days commencing on the day of
wounding. Prior to treatment, wounds are gently cleansed with
sterile saline and gauze sponges.
[1615] Wounds are visually examined and photographed at a fixed
distance at the day of surgery and at two day intervals thereafter.
Wound closure is determined by daily measurement on days 1-5 and on
day 8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[1616] An albumin fusion protein of the invention is administered
using at a range different doses from 4 mg to 500 mg per wound per
day for 8 days in vehicle. Vehicle control groups received 50 mL of
vehicle solution.
[1617] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology and
immunohistochemistry. Tissue specimens are placed in 10% neutral
buffered formalin in tissue cassettes between biopsy sponges for
further processing.
[1618] Three groups of 10 animals each (5 diabetic and 5
non-diabetic controls) are evaluated: 1) Vehicle placebo control,
2) untreated group, and 3) treated group.
[1619] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total square area of
the wound. Contraction is then estimated by establishing the
differences between the initial wound area (day 0) and that of post
treatment (day 8). The wound area on day I is 64 mm.sup.2, the
corresponding size of the dermal punch. Calculations are made using
the following formula:
[Open area on day 8]-[Open area on day 1]/[Open area on day 1]
[1620] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using a Reichert-Jung microtome. Routine
hematoxylin-eosin (H&E) staining is performed on cross-sections
of bisected wounds. Histologic examination of the wounds are used
to assess whether the healing process and the morphologic
appearance of the repaired skin is altered by treatment with an
albumin fusion protein of the invention. This assessment included
verification of the presence of cell accumulation, inflammatory
cells, capillaries, fibroblasts, re-epithelialization and epidermal
maturity (Greenhalgh, D. G. et al, Am. J. Pathol. 136:1235 (1990)).
A calibrated lens micrometer is used by a blinded observer.
[1621] Tissue sections are also stained immunohistochemically with
a polyclonal rabbit anti-human keratin antibody using ABC Elite
detection system. Human skin is used as a positive tissue control
while non-immune IgG is used as a negative control. Keratinocyte
growth is determined by evaluating the extent of
reepithelialization of the wound using a calibrated lens
micrometer.
[1622] Proliferating cell nuclear antigen/cyclin (PCNA) in skin
specimens is demonstrated by using anti-PCNA antibody (1:50) with
an ABC Elite detection system. Human colon cancer served as a
positive tissue control and human brain tissue is used as a
negative tissue control. Each specimen included a section with
omission of the primary antibody and substitution with non-immune
mouse IgG. Ranking of these sections is based on the extent of
proliferation on a scale of 0-8, the lower side of the scale
reflecting slight proliferation to the higher side reflecting
intense proliferation.
[1623] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[1624] Steroid Impaired Rat Model
[1625] The inhibition of wound healing by steroids has been well
documented in various in vitro and in vivo systems (Wahl,
Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid
Action: Basic and Clinical Aspects. 280-302 (1989); Wahl et al., J.
Immunol. 115: 476-481 (1975); Werb et al J. Exp. Med. 147:1684-1694
(1978)), Glucocorticoids retard wound healing by inhibiting
angiogenesis, decreasing vascular permeability (Ebert et al., An.
Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and
collagen synthesis (Beck er din Growth Factors. 5: 295-304 (1991);
Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a
transient reduction of circulating monocytes (Haynes et al., J.
Clin. Invest. 61: 703-797 (1978); Wahl, "Glucocorticoids and wound
healing", In: Antiinflammatory Steroid Action: Basic and Clinical
Aspects, Academic Press, New York, pp. 280-302 (1989)). The
systemic administration of steroids to impaired wound healing is a
well establish phenomenon in rats (Beck et al, Growth Factors. 5:
295-304 (1991); Haynes et al, J. Clin. Invest. 61: 703797 (1978);
Wahl, "Glucocorticoids and wound healing", In: Antiinflammatory
Steroid Action Basic and Clinical Aspects, Academic Press, New
York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA
86: 2229-2233 (1989)).
[1626] To demonstrate that an albumin fusion protein of the
invention can accelerate the healing process, the effects of
multiple topical applications of the fusion protein on full
thickness excisional skin wounds in rats in which healing has been
impaired by the systemic administration of methylprednisolone is
assessed.
[1627] Young adult male Sprague Dawley rats weighing 250-300 g
(Charles River Laboratories) are used in this example. The animals
are purchased at 8 weeks of age and are 9 weeks old at the
beginning of the study. The healing response of rats is impaired by
the systemic administration of methylprednisolone (17 mg/kg/rat
intramuscularly) at the time of wounding. Animals are individually
housed and received food and water ad libitum. All manipulations
are performed using aseptic techniques. This study is conducted
according to the rules and guidelines of Human Genome Sciences,
Inc. Institutional Animal Care and Use Committee and the Guidelines
for the Care and Use of Laboratory Animals.
[1628] The wounding protocol is followed according to that
described above. On the day of wounding, animals are anesthetized
with an intramuscular injection of ketamine (50 mg/kg) and xylazine
(5 mg/kg). The dorsal region of the animal is shaved and the skin
washed with 70% ethanol and iodine solutions. The surgical area is
dried with sterile gauze prior to wounding. An 8 mm full-thickness
wound is created using a Keyes tissue punch. The wounds are left
open for the duration of the experiment. Applications of the
testing materials are given topically once a day for 7 consecutive
days commencing on the day of wounding and subsequent to
methylprednisolone administration. Prior to treatment, wounds are
gently cleansed with sterile saline and gauze sponges.
[1629] Wounds are visually examined and photographed at a fixed
distance at the day of wounding and at the end of treatment. Wound
closure is determined by daily measurement on days 1-5 and on day
8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[1630] The fusion protein of the invention is administered using at
a range different doses, from 4 mg to 500 mg per wound per day for
8 days in vehicle. Vehicle control groups received 50 mL of vehicle
solution.
[1631] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology. Tissue specimens
are placed in 10% neutral buffered formalin in tissue cassettes
between biopsy sponges for further processing.
[1632] Three groups of 10 animals each (5 with methylprednisolone
and 5 without glucocorticoid) are evaluated: 1) Untreated group 2)
Vehicle placebo control 3) treated groups.
[1633] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total area of the
wound. Closure is then estimated by establishing the differences
between the initial wound area (day 0) and that of post treatment
(day 8). The wound area on day 1 is 64 mm.sup.2, the corresponding
size of the dermal punch. Calculations are made using the following
formula:
[Open area on day 8]-[Open area on day 1]/[Open area on day 1]
[1634] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using an Olympus microtome. Routine hematoxylin-eosin
(H&E) staining is performed on cross-sections of bisected
wounds. Histologic examination of the wounds allows assessment of
whether the healing process and the morphologic appearance of the
repaired skin is improved by treatment with an albumin fusion
protein of the invention. A calibrated lens micrometer is used by a
blinded observer to determine the distance of the wound gap.
[1635] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[1636] The studies described in this example tested activity of an
albumin fusion protein of the invention. However, one skilled in
the art could easily modify the exemplified studies to test the
activity of fusion proteins and polynucleotides of the invention
(e.g., gene therapy).
Example 52
Lymphedema Animal Model
[1637] The purpose of this experimental approach is to create an
appropriate and consistent lymphedema model for testing the
therapeutic effects of an albumin fusion protein of the invention
in lymphangiogenesis and re-establishment of the lymphatic
circulatory system in the rat hind limb. Effectiveness is measured
by swelling volume of the affected limb, quantification of the
amount of lymphatic vasculature, total blood plasma protein, and
histopathology. Acute lymphedema is observed for 7-10 days, Perhaps
more importantly, the chronic progress of the edema is followed for
up to 3-4 weeks.
[1638] Prior to beginning surgery, blood sample is drawn for
protein concentration analysis. Male rats weighing approximately
350 g are dosed with Pentobarbital. Subsequently, the right legs
are shaved from knee to hip. The shaved area is swabbed with gauze
soaked in 70% EtOH. Blood is drawn for serum total protein testing.
Circumference and volumetric measurements are made prior to
injecting dye into paws after marking 2 measurement levels (0.5 cm
above heel, at mid-pt of dorsal paw). The intradermal dorsum of
both right and left paws are injected with 0.05 rat of 3% Evan's
Blue. Circumference and volumetric measurements are then made
following injection of dye into paws.
[1639] Using the knee joint as a landmark, a mid-leg inguinal
incision is made circumferentially allowing the femoral vessels to
be located. Forceps and hemostats are used to dissect and separate
the skin flaps. After locating the femoral vessels, the lymphatic
vessel that runs along side and underneath the vessel(s) is
located. The main lymphatic vessels in this area are then
electrically coagulated or suture ligated.
[1640] Using a microscope, muscles in hack of the leg (near the
semitendinosis and adductors) are bluntly dissected. The popliteal
lymph node is then located. The 2 proximal and 2 distal lymphatic
vessels and distal blood supply of the popliteal node are then
ligated by suturing. The popliteal lymph node, and any accompanying
adipose tissue, is then removed by cutting connective tissues.
[1641] Care is taken to control any mild bleeding resulting from
this procedure. After lymphatics are occluded, the skin flaps are
sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin
edges are sealed to the underlying muscle tissue while leaving a
gap of .about.0.5 cm around the leg. Skin also may be anchored by
suturing to underlying muscle when necessary.
[1642] To avoid infection, animals are housed individually with
mesh (no bedding). Recovering animals are checked daily through the
optimal edematous peak, which typically occurred by day 5-7. The
plateau edematous peak are then observed. To evaluate the intensity
of the lymphedema, the circumference and volumes of 2 designated
places on each paw before operation and daily for 7 days are
measured. The effect of plasma proteins on lymphedema is determined
and whether protein analysis is a useful testing perimeter is also
investigated. The weights of both control and edematous limbs are
evaluated at 2 places. Analysis is performed in a blind manner.
[1643] Circumference Measurements: Under brief gas anesthetic to
prevent limb movement, a cloth tape is used to measure limb
circumference, Measurements are done at the ankle bone and dorsal
paw by 2 different people and those 2 readings are averaged.
Readings are taken from both control and edematous limbs.
[1644] Volumetric Measurements: On the day of surgery, animals are
anesthetized with Pentobarbital and are tested prior to surgery.
For daily volumetrics animals are under brief halothane anesthetic
(rapid immobilization and quick recovery), and both legs are shaved
and equally marked using waterproof marker on legs. Legs are first
dipped in water, then dipped into instrument to each marked level
then measured by Buxco edema software (Chen/Victor). Data is
recorded by one person, while the other is dipping the limb to
marked area.
[1645] Blood plasma protein measurements: Blood is drawn, spun, and
serum separated prior to surgery and then at conclusion for total
protein and Ca2.sup.+ comparison.
[1646] Limb Weight Comparison: After drawing blood, the animal is
prepared for tissue collection. The limbs are amputated using a
quillitine, then both experimental and control legs are cut at the
ligature and weighed. A second weighing is done as the
tibio-cacaneal joint is disarticulated and the foot is weighed.
[1647] Histological Preparations: The transverse muscle located
behind the knee (popliteal) area is dissected and arranged in a
metal mold, filled with freezeGel, dipped into cold methylbutane,
placed into labeled sample bags at -80EC until sectioning. Upon
sectioning, the muscle is observed under fluorescent microscopy for
lymphatics.
[1648] The studies described in this example tasted activity of
fusion proteins of the invention. However, one skilled in the art
could easily modify the exemplified studies to test the activity of
fusion protein and polynucleotides of the invention (e.g., gene
therapy).
Example 53
T Cell Proliferation Assay
[1649] A CD3-induced proliferation assay is performed on PBMCs and
is measured by the uptake of .sup.3H-thymidine. The assay is
performed as follows. Ninety-six well plates are coated with 100
.mu.l/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched
control mAb (1333.1) overnight at 4 degrees C. (1 .mu.g/ml in 0.05M
bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC
are isolated by F/H gradient centrifugation from human peripheral
blood and added to quadruplicate wells (5.times.10.sup.4/well) of
mAb coated plates in RPMI containing 10% FCS and P/S in the
presence of varying concentrations of an albumin fusion protein of
the invention (including fusion proteins containing fragments or
variants of Therapeutic proteins and/or albumin or fragments or
variants of albumin) (total volume 200 tit), Relevant protein
buffer and medium alone are controls. After 48 hr. culture at 37
degrees C., plates are spun for 2 min. at 1000 rpm and 100 .mu.l of
supernatant is removed and stored 20 degrees C. for measurement of
IL-2 (or other cytokines) if effect on proliferation is observed.
Wells are supplemented with 100 ul of medium containing 0.5 uCi of
.sup.3H-thymidine and cultured at 37 degrees C. for 18-24 hr. Wells
are harvested and incorporation of .sup.5H-thymidine used as a
measure of proliferation. Anti-CD3 alone is the positive control
for proliferation. IL-2 (100 U/ml) is also used as a control which
enhances proliferation. Control antibody which does not induce
proliferation of T cells is used as the negative control for the
effects of fusion proteins of the invention.
Example 54
Effect of Fusion Proteins of the Invention on the Expression of MHC
Class II, Costimulatory and Adhesion Molecules and Cell
Differentiation of Monocytes and Monocyte-Derived Human Dendritic
Cells
[1650] Dendritic cells are generated by the expansion of
proliferating precursors found in the peripheral blood; adherent
PBMC or elutriated monocytic fractions are cultured for 7-10 days
with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells
have the characteristic phenotype of immature cells (expression of
CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with
activating factors, such as TNF-.alpha., causes a rapid change in
surface phenotype (increased expression of MHC class I and II,
costimulatory and adhesion molecules, downregulation of
FC.gamma.RII, upregulation of CD83). These changes correlate with
increased antigen-presenting capacity and with functional
maturation of the dendritic cells.
[1651] FACS analysis of surface antigens is performed as follows.
Cells are treated 1-3 days with increasing concentrations of an
albumin fusion protein of the invention or LPS (positive control),
washed with PBS containing 1% BSA and 0.02 mM sodium azide, and
then incubated with 1:20 dilution of appropriate FITC- or
PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C.
After an additional wash, the labeled cells are analyzed by flow
cytometry on a FACScan (Becton Dickinson).
[1652] Effect on the Production of Cytokines.
[1653] Cytokines generated by dendritic cells, in particular IL-12,
are important in the initiation of T-cell dependent immune
responses, IL-12 strongly influences the development of Th1 helper
T-cell immune response, and induces cytotoxic T and NK cell
function. An ELISA is used to measure the IL-12 release as follows.
Dendritic cells (10.sup.6/ml) are treated with increasing
concentrations of an albumin fusion protein of the invention for 24
hours. LPS (100 ng/ml) is added to the cell culture as positive
control, Supernatants from the cell cultures are then collected and
analyzed for IL-12 content using commercial ELISA kit (e.g., R
& D Systems (Minneapolis, Minn.)). The standard protocols
provided with the kits are used.
[1654] Effect on the Expression of MHC Class II Costimulatory and
Adhesion Molecules.
[1655] Three major families of cell surface antigens can be
identified on monocytes: adhesion molecules, molecules involved in
antigen presentation, and Fc receptor. Modulation of the expression
of MHC class II antigens and other costimulatory molecules, such as
137 and ICAM-1, may result in changes in the antigen presenting
capacity of monocytes and ability to induce T cell activation.
Increased expression of Fc receptors may correlate with improved
monocyte cytotoxic activity, cytokine release and phagocytosis.
[1656] FACS analysis is used to examine the surface antigens as
follows. Monocytes are treated 1-5 days with increasing
concentrations of an albumin fusion protein of the invention or LPS
(positive control), washed with PBS containing 1% BSA and 0.02 mM
sodium aside, and then incubated with 1:20 dilution of appropriate
FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4
degrees C. After an additional wash, the labeled cells are analyzed
by flow cytometry on a FACScan (Becton Dickinson).
[1657] Monocyte Activation and/or Increased Survival.
[1658] Assays for molecules that activate (or alternatively,
inactivate) monocytes and/or increase monocyte survival (or
alternatively, decrease monocyte survival) are known in the art and
may routinely be applied to determine whether a molecule of the
invention functions as an inhibitor or activator of monocytes.
Albumin fusion proteins of the invention can be screened using the
three assays described below. For each of these assays, Peripheral
blood mononuclear cells (PBMC) are purified from single donor
leukopacks (American Red Cross, Baltimore, Md.) by centrifugation
through a Histopaque gradient (Sigma). Monocytes are isolated from
PBMC by counterflow centrifugal elutriation.
[1659] Monocyte Survival Assay.
[1660] Human peripheral blood monocytes progressively lose
viability when cultured in absence of serum or other stimuli. Their
death results from internally regulated processes (apoptosis).
Addition to the culture of activating factors, such as TNF-alpha
dramatically improves cell survival and prevents DNA fragmentation.
Propidium iodide (PI) staining is used to measure apoptosis as
follows. Monocytes are cultured for 48 hours in polypropylene tubes
in serum-free medium (positive control), in the presence of 100
ng/ml TNF-alpha (negative control), and in the presence of varying
concentrations of the fusion protein to be tested. Cells are
suspended at a concentration of 2.times.10.sup.6/ml in PBS
containing PI at a final concentration of 5 .mu.g/ml, and then
incubated at room temperature for 5 minutes before FACScan
analysis. PI uptake has been demonstrated to correlate with DNA
fragmentation in this experimental paradigm.
[1661] Effect on Cytokine Release.
[1662] An important function of monocytes/macrophages is their
regulatory activity on other cellular populations of the immune
system through the release of cytokines after stimulation. An ELISA
to measure cytokine release is performed as follows. Human
monocytes are incubated at a density of 5.times.10.sup.5 cells/ml
with increasing concentrations of an albumin fusion protein of the
invention and under the same conditions, but in the absence of the
fusion protein. For IL-12 production, the cells are primed
overnight with IFN (100 U/ml) in the presence of the fusion
protein. LPS (10 ng/ml) is then added. Conditioned media are
collected after 24 h and kept frozen until use. Measurement of
TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a
commercially available ELISA kit (e.g., R & D Systems
(Minneapolis, Minn.)) and applying the standard protocols provided
with the kit.
[1663] Oxidative Burst.
[1664] Purified monocytes are plated in 96-w plate at
2-1.times.10.sup.5 cell/well. Increasing concentrations of an
albumin fusion protein of the invention are added to the wells in a
total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine
and antibiotics). After 3 days incubation, the plates are
centrifuged and the medium is removed from the wells. To the
macrophage monolayers, 0.2 ml per well of phenol red solution (140
mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose,
0.50 mM phenol red and 19 U/ml of HRPO) is added, together with the
stimulant (200 nM PMA). The plates are incubated at 37.degree. C.
for 2 hours and the reaction is stopped by adding 20 .mu.l 1N NaOH
per well. The absorbance is read at 610 nm. To calculate the amount
of H.sub.2O.sub.2 produced by the macrophages, a standard curve of
a H.sub.2O.sub.2 solution of known molarity is performed for each
experiment.
Example 55
Biological Effects of Fusion Proteins of the Invention
[1665] Astrocyte and Neuronal Assays.
[1666] Albumin fusion proteins of the invention can be tested for
activity in promoting the survival, neurite outgrowth, or
phenotypic differentiation of cortical neuronal cells and for
inducing the proliferation of glial fibrillary acidic protein
immunopositive cells, astrocytes. The selection of cortical cells
for the bioassay is hated on the prevalent expression of FGF-1 and
FGF-2 in cortical structures and on the previously reported
enhancement of cortical neuronal survival resulting from FGF-2
treatment. A thymidine incorporation assay, for example, can be
used to elucidate an albumin fusion protein of the invention's
activity on these cells.
[1667] Moreover, previous reports describing the biological effects
of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro
have demonstrated increases in both neuron survival and neurite
outgrowth (Walicke et al., "Fibroblast growth factor promotes
survival of dissociated hippocampal neurons and enhances neurite
extension." Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay
herein incorporated by reference in its entirety). However, reports
from experiments done on PC-12 cells suggest that these two
responses are not necessarily synonymous and may depend on not only
which FGF is being tested but also on which receptor(s) are
expressed on the target cells. Using the primary conical neuronal
culture paradigm, the ability of an albumin fusion protein of the
invention to induce neurite outgrowth can be compared to the
response achieved with FGF-2 using, for example, a thymidine
incorporation assay.
[1668] Fibroblast and Endothelial Cell Assays.
[1669] Human lung fibroblasts are obtained from Clonetics (San
Diego, Calif.) and maintained in growth media from Clonetics.
Dermal microvascular endothelial cells are obtained from Cell.
Applications (San Diego, Calif.). For proliferation assays, the
human lung fibroblasts and dermal microvascular endothelial cells
can be cultured at 5,000 cells/well in a 96-well plate for one day
in growth medium. The cells are then incubated for one day in 0.1%
BSA basal medium. After replacing the medium with fresh 0.1% BSA
medium, the cells are incubated with the test fusion protein of the
invention proteins for 3 days. Alamar Blue (Alamar Biosciences,
Sacramento, Calif.) is added to each well to a final concentration
of 10%. The cells are incubated for 4 hr. Cell viability is
measured by reading in a CytoFluor fluorescence reader. For the
PGE.sub.2 assays, the human lung fibroblasts are cultured at 5,000
cells/well in a 96-well plate for one day. After a medium change to
0.1% BSA basal medium, the cells are incubated with FGF-2 or fusion
protein of the invention with or without IL-let for 24 hours. The
supernatants are collected and assayed for PGE.sub.2 by EIA kit
(Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung
fibroblasts are cultured at 5,000 cells/well in a 96-well plate for
one day. After a medium change to 0.1% BSA basal medium, the cells
are incubated with FOP 2 or with or without an albumin fusion
protein of the invention and/or IL-1.alpha. for 24 hours. The
supernatants are collected and assayed for IL-6 by ELISA kit
(Endogen, Cambridge, Mass.).
[1670] Human lung fibroblasts are cultured with FGF-2 or an albumin
fusion protein of the invention for 3 days in basal medium before
the addition of Alamar Blue to assess effects on growth of the
fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which
can be used to compare stimulation with the fusion protein of the
invention.
[1671] Cell Proliferation Based on [3H]Thymidine Incorporation
[1672] The following [3H]-Thymidine incorporation assay can be used
to measure the effect of a Therapeutic proteins, e.g., growth
factor proteins, on the proliferation of cells such as fibroblast
cells, epithelial cells or immature muscle cells.
[1673] Sub-confluent cultures are arrested in G1 phase by an 18 h
incubation in serum-free medium. Therapeutic proteins are then
added for 24 h and during the last 4 h, the cultures are labeled
with [3H]thymidine, at a final concentration of 0.33 .mu.M (25
Ci/mmol, Amersham, Arlington Heights, Ill.). The incorporated
[311]thymidine is precipitated with ice-cold 10% trichloroacetic
acid for 24 h. Subsequently, the cells are rinsed sequentially with
ice-cold 10% trichloroacetic acid and then with ice-cold water.
Following lysis in 0.5 M NaOH, the lysates and PBS rinses (500 ml)
are pooled, and the amount of radioactivity is measured.
[1674] Parkinson Models.
[1675] The loss of motor function in Parkinson's disease is
attributed to a deficiency of striatal dopamine resulting from the
degeneration of the nigrostriatal dopaminergic projection neurons.
An animal model for Parkinson's that has been extensively
characterized involves the systemic administration of 1-methyl-4
phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is
taken-up by astrocytes and catabolized by monoamine oxidase B to
1-methyl-4-phenyl pyridine (MPP.sup.+) and released. Subsequently,
MPP.sup.- is actively accumulated in dopaminergic neurons by the
high-affinity reuptake transporter for dopamine. MPP.sup.+ is then
concentrated in mitochondria by the electrochemical gradient and
selectively inhibits nicotidamide adenine disphosphate ubiquinone
oxidoreductionase (complex 1), thereby interfering with electron
transport and eventually generating oxygen radicals.
[1676] It has been demonstrated in tissue culture paradigms that
FGF-2 (basic FOP) has trophic activity towards nigral dopaminergic
neurons (Ferrari et al., Dev. Biol. 1989), Recently, Dr. Unsicker's
group has demonstrated that administering FGF-2 in gel foam
implants in the striatum results in the near complete protection of
nigral dopaminergic neurons from the toxicity associated with MPTP
exposure (Otto and Unsicker, J. Neuroscience, 1990).
[1677] Based on the data with FGF-2, an albumin fusion protein of
the invention can be evaluated to determine whether it has an
action similar to that of FGF-2 in enhancing dopaminergic neuronal
survival in vitro and it can also be tested in vivo for protection
of dopaminergic neurons in the striatum from the damage associated
with MPTP treatment. The potential effect of an albumin fusion
protein of the invention is first examined in vitro in a
dopaminergic neuronal cell culture paradigm. The cultures are
prepared by dissecting the midbrain floor plate nom gestation day
14 Wistar rat embryos. The tissue is dissociated with trypsin and
seeded at a density of 200,000 cells/cm.sup.2 on
polyorthinine-laminin coated glass coverslips. The cells are
maintained in Dulbecco's Modified Eagle's medium and F12 medium
containing hormonal supplements (N1). The cultures are fixed with
paraformaldehyde after 8 days in vitro and are processed for
tyrosine hydroxylase, a specific marker for dopaminergic neurons,
immunohistochemical staining. Dissociated cell cultures are
prepared from embryonic rats. The culture medium is changed every
third day and the factors are also added at that time.
[1678] Since the dopaminergic neurons are isolated from animals at
gestation day 14, a developmental time which is past the stage when
the dopaminergic precursor cells are proliferating, an increase in
the number of tyrosine hydroxylase immunopositive neurons would
represent an increase in the number of dopaminergic neurons
surviving in vitro. Therefore, if a therapeutic protein of the
invention acts to prolong the survival of dopaminergic neurons, it
would suggest that the fusion protein may be involved in
Parkinson's Disease.
Example 56
The Effect of Albumin Fusion Proteins of the Invention on the
Growth of Vascular Endothelial Cells
[1679] On day 1, human umbilical vein endothelial cells (HUVEC) are
seeded at 2-5.times.10.sup.4 cells/35 mm dish density in M199
medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin,
and 50 units/ml endothelial cell growth supplements (ECUS,
Biotechnique, Inc.). On day 2, the medium is replaced with M199
containing 10% FBS, S units/ml heparin. An albumin fusion protein
of the invention, and positive controls, such as VEGF and basic EGF
(bFGF) are added, at varying concentrations. On days 4 and 6, the
medium is replaced. On day 8, cell number is determined with a
Coulter Counter.
[1680] An increase in the number of HUVEC cells indicates that the
fusion protein may proliferate vascular endothelial cells, while a
decrease in the number of HUVEC cells indicates that the fusion
protein inhibits vascular endothelial cells.
Example 57
Rat Corneal Wound Healing Model
[1681] This animal model shows the effect of an albumin fusion
protein of the invention on neovascularization. The experimental
protocol includes:
[1682] Making a 1-1.5 mm long incision from the center of cornea
into she Animal layer.
[1683] Inserting a spatula below the lip of the incision facing the
outer corner of the eye.
[1684] Making a pocket (its base is 1-1.5 mm form the edge of the
eye).
[1685] Positioning a pellet, containing 50 ng-5 ug of an albumin
fusion protein of the invention, within the pocket.
[1686] Treatment with an albumin fusion protein of the invention
can also be applied topically to the corneal wounds in a dosage
range of 20 mg-500 mg (daily treatment for five days).
Example 58
Suppression of TNF Alpha-Induced Adhesion Molecule Expression by an
Albumin Fusion Protein of the Invention
[1687] The recruitment of lymphocytes to areas of inflammation and
angiogenesis involves specific receptor-ligand interactions between
cell surface adhesion molecules (CAMs) on lymphocytes and the
vascular endothelium. The adhesion process, in both normal and
pathological settings, follows a multi-step cascade that involves
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1
(E-selectin) expression on endothelial cells (EC). The expression
of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the
local vasculature and extravasate into the local tissue during the
development of an inflammatory response. The local concentration of
cytokines and growth factor participate in the modulation of the
expression of these CAMs.
[1688] Tumor necrosis factor alpha (TNF-a), a potent
proinflammatory cytokine, is a stimulator of all three CAMs on
endothelial cells and may be involved in a wide variety of
inflammatory responses, often resulting in a pathological
outcome.
[1689] The potential of an albumin fusion protein of the invention
to mediate a suppression of TNF-a induced CAM expression can be
examined. A modified ELISA assay which uses ECs as a solid phase
absorbent is employed to measure the amount of CAM expression on
TNF-a treated ECs when co-stimulated with a member of the FGF
family of proteins.
[1690] To perform the experiment, human umbilical vein endothelial
cell (HUVEC) cultures are obtained from pooled cord harvests and
maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.)
supplemented with 10% FCS and 1% penicillin/streptomycin in a 37
degree C. humidified incubator containing 5% CO.sub.2. HUVECs are
seeded in 96-well plates at concentrations of 1.times.10.sup.4
cells/well in EGM medium at 37 degree C. for 18-24 hrs or until
continent. The monolayers are subsequently washed 3 times with a
serum-free solution of RPMI-1640 supplemented with 100 U/ml
penicillin and 100 mg/ml streptomycin, and treated with a given
cytokine and/or growth factor(s) for 24 h at 37 degree C. Following
incubation, the cells are then evaluated for CAM expression.
[1691] Human Umbilical Vein Endothelial cells (HUVECs) are grown in
a standard 96 well plate to confluence. Growth medium is removed
from the cells and replaced with 90 ul of 199 Medium (1091 FBS).
Samples for testing and positive or negative controls are added to
the plate in triplicate (in 10 ul volumes). Plates are incubated at
37 degree C. for either 5 h (selectin and integrin expression) or
24 h (integrin expression only), Plates are aspirated to remove
medium and 100 .mu.l of 0.1% paraformaldehyde-PBS (with Ca.sup.++
and Mg.sup.++) is added to each well. Plates are held at 4.degree.
C. for 30 min.
[1692] Fixative is then removed from the wells and wells are washed
1.times. with PBS(+Ca,Mg)+0.5% BSA and drained, Do not allow the
wells to dry. Add 10 .mu.l of diluted primary antibody to the test
and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and
Anti-E-selectin-Biotin are used at a concentration of 10 .mu.g/ml
(1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at
37.degree. C. for 30 min. in a humidified environment. Wells are
washed .times.3 with PBS(+Ca,Mg)+0.5% BSA.
[1693] Then add 20 .mu.l of diluted ExtrAvidin-Alkaline Phosphotase
(1:5,000 dilution) to each well and incubated at 37.degree. C. for
30 min. Wells are washed .times.3 with PBS(+Ca,Mg)+0.5% BSA. 1
tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of
glycine buffer (pH 10.4). 100 .mu.l of pNPP substrate in glycine
buffer is added to each test well. Standard wells in triplicate are
prepared from the working dilution of the ExtrAvidin-Alkaline
Phosphotase in glycine buffer: 1:5,000
(10.degree.)>10.sup.-0.5>10.sup.-4>10.sup.-1.5. 5 .mu.l of
each dilution is added to triplicate wells and the resulting AP
content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100
.mu.l of pNNP reagent must then be added to each of the standard
wells. The plate must be incubated at 37.degree. C. for 4 h. A
volume of 50 .mu.l of 3M NaOH is added to all wells. The results
are quantified on a plate reader at 405 nm. The background
subtraction option is used on blank wells filled with glycine
buffer only. The template is set up to indicate the concentration
of AP-conjugate in each standard well [5.50 ng; 1.74 tug; 0.55 ng;
0.18 ng]. Results are indicated as amount of bound AP-conjugate in
each sample.
Example 59
Construction of GAS Reporter Construct
[1694] One signal transduction pathway involved in the
differentiation and proliferation of cells is called the Jaks-STATs
pathway. Activated proteins in the Jaks-STATs pathway bind to gamma
activation site "GAS" elements or interferon-sensitive responsive
element ("ISRE"), located in the promoter of many genes. The
binding of a protein to these elements alter the expression of the
associated gene.
[1695] GAS and ISRE elements are recognized by a class of
transcription factors called Signal Transducers and Activators of
Transcription, or "STATs." There are six members of the STATS
family. Stat1 and Stat3 are present in many cell types, as is Stat2
(as response to IFN-alpha is widespread). Stat4 is more restricted
and is not in many cell types though it has been found in T helper
class I, cells after treatment with IL-12. Stat5 was originally
called mammary growth factor, but has been found at higher
concentrations in other cells including myeloid cells. It can be
activated in tissue culture cells by many cytokines.
[1696] The STATS are activated to translocate from the cytoplasm to
the nucleus upon tyrosine phosphorylation by a set of kinases known
as the Janus Kinase ("Jaks") family. Jaks represent a distinct
family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2,
and Jak3. These kinases display significant sequence similarity and
are generally catalytically inactive in resting cells.
[1697] The Jaks are activated by a wide range of receptors
summarized in the Table below. (Adapted from review by Sandler and
Darnell, Ann. Rev. Biochem. 64:621-51 (1995)). A cytokine receptor
family, capable of activating Yaks, is divided into two groups: (a)
Class I includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9,
IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and
thrombopoietin; and (b) Class 2 includes IFN-.alpha., IFN-g, and
IL-10. The Class 1 receptors share a conserved cysteine motif (a
set of four conserved cysteines and one tryptophan) and a WSXWS
motif (a membrane proximal region encoding Trp-Ser-Xaa-Trp-Ser (SEQ
NO: 398)).
[1698] Thus, on binding of a ligand to a receptor, Jaks are
activated, which in turn activate STATs, which then translocate and
bind to GAS elements. This entire process is encompassed in the
Jaks-STATs signal transduction pathway. Therefore, activation of
the Jaks-STATs pathway, reflected by the binding of the GAS or the
ISRE element, can be used to indicate proteins involved in the
proliferation and differentiation of cells. For example, growth
factors and cytokines are known to activate the Jaks-STATs pathway
(See Table 4, below). Thus, by using GAS elements linked to
reporter molecules, activators of the Jaks-STATs pathway can be
identified.
TABLE-US-00025 TABLE 4 JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS
(elements) or ISRE IFN family IFN-a/B + + - - 1, 2, 3 ISRE IFN-g +
+ - 1 GAS (IRF1 > Lys6 > IFP) Il-10 + ? ? - 1, 3 gp130 family
IL-6 (Pleiotropic) + + + ? 1, 3 GAS(IRF1 > Lys6 > IFP)
Il-11(Pleiotropic) ? + ? ? 1, 3 OnM(Pleiotropic) ? + + ? 1, 3
LIF(Pleiotropic) ? + + ? 1, 3 CNTF(Pleiotropic) -/+ + + ? 1, 3
G-CSF(Pleiotropic) ? + ? ? 1, 3 IL-12(Pleiotropic) + - + + 1, 3 g-C
family IL-2 (lymphocytes) - + - + 1, 3, 5 GAS IL-4 (lymph/myeloid)
- + - + 6 GAS(IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) - +
- + 5 GAS IL-9 (lymphocytes) - + - + 5 GAS IL-13 (lymphocyte) - + ?
? 6 GAS IL-15 ? + ? + 5 GAS gp140 family IL-3 (myeloid) - - + - 5
GAS(IRF1 > IFP >> Ly6) IL-5 (myeloid) - - + - 5 GAS GM-CSF
(myeloid) - - + - 5 GAS Growth hormone family GH ? - + - 5 PRL ?
+/- + - 1, 3, 5 EPO ? - + - 5 GAS(B-CAS > IRF1 = IFP >>
Ly6) Receptor Tyrosine Kinases EGF ? + + - 1, 3 GAS (IRF1) PDGF ? +
+ - 1, 3 CSF-1 ? + + - 1, 3 GAS(not IRF1)
[1699] To construct a synthetic GAS containing promoter element,
which is used in the Biological Assays described in Examples 62-64,
a PCR based strategy is employed to generate a GAS-SV40 promoter
sequence. The 5' primer contains four tandem copies of the GAS
binding site found in the IRF1 promoter and previously demonstrated
to bind STATS upon induction with a range of cytokines (Rothman et
al., Immunity 1:457-468 (1994).), although other GAS or ISRE
elements can be used instead. The 5' primer also contains 18 bp of
sequence complementary to the SV40 early promoter sequence and is
flanked with an XhoI site. The sequence of the 5' primer is:
TABLE-US-00026 (SEQ ID NO: 399)
5':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATT
TCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3'
[1700] The downstream primer is complementary to the SV40 promoter
and is flanked with a Hind III site:
5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:400)
[1701] PCR amplification is performed using the SV40 promoter
template present in the B-gal:promoter plasmid obtained from
Clontech. The resulting PCR fragment is digested with XhoI/Hind III
and subcloned into BLSK2-. (Stratagene.) Sequencing with forward
and reverse primers confirms that the insert contains the following
sequence:
TABLE-US-00027 (SEQ ID NO: 401)
5':CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCC
CCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAA
CCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCC
CAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATT
TATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGT
AGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3'
[1702] With this GAS promoter element linked to the SV40 promoter,
a GAS:SEAP2 reporter construct is next engineered. Here, the
reporter molecule is a secreted alkaline phosphatase, or "SEAP."
Clearly, however, any reporter molecule can be instead of SEAP, in
this or in any of the other Examples. Well known reporter molecules
that can be used instead of SEAP include chloramphenicol
acetyltransferase (CAT), luciferase, alkaline phosphatase,
B-galactosidase, green fluorescent protein (GFP), or any protein
detectable by an antibody.
[1703] The above sequence confirmed synthetic GAS-SV40 promoter
element is subcloned into the pSEAP-Promoter vector obtained from
Clontech using HindIII and XhoI, effectively replacing the SV40
promoter with the amplified GAS:SV40 promoter element, to create
the GAS-SEAP vector. However, this vector does not contain a
neomycin resistance gene, and therefore, is not preferred for
mammalian expression systems.
[1704] Thus, in order to generate mammalian stable cell lines
expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed
from the GAS-SEAP vector using SalI and NotI, and inserted into a
backbone vector containing the neomycin resistance gene, such as
pGFP-1 (Clontech), using these restriction sites in the multiple
cloning site, to create the GAS-SEAP/Neo vector. Once this vector
is transfected into mammalian cells, this vector can then be used
as a reporter molecule for GAS binding as described in Examples
62-64.
[1705] Other constructs can be made using the above description and
replacing GAS with a different promoter sequence. For example,
construction of reporter molecules containing EGR and NF-KB
promoter sequences are described in Examples 62-66. However, many
other promoters can be substituted using the protocols described in
these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin
promoters can be substituted, alone or in combination (e.g.,
GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, or NF-KB/GAS). Similarly,
other cell lines can be used to test reporter construct activity,
such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell),
Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.
Example 60
Assay for SEAP Activity
[1706] As a reporter molecule for the assays described in examples
disclosed herein, SEAP activity is assayed using the Tropix
Phospho-light Kit (Cat. BP-400) according to the following general
procedure. The Tropix Phospho-light Kit supplies the Dilution,
Assay, and Reaction Buffers used below.
[1707] Prime a dispenser with the 2.5.times. Dilution Buffer and
dispense 15 ul of 2.5.times. dilution buffer into Optiplates
containing 35 ul of a solution containing an albumin fusion protein
of the invention. Seal the plates with a plastic sealer and
incubate at 65 degree C. for 30 min. Separate the Optipiates to
avoid uneven heating.
[1708] Cool the samples to room temperature for 15 minutes. Empty
the dispenser and prime with the Assay Buffer. Add 50 ml Assay
Buffer and incubate at room temperature 5 min. Empty the dispenser
and prime with the Reaction Buffer (see the Table below). Add 50
III Reaction Buffer and incubate at room temperature for 20
minutes. Since the intensity of the chemiluminescent signal is time
dependent, and it takes about 10 minutes to read 5 plates on a
luminometer, thus one should treat 5 plates at each time and start
the second set 10 minutes later.
[1709] Read the relative light unit in the luminometer. Set H12 as
blank, and print the results. An increase in chemiluminescence
indicates reporter activity.
TABLE-US-00028 TABLE 5 Rxn buffer diluent # of plates (ml) CSPD
(ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16
90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22
120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28
150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34
180 9 35 185 9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40
210 10.5 41 215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235
11.75 46 240 12 47 245 12.25 48 250 12.5 49 255 12.75 50 260 13
Example 61
Assay Identifying Neuronal Activity
[1710] When cells undergo differentiation and proliferation, a
group of genes are activated through many different signal
transduction pathways. One of these genes, EGR1 (early growth
response gene 1), is induced in various tissues and cell types upon
activation. The promoter of EGR 1 is responsible for such
induction. Using the EGR 1 promoter linked to reporter molecules,
the ability of fusion proteins of the invention to activate cells
can be assessed.
[1711] Particularly, the following protocol is used to assess
neuronal activity in PC12 cell lines. PC12 cells (rat
phenochromocytoma cells) are known to proliferate and/or
differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF
(epidermal growth factor). The EGR1 gene expression is activated
during this treatment. Thus, by stably transfecting PC12 cells with
a construct containing an EGR promoter linked to SEAP reporter,
activation of PC12 cells by an albumin fusion protein of the
present invention can be assessed.
[1712] The EGR/SEAP reporter construct can be assembled by the
following protocol. The EGR-1 promoter sequence (-633 to +1)
(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified
from human genomic DNA using the following primers:
TABLE-US-00029 First primer: (SEQ ID NO: 402) 5'
GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3' Second primer: (SEQ ID NO: 403)
5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3'
[1713] Using the GAS:SEAP/Neo vector produced in Example 59, EGR1
amplified product can then be inserted into this vector. Linearize
the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII,
removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product
with these same enzymes. Ligate the vector and the EGR 1
promoter.
[1714] To prepare 96 well-plates for cell culture, two mls of a
coating solution (1:30 dilution of collagen type I (Upstate Biotech
Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per
one 10 cm plate or 50 ml per well of the 96-well plate, and allowed
to air dry for 2 hr.
[1715] PC12 cells are routinely grown in RPMI-1640 medium (Bio
Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat.
#12449-78P), 5% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin and 100 ug/ml
streptomycin on a precoated 10 cm tissue culture dish. One to four
split is done every three to four days. Cells are removed front the
plates by scraping and resuspended with pipetting up and down for
more than 15 times.
[1716] Transfect the EGR/SEAP/Neo construct into PC12 using
techniques known in the art. EGR-SEAP/PC12 stable cells are
obtained by growing the cells in 390 ug/ml G418. The G418-free
medium is used for routine growth but every one to two months, the
cells should be re-grown in 300 ug/ml G418 for couple of
passages.
[1717] To assay for neuronal activity, a 10 cm plate with cells
around 70 to 80% confluent is screened by removing the old medium.
Wash the cells once with PBS (Phosphate buffered saline). Then
starve the cells in low serum medium (RPMI-1640 containing 1% horse
serum and 0.5% FBS with antibiotics) overnight.
[1718] The next morning, remove the medium and wash the cells with
PBS. Scrape off the cells from the plate, suspend the cells well in
2 ml low serum medium. Count the cell number and add more low serum
medium to reach final cell density as 5.times.10.sup.5
cells/ml.
[1719] Add 200 ul of the cell suspension to each well of 96-well
plate (equivalent to 1.times.10.sup.5 cells/well). Add a series of
different concentrations of an albumin fusion protein of the
invention, 37 degree C. for 48 to 72 hr. As a positive control, a
growth factor known to activate PC12 cells through EGR can be used,
such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold
induction of SEAP is typically seen in the positive control wells.
SEAP assay may be routinely performed using techniques known in the
art and/or as described in Example 60.
Example 62
Assay for T-cell Activity
[1720] The following protocol is used to assess T-cell activity by
identifying factors, and determining whether an albumin fusion
protein of the invention proliferates and/or differentiates
T-cells. T-cell activity is assessed using the GAS/SEAP/Neo
construct produced in Example 59. Thus, factors that increase SLAP
activity indicate the ability to activate the Jaks-STATS signal
transduction pathway. The T-cell used in this assay is Jurkat
T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC
Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No.
CRL-1582) cells can also be used.
[1721] Jurkat T-cells are lymphoblastic CO.sub.4+ Th1 helper cells.
In order to generate stable cell lines, approximately 2 million
Jurkat cells are transfected with the GAS-SLAP/neo vector using
DMRIE-C (Life Technologies) (transfection procedure described
below). The transfected cells are seeded to a density of
approximately 20,000 cells per well and transfectants resistant to
1 mg/ml genticin selected. Resistant colonies are expanded and then
tested for their response to increasing concentrations of
interferon gamma. The dose response of a selected clone is
demonstrated.
[1722] Specifically, the following protocol will yield sufficient
cells for 75 wells containing 200 ul of cells. Thus, it is either
scaled up, or performed in multiple to generate sufficient cells
for multiple 96 well plates. Jurkat cells are maintained in
RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life
Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml
OPTI-MEM containing 50 ul of DMRIE-C and incubate at room
temperature for 15-45 mins.
[1723] During the incubation period, count cell concentration, spin
down the required number of cells (10.sup.7 per transfection), and
resuspend in OPTI-MEM to a final concentration of 10.sup.7
cells/ml. Then add 1 ml of 1.times.10.sup.7 cells in OPTI-MEM to
T25 flask and incubate at 37 degree C. for 6 hrs. After the
incubation, add 10 ml of RPMI+15% serum.
[1724] The Jurkat:GAS-SEAP stable reporter lines are maintained in
RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are
treated with varying concentrations of one or more fusion proteins
of the present invention.
[1725] On the day of treatment with the fusion protein, the cells
should be washed and resuspended in fresh RPMI+10% serum to a
density of 500,000 cells per ml. The exact number of cells required
will depend on the number of fusion proteins and the number of
different concentrations of fusion proteins being screened. For one
96 well plate, approximately 10 million cells (for 10 plates, 100
million cells) are required.
[1726] The well dishes containing Jurkat cells treated with the
fusion protein are placed in an incubator for 48 hrs (note: this
time is variable between 48-72 hrs). 35 ul samples from each well
are then transferred to an opaque 96 well plate using a 12 channel
pipette. The opaque plates should be covered (using sellophene
covers) and stored at -20 degree C. until SEAP assays are performed
according to Example 60. The plates containing the remaining
treated cells are placed at 4 degree C. and serve as a source of
material for repeating the assay on a specific well if desired.
[1727] As a positive control, 100 Unit/ml interferon gamma can be
used which is known to activate Jurkat T cells. Over 30 fold
induction is typically observed in the positive control wells.
[1728] The above protocol may be used in the generation of both
transient, as well as, stable transfected cells, which would be
apparent to those of skill in the art.
Example 63
Assay for T-Cell Activity
[1729] NF-KB (Nuclear Factor KB) is a transcription factor
activated by a wide variety of agents including the inflammatory
cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and
lymphotoxin-beta, by exposure to LPS or thrombin, and by expression
of certain viral gene products. As a transcription factor, NF-KB
regulates the expression of genes involved in immune cell
activation, control of apoptosis (NF-KB appears to shield cells
from apoptosis), B and T-cell development, anti-viral and
antimicrobial responses, and multiple stress responses.
[1730] In non-stimulated conditions, NF-KB is retained in the
cytoplasm with t-KB (Inhibitor KB). However, upon stimulation, I-KB
is phosphorylated and degraded, causing NF-KB to shuttle to the
nucleus, thereby activating transcription of target genes. Target
genes activated by NF-KB include GM-CSF, ICAM-1 and class I
MHC.
[1731] Due to its central role and ability to respond to a range of
stimuli, reporter constructs utilizing the NF-KB promoter element
are used to screen the fusion protein. Activators or inhibitors of
NF-KB would be useful in treating, preventing, and/or diagnosing
diseases. For example, inhibitors of NF-KB could be used to treat
those diseases related to the acute or chronic activation of NF-KB,
such as rheumatoid arthritis.
[1732] To construct a vector containing the NF-KB promoter element,
a PCR based strategy is employed. The upstream primer contains four
tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID
NO:404), 18 bp of sequence complementary to the 5' end of the SV40
early promoter sequence, and is flanked with an XhoI site:
TABLE-US-00030 (SEQ ID NO: 405)
5':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACT
TTCCGGGACTTTCCATCCTGCCATCTCAATTAG:3'
[1733] The downstream primer is complementary to the 3' end of the
SV40 promoter and is flanked with a Hind III site:
5':GCGGCAAGCTTTTTCAAAGCCTAGGC:3' (SEQ ID NO: 400)
[1734] PCR amplification is performed using the SV40 promoter
template present in the pB-gal:promoter plasmid obtained from
Clontech. The resulting PCR fragment is digested with XhoI and Hind
III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7
and T3 primers confirms the insert contains the following
sequence:
TABLE-US-00031 (SEQ ID NO: 406)
5':CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGA
CTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCC
TAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTC
TCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG
GCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTT
TTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3'
[1735] Next, replace the SV40 minimal promoter element present in
the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40
fragment using XhoI and HindIII. However, this vector does not
contain a neomycin resistance gene, and therefore, is not preferred
for mammalian expression systems.
[1736] In order to generate stable mammalian cell lines, the
NF-KB/SV40/SEAP cassette is removed front the above NF-KB/SEAP
vector using restriction enzymes SalI and NotI, and inserted into a
vector containing neomycin resistance. Particularly, the
NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech),
replacing the OFF gene, after restricting pGFP-1 with Sail and
NotI.
[1737] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat
T-cells are created and maintained according to the protocol
described in Example 60. Similarly, the method for assaying fusion
proteins with these stable Jurkat T-cells is also described in
Example 60. As a positive control, exogenous TNF alpha (0.1, 1, 10
ng) is added to wells H9, H10, and H11, with a 5-10 fold activation
typically observed.
Example 641
Assay Identifying Myeloid Activity
[1738] The following protocol is used to assess myeloid activity of
an albumin fusion protein of the present invention by determining
whether the fusion protein proliferates and/or differentiates
myeloid cells. Myeloid cell activity is assessed using the
GAS/SEAP/Neo construct produced in Example 59. Thus, factors that
increase SLAP activity indicate the ability to activate the
Jaks-STATS signal transduction pathway. The myeloid cell used in
this assay is U937, a pre-monocyte cell line, although TF-1, HL60,
or KG1 can be used.
[1739] To transiently transfect U937 cells with the GAS/SEAP/Neo
construct produced in Example 59, a DEAE-Dextran method (Kharbanda
et, al 1994, Cell Growth & Differentiation, 5:259-265) is used.
First, harvest 2.times.10.sup.7 U937 cells and wash with PBS. The
U937 cells are usually grown in RPMI 1640 medium containing 10%
heat-inactivated fetal bovine serum (HIS) supplemented with 100
units/ml penicillin and 100 mg/ml streptomycin.
[1740] Next, suspend the cells in 1 ml of 20 mM Tris.HCl (pH 7.4)
buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid
DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na.sub.2HPO.sub.4.7H.sub.2O, 1
mM MgCl.sub.2, and 675 uM CaCl.sub.2. Incubate at 37 degrees C. for
45 min.
[1741] Wash the cells with RPMI 1640 medium containing 10% FBS and
then resuspend in 10 ml complete medium and incubate at 37 degree
C. for 36 hr.
[1742] The GAS-SEAP/U937 stable cells are obtained by growing the
cells in 400 ug/ml G418. The G418-free medium is used for routine
growth but every one to two months, the cells should be re-grown in
400 ug/ml G418 for couple of passages.
[1743] These cells are tested by harvesting 1.times.10.sup.8 cells
(this is enough for ten 96-well plates assay) and wash with PBS.
Suspend the cells in 200 ml above described growth medium, with a
final density of 5.times.10.sup.5 cells/ml. Plate 200 ul cells per
well in the 96-well plate (or 1.times.10.sup.5 cells/well).
[1744] Add different concentrations of the fusion protein. Incubate
at 37 degree C for 48 to 72 hr. As a positive control, 100 Unit/ml
interferon gamma can be used which is known to activate U937 cells.
Over 30 fold induction is typically observed in the positive
control wells. SEAP assay the supernatant according to methods
known in the art and/or the protocol described in Example 60.
Example 65
Assay Identifying Changes in Small Molecule Concentration and
Membrane Permeability
[1745] Binding of a ligand to a receptor is known to alter
intracellular levels of small molecules, such as calcium,
potassium, sodium, and pH, as well as alter membrane potential.
These alterations can be measured in an assay to identify fusion
proteins which bind to receptors of a particular cell. Although the
following protocol describes an assay for calcium, this protocol
can easily be modified to detect changes in potassium, sodium, pH,
membrane potential, or any other small molecule which is detectable
by a fluorescent probe.
[1746] The following assay uses Fluorometric Imaging Plate Reader
("FLIPR") to measure changes in fluorescent molecules (Molecular
Probes) that bind small molecules. Clearly, any fluorescent
molecule detecting a small molecule can be used instead of the
calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.;
catalog no. F-14202), used here.
[1747] For adherent cells, seed the cells at 10,000-20,000
cells/well in a Co-star black 96-well plate with cleat bottom. The
plate is incubated in a CO.sub.2 incubator for 20 hours. The
adherent cells are washed two times in Biotek washer with 200 ul of
HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after
the final wash.
[1748] A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic
acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4
is added to each well. The plate is incubated at 37 degrees C. in a
CO.sub.2 incubator for 60 min. The plate is washed four times in
the Biota washer with HBSS leaving 100 ul of buffer.
[1749] For non-adherent cells, the cells are spun down from culture
media. Cells are re-suspended to 2-5.times.10.sup.6 cells/ml with
HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in
10% pluronic acid DMSO is added to each ml of cell suspension. The
tube is then placed in a 37 degrees C. water bath far 30-60 min.
The cells are washed twice with HBSS, resuspended to
1.times.10.sup.6 cells/ml, and dispensed into a microplate, 100
ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate
is then washed once in Denley Cell Wash with 200 ul, followed by an
aspiration step to 100 ul final volume.
[1750] For a non-cell based assay, each well contains a fluorescent
molecule, such as fluo-4. The fusion protein of the invention is
added to the well, and a change in fluorescence is detected.
[1751] To measure the fluorescence of intracellular calcium, the
FL/PR is set for the following parameters: (1) System gain is
300-100 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is
F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6)
Sample addition is 50 ul. Increased emission at 530 nm indicates an
extracellular signaling event caused by an albumin fusion protein
of the present invention or a molecule induced by an albumin fusion
protein of the present invention, which has resulted in an increase
in the intracellular Ca.sup.++ concentration.
Example 66
Assay Identifying Tyrosine Kinase Activity
[1752] The Protein Tyrosine Kinases (PTK) represent a diverse group
of transmembrane and cytoplasmic kinases. Within the Receptor
Protein Tyrosine Kinase (RPTK) group are receptors for a range of
mitogenic and metabolic growth factors including the PDGF, FGF,
EGF, NGF, HGF and Insulin receptor subfamilies. In addition there
are a large family of RPTKs for which the corresponding ligand is
unknown. Uganda for RPTKs include mainly secreted small proteins,
but also membrane-bound and extracellular matrix proteins.
[1753] Activation of RPTK by ligands involves ligand-mediated
receptor dimerization, resulting in transphosphorylation of the
receptor subunits and activation of the cytoplasmic tyrosine
kinases. The cytoplasmic tyrosine kinases include receptor
associated tyrosine kinases of the src-family (e.g., src, yes, lck,
lyn, fyn) and non-receptor linked and cytosotic protein tyrosine
kinases, such as the Jak family, members of which mediate signal
transduction triggered by the cytokine superfamily of receptors
(e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
[1754] Because of the wide range of known factors capable of
stimulating tyrosine kinase activity, identifying whether an
albumin fusion protein of the present invention or a molecule
induced by a fusion protein of the present invention is capable of
activating tyrosine kinase signal transduction pathways is of
interest. Therefore, the following protocol is designed to identify
such molecules capable of activating the tyrosine kinase signal
transduction pathways.
[1755] Seed target cells (e.g., primary keratinocytes) at a density
of approximately 25,000 cells per well in a 96 well Loprodyne
Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.).
The plates are sterilized with two 30 minute rinses with 100%
ethanol, rinsed with water and dried overnight. Some plates are
coated for 2 hr with 100 ml of cell culture grade type I collagen
(50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can
be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel
purchased from Becton Dickinson (Bedford, Mass.), or calf serum,
rinsed with PBS and stored at 4 degree C. Cell growth on these
plates is assayed by seeding 5,000 cells/well in growth medium and
indirect quantitation of cell number through use of alamarBlue as
described by the manufacturer Atamar Biosciences, Inc. (Sacramento,
Calif.) after 48 hr. Falcon plate covers #3071 from Becton
Dickinson (Bedford, Mass.) are used to cover the Loprodyne Silent
Screen Plates. Falcon Microtest III cell culture plates can also be
used in some proliferation experiments.
[1756] To prepare extracts, A431 cells are seeded onto the nylon
membranes of Loprodyne plates (20,000/200 ml/well) and cultured
overnight in complete medium. Cells are quiesced by incubation in
serum-free basal medium for 24 hr. After 5-20 minutes treatment
with EGF (60 ng/ml) or a different concentrations of an albumin
fusion protein of the invention, the medium was removed and 100 ml
of exuaction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton
X-100, 0.1% SOS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of
protease inhibitors (#1836170) obtained from Boeheringer Mannheim
(Indianapolis, Ind.)) is added to each well and the plate is shaken
on a rotating shaker for 5 minutes at 4.degree. C. The plate is
then placed in a vacuum transfer manifold and the extract filtered
through the 0.45 min membrane bottoms of each well using house
vacuum, Extracts are collected in a 96-well catch/assay plate in
the bottom of the vacuum manifold and immediately placed on ice. To
obtain extracts clarified by centrifugation, the content of each
well, after detergent solubilization for 5 minutes, is removed and
centrifuged for 15 minutes at 4 degree C. at 16,000.times.g.
[1757] Test the filtered extracts for levels of tyrosine kinase
activity. Although many methods of detecting tyrosine kinase
activity are known, one method is described here.
[1758] Generally, the tyrosine kinase activity of an albumin fusion
protein of the invention is evaluated by determining its ability to
phosphorylate a tyrosine residue on a specific substrate (a
biotinylated peptide). Biotinylated peptides that can be used for
this purpose include PSK1 (corresponding to amino acids 6-20 of the
cell division kinase cdc2-p34) and PSK2 (corresponding to amino
acids 1-17 of gastrin). Both peptides are substrates for a range of
tyrosine kinases and are available from Boehringer Mannheim.
[1759] The tyrosine kinase reaction is set up by adding the
following components in order. First, add 10 ul of 5 uM
Biotinylated Peptide, then 10 ul ATP/Mg.sub.2+ (5 mM ATP/50 mM
MgCl.sub.2), then 10 ul of 5.times. Assay Buffer (40 mM imidazole
hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100
mM MgCl.sub.2, 5 mM MnCl.sub.2, 0.5 mg/ml BSA), then 5 ul of Sodium
Vanadate (1 mM), and then 5 ul of water. Mix the components gently
and preincubate the reaction mix at 30 degree C. for 2 min. Initial
the reaction by adding 10 ul of the control enzyme or the filtered
supernatant.
[1760] The tyrosine kinase assay reaction is then terminated by
adding 10 ul of 120 mm EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 ul
aliquot of reaction mixture to a microtiter plate (MTP) module and
incubating at 37 degree C. for 20 min. This allows the streptavidin
coated 96 well plate to associate with the biotinylated peptide.
Wash the MTP module with 300 ul/well of PBS four times. Next add 75
ul of anti-phosphotyrosine antibody conjugated to horse radish
peroxidase (anti-P-Tyr-POD (0.5 u/ml)) to each well and incubate at
37 degree C. for one hour. Wash the well as above.
[1761] Next add 100 .mu.l of peroxidase substrate solution
(Boehringer Mannheim) and incubate at room temperature for at least
5 mins (up to 30 min). Measure the absorbance of the sample at 405
nm by using ELISA reader. The level of bound peroxidase activity is
quantitated using an ELISA reader and reflects the level of
tyrosine kinase activity,
Example 67
Assay Identifying Phosphorylation Activity
[1762] As a potential alternative and/or complement to the assay of
protein tyrosine kinase activity described in Example 66, an assay
which detects activation (phosphorylation) of major intracellular
signal transduction intermediates can also be used. For example, as
described below one particular assay can detect tyrosine
phosphorylation of the Erk-1 and Erk-2 kinases. However,
phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map
kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase
(MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,
phosphotyrosine, or phosphothreonine molecule, can be detected by
substituting these molecules for Erk-1 or Erk-2 in the following
assay.
[1763] Specifically, assay plates are made by coating the wells of
a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr
at room temp, (RT). The plates are then rinsed with PBS and blocked
with 3% BSA/PBS for 1 hr at RT. The protein G plates are then
treated with 2 commercial monoclonal antibodies (100 ng/well)
against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology).
(To detect other molecules, this step can easily be modified by
substituting a monoclonal antibody detecting any of the above
described molecules.) After 3-5 rinses with PBS, the plates are
stored at 4 degree C. until use.
[1764] A431 cells are seeded at 20,000/well in a 96-well Loprodyne
filterplate and cultured overnight in growth medium. The cells are
then starved for 48 hr in basal medium (DMEM) and then treated with
EGF (6 ng/well) or varying concentrations of the fusion protein of
the invention for 5-20 minutes. The cells are then solubilized and
extracts filtered directly into the assay plate.
[1765] After incubation with the extract for 1 hr at RT, the wells
are again rinsed. As a positive control, a commercial preparation
of MAP kinase (10 ng/well) is used in place of A431 extract. Plates
are then treated with a commercial polyclonal (rabbit) antibody (1
ug/ml) which specifically recognizes the phosphorylated epitope of
the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is
biotinylated by standard procedures. The bound polyclonal antibody
is then quantitated by successive incubations with
Europium-streptavidin and Europium fluorescence enhancing reagent
in the Wallac DELFIA instrument (time-resolved fluorescence). An
increased fluorescent signal over background indicates a
phosphorylation by the fusion protein of the present invention or a
molecule induced by an albumin fusion protein of the present
invention.
Example 68
Assay for the Stimulation of Bone Marrow CD34+ Cell
Proliferation
[1766] This assay is based on the ability of human CD34+ to
proliferate in the presence of hematopoietic growth factors and
evaluates the ability of fusion proteins of the invention to
stimulate proliferation of CD34+ cells.
[1767] It has been previously shown that most mature precursors
will respond to only a single signal. More immature precursors
require at least two signals to respond. Therefore, to test the
effect of fusion proteins of the invention on hematopoietic
activity of a wide range of progenitor cells, the assay contains a
given fusion protein of the invention in the presence or absence of
hematopoietic growth factors. Isolated cells are cultured for 5
days in the presence of Stem Cell Factor (SCF) in combination with
tested sample. SCF alone has a very limited effect on the
proliferation of bone marrow (BM) cells, acting in such conditions
only as a "survival" factor. However, combined with any factor
exhibiting stimulatory effect on these cells (e.g., IL-3), SCF will
cause a synergistic effect. Therefore, if the tested fusion protein
has a stimulatory effect on hematopoietic progenitors, such
activity can be easily detected. Since normal BM cells have a low
level of cycling cells, it is likely that any inhibitory effect of
a given fusion protein might not be detected. Accordingly, assays
for an inhibitory effect on progenitors is preferably tested in
cells that are first subjected to in vitro stimulation with
SCF+IL+3, and then contacted with the compound that is being
evaluated for inhibition of such induced proliferation.
[1768] Briefly, CD34+ cells are isolated using methods known in the
art. The cells are thawed and resuspended in medium (QBSF 60
serum-free medium with 1% L-glutamine (500 ml) Quality Biological,
Inc., Gaithersburg, Md. Cain 160-204-101). After several gentle
centrifugation steps at 200.times.g, cells are allowed to rest for
one hour. The cell count is adjusted to 2.5.times.10.sup.5
cells/ml. During this time, 100 .mu.l of sterile water is added to
the peripheral wells of a 96-well plate. The cytokines that can be
tested with an albumin fusion protein of the invention in this
assay is rhSCF (R&D Systems, Minneapolis, Minn., Cat#255-SC) at
50 ng/ml atone and in combination with rhSCF and rhIL-3 (R&D
Systems, Minneapolis, Minn., Cat#203-ML) at 30 ng/ml. After one
hour, 10 .mu.l of prepared cytokines, varying concentrations of an
albumin fusion protein of the invention, and 20 .mu.l of diluted
cells are added to the media which is already present in the wells
to allow for a final total volume of 100. The plates are then
placed in a 37.degree. C./5% CO.sub.2 incubator for five days.
[1769] Eighteen hours before the assay is harvested, 0.5
.mu.Ci/well of [3H] Thymidine is added in a 10 .mu.l volume to each
well to determine the proliferation rate. The experiment is
terminated by harvesting the cells from each 96-well plate to a
filtermat using the Tomtec Harvester 96. After harvesting, the
filtermats are dried, trimmed and placed into OmniFilter assemblies
consisting of one OmniFilter plate and one OmniFilter Tray. 60
.mu.l Microscint is added to each well and the plate sealed with
TopScal-A press-on sealing film A bar code 15 sticker is affixed to
the first plate for counting. The sealed plates are then loaded and
the level of radioactivity determined via the Packard Top Count and
the printed data collected for analysis. The level of radioactivity
reflects the amount of cell proliferation.
[1770] The studies described n this example test the activity of a
given fusion protein to stimulate bone marrow CD34+ cell
proliferation. One skilled in the art could easily modify the
exemplified studies to test the activity of fusion proteins and
polynucleotides of the invention (e.g., gene therapy) as well as
agonists and antagonists thereof. The ability of an albumin fusion
protein of the invention to stimulate the proliferation of bone
marrow CD34+ cells indicates that the albumin fusion protein and/or
polynucleotides corresponding to the fusion protein are useful for
the diagnosis and treatment of disorders affecting the immune
system and hematopoiesis. Representative uses are described in the
"Immune Activity" and "Infectious Disease" sections above, and
elsewhere herein.
Example 69
Assay for Extracellular Matrix Enhanced Cell Response (EMECR)
[1771] The objective of the Extracellular Matrix Enhanced Cell
Response (EMECR) assay is to evaluate the ability of fusion
proteins of the invention to act on hematopoietic stem cells in the
context of the extracellular matrix (ECM) induced signal.
[1772] Cells respond to the regulatory factors in the context of
signal(s) received from the surrounding microenvironment. For
example, fibroblasts, and endothelial and epithelial stem cells
tail to replicate in the absence of signals from the ECM.
Hematopoietic stem cells can undergo self-renewal in the bone
marrow, but not in in vitro suspension culture. The ability of
stein cells to undergo self-renewal in vitro is dependent upon
theft interaction with the stromal cells and the ECM protein
fibronectin (fn). Adhesion of cells to fn is mediated by the
.alpha..sub.5..beta..sub.1 and .alpha..sub.4..beta..sub.1 integrin
receptors, which are expressed by human and mouse hematopoietic
stem cells. The factor(s) which integrate with the ECM environment
and are responsible for stimulating stem cell self-renewal have a
not yet been identified. Discovery of such factors should be of
great interest in gene therapy and bone marrow transplant
applications
[1773] Briefly, polystyrene, non tissue culture treated, 96-well
plates are coated with in fragment at a coating concentration of
0.2 .mu.g/cm.sup.2. Mouse bone marrow cells are plated (1,000
cells/well) in 0.2 ml of serum-free medium. Cells cultured in the
presence of IL-3 (5 ng/ml)+SCF (50 ng/ml) would serve as the
positive control, conditions under which little self-renewal but
pronounced differentiation of the stem cells is to be expected.
Albumin fusion proteins of the invention are tested with
appropriate negative controls in the presence and absence of SCF
(5.0 ng/ml), where volume of the administed composition containing
the albumin fusion protein of the invention represents 10% of the
total assay volume. The plated cells are then allowed to grow by
incubating in a low oxygen environment (5% CO.sub.2, 7% O.sub.2,
and 88% N.sub.2) tissue culture incubator for 7 days. The number of
proliferating cells within the wells is then quantitated by
measuring thymidine incorporation into cellular DNA. Verification
of the positive hits in the assay will require phenotypic
characterization of the cells, which Can be accomplished by scaling
up of the culture system and using appropriate antibody reagents
against cell surface antigens and FACScan.
[1774] If a particular fusion protein of the present invention is
found to be a stimulator of hematopoietic progenitors, the fusion
protein and polynucleotides corresponding to the fusion protein may
be useful for example, in the diagnosis and treatment of disorders
affecting the immune system and hematopoiesis. Representative uses
are described in the "Immune Activity" and "Infectious Disease"
sections above, and elsewhere herein. The fusion protein may also
be useful in the expansion of stem cells and committed progenitors
of various blood lineages, and in the differentiation and/or
proliferation of various cell types.
[1775] Additionally, the albumin fusion proteins of the invention
and polynucleotides encoding albumin fusion proteins of the
invention, may also be employed to inhibit the proliferation and
differentiation of hematopoietic cells and therefore may be
employed to protect bone marrow stem cells from chemotherapeutic
agents during chemotherapy. This antiproliferative effect may allow
administration of higher doses of chemotherapeutic agents and,
therefore, more effective chemotherapeutic treatment.
[1776] Moreover, fusion proteins of the invention and
polynucleotides encoding albumin fusion proteins of the invention
may also be useful for the treatment and diagnosis of hematopoietic
related disorders such as, anemia, pancytopenia, leukopenia,
thrombocytopenia or leukemia, since stromal cells are important in
the production of cells of hematopoietic lineages. The uses include
bone marrow cell ex-vivo culture, bone marrow transplantation, bone
marrow reconstitution, radiotherapy or chemotherapy of
neoplasia.
Example 70
Human Dermal Fibroblast and Aortic Smooth Muscle Cell
Proliferation
[1777] An albumin fusion protein of the invention is added to
cultures of normal human dermal fibroblasts (NHDF) and human aortic
smooth muscle cells (AoSMC) and two co-assays are performed with
each sample. The first assay examines the effect of the fusion
protein on the proliferation of normal human dermal fibroblasts
(NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of
fibroblasts or smooth muscle cells is a part of several
pathological processes, including fibrosis, and restenosis. The
second assay examines IL6 production by both NHDF and SMC. IL6
production is art indication of functional activation. Activated
cells will have increased production of a number of cytokines and
other factors, which can result in a proinflammatory or
immunomodulatory outcome. Assays are run with and without co-TNFa
stimulation, in order to check for costimulatory or inhibitory
activity.
[1778] Briefly, on day 1, 96-well black plates are set up with 1000
cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 .mu.l culture
media. NHDF culture media contains: Clonetics FB basal media, 1
mg/ml hFGF, 5 mg/ml insulin, 50 mg/ml gentamycin, 2% FBS, while
AoSMC culture media contains Genetics SM basal media, 0.5 .mu.g/ml
hFGF, 5 mg/ml insulin, 1 .mu.g/ml hFGF, 50 mg/ml gentamycin, 50
.mu.g/ml Amphotericin B, 5% FBS. After incubation at 37.degree. C.
for at least 4-5 hours culture media is aspirated and replaced with
growth arrest media. Growth arrest media for NHDF contains
fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth
arrest media for AoSMC contains SM basal media, 50 mg/ml
gentamycin, 50 .mu.g/ml Amphotericin II, 0.4% FBS. Incubate at
37.degree. C. until day 2.
[1779] On day 2, serial dilutions and templates of an albumin
fusion protein of the invention are designed such that they always
include media controls and known-protein controls. For both
stimulation and inhibition experiments, proteins are diluted in
growth arrest media. For inhibition experiments. TNFa is added to a
final concentration of 2 ng/ml (NHDF) or 5 ng/ml (AoSMC). Add 1/3
vol media containing controls or an albumin fusion protein of the
invention and incubate at 37 degrees C./5% CO.sub.2 until day
5.
[1780] Transfer 60 .mu.l from each well to another labeled 96-well
plate, cover with a plate-sealer, and store at 4 degrees C. until
Day 6 (for IL6 ELISA). To the remaining 100 .mu.l in the cell
culture plate, aseptically add Alamar Blue in an amount equal to
10% of the culture volume (10 .mu.l). Return plates to incubator
for 3 to 4 hours. Then measure fluorescence with excitation at 530
nm and emission at 590 nm using the CytoFluor. This yields the
growth stimulation/inhibition data.
[1781] On day 5, the IL6 ELISA is performed by coating a 96 well
plate with 50-100 ul/well of Anti-Human IL6 Monoclonal antibody
diluted in PBS, pH 7.4, incubate ON at room temperature.
[1782] On day 6, empty the plates into the sink and blot on paper
towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the
plates with 200 .mu.l/well of Pierce Super Block blocking buffer in
PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05%
Tween-20). Blot plates on paper towels. Then add 50 .mu.l/well of
diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50
mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0
ng/ml). Add duplicate samples to top row of plate. Cover the plates
and incubate for 2 hours at RT on shaker.
[1783] Plates are washed with wash buffer and blotted on paper
towels, Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and
add 100 .mu.l/well. Cover the plate and incubate 1 h at RT. Plates
are again washed with wash buffer and blotted on paper towels.
[1784] Add 100 .mu.l/well of Enhancement Solution. Shake for 5
minutes. Read the plate on the Wallac DELFIA Fluorometer. Readings
from triplicate samples in each assay were tabulated and
averaged.
[1785] A positive result in this assay suggests AoSMC cell
proliferation and that the albumin fusion protein may be involved
in dermal fibroblast proliferation and/or smooth muscle cell
proliferation. A positive result also suggests many potential uses
of the fusion protein and polynucleotides encoding the albumin
fusion protein. For example, inflammation and immune responses,
wound healing, and angiogenesis, as detailed throughout this
specification. Particularly, fusion proteins may be used in wound
healing and dermal regeneration, as well as the promotion of
vasculogenesis, both of the blood vessels and lymphatics. The
growth of vessels can be used in the treatment of, for example,
cardiovascular diseases. Additionally, fusion proteins showing
antagonistic activity in this assay may be useful in treating
diseases, disorders, and/or conditions which involve angiogenesis
by acting as an anti-vascular agent (e.g., anti-angiogenesis).
These diseases, disorders, and/or conditions are known in the art
and/or are described herein, such as, for example, malignancies,
solid tumors, benign tumors, for example hemangiomas, acoustic
neuromas, neurofibromas, trachomas, and pyogenic granulomas;
artheroscleric plaques; ocular angiogenic diseases, for example,
diabetic retinopathy, retinopathy of prematurity, macular
degeneration, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and
Pterygia (abnormal blood vessel growth) of the eye; rheumatoid
arthritis; psoriasis; delayed wound healing; endometriosis;
vasculogenesis; granulations; hypertrophic scars (keloids);
nonunion fractures; scleroderma; trachoma; vascular adhesions;
myocardial angiogenesis; corollary collaterals; cerebral
collaterals; arteriovenous malformations; ischemic limb
angiogenesis; Osier-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular
dysplasia; wound granulation; Crohn's disease; and atherosclerosis.
Moreover, albumin fusion proteins that act as antagonists in this
assay may be useful in treating anti-hyperproliferative diseases
and/or anti inflammatory known in the art and/or described
herein.
Example 71
Cellular Adhesion Molecule (CAM) Expression on Endothelial
Cells
[1786] The recruitment of lymphocytes to areas of inflammation and
angiogenesis involves specific receptor-ligand interactions between
cell surface adhesion molecules (CAMs) on lymphocytes and the
vascular endothelium. The adhesion process, in both normal and
pathological settings, follows a multi-step cascade that involves
intercellular adhesion molecule-1 (VCAM-1), vascular cell adhesion
molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1
(E-selectin) expression on endothelial cells (EC). The expression
of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the
local vasculature and extravasate into the local tissue during the
development of an inflammatory response. The local concentration of
cytokines and growth factor participate in the modulation of the
expression of these CAMs.
[1787] Briefly, endothelial cells (e.g. Human Umbilical Vein
Endothelial cells (HUVECs)) are grown in a standard 96 well plate
to confluence, growth medium is removed from the cells and replaced
with 100 .mu.l of 199 Medium (10% fetal bovine serum (PBS)).
Samples for testing (containing an albumin fusion protein of the
invention) and positive or negative controls are added to the plate
in triplicate (in 10 .mu.l volumes). Plates are then incubated at
37.degree. C. for either 5 h (selectin and integrin expression) or
24 h (integrin expression only). Plates are aspirated to remove
medium and 100 .mu.l of 0.1% paraformaldehyde-PBS(with Ca++ and
Mg++) is added to each well. Plates are held at 4.degree. C. for 30
min. Fixative is removed from the wells and wells are washed
1.times. with PBS(+Ca,Mg) 0.5% BSA and drained. 10 .mu.l of diluted
primary antibody is added to the test and control wells.
Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin
are used at a concentration of 10 .mu.g/ml. (1:10 dilution of 0.1
mg/ml stock antibody). Cells are incubated at 37.degree. C. for 30
min. in a humidified environment. Wells are washed three times with
PBS(+Ca,Mg)+0.5% BSA. 20 .mu.l of diluted ExtrAvidin-Alkaline
Phosphatase (1:5,000 dilution, referred to herein as the working
dilution) are added to each well and incubated at 37.degree. C. for
30 min. Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA.
Dissolve 1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of
glycine buffer (pH 10.4). 100 .mu.l of pNPP substrate in glycine
buffer is added to each test well. Standard wells in triplicate are
prepared from the working dilution of the ExtrAvidin-Alkaline
Phosphotase in glycine buffer: 1:5,000
(10.sup.0)>10.sup.-0.5>10.sup.-1>10.sup.-1.5. 5 .mu.l of
each dilution is added to triplicate wells and the resulting AP
content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100
.mu.l of pNNP reagent is then added to each of the standard wells.
The plate is incubated at 37.degree. C. for 4 h. A volume of 50
.mu.l of 3M NaOH is added to all wells. The plate is read on a
plate reader at 405 nm using the background subtraction option on
blank wells filled with glycine buffer only. Additionally, the
template is set up to indicate the concentration of AP-conjugate in
each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.13 ng]. Results
are indicated as amount of bound AP-conjugate in each sample.
Example 72
Alamar Blue Endothelial Cells Proliferation Assay
[1788] This assay may be used to quantitatively determine protein
mediated inhibition of bFGF-induced proliferation of Bovine
Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells
(BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs).
This assay incorporates a fluorometric growth indicator based on
detection of metabolic activity. A standard Alamar Blue
Proliferation Assay is prepared in EGM-2MV with 10 ng/ml of bFGF
added as a source of endothelial cell stimulation. This assay may
be used with a variety of endothelial cells with slight changes in
growth medium and cell concentration. Dilutions of protein batches
to be tested are diluted as appropriate. Serum-free medium (GIBCO
SFM) without bFGF is used as a non-stimulated control and
Angiostatin or TSP-1 are included as a known inhibitory
controls.
[1789] Briefly, LEC, BAECs or UTMECs are seeded in growth media at
a density of 5000 to 2000 cells/well in a 96 well plate and placed
at 37 degrees C. overnight. After the overnight incubation of the
cells, the growth media is removed and replaced with GIBCO EC-SFM.
The cells are treated with the appropriate dilutions of an albumin
fusion protein of the invention or control protein sample(s)
(prepared in SEM) in triplicate wells with additional bFGF to a
concentration of 10 ng/ml. Once the cells have been treated with
the samples, the plate(s) is/are placed back in the 37.degree. C.
incubator for three days. After three days 10 ml of stock alamar
blue (Biosource Cat# DAL1100) is added to each well and the
plate(s) is/are placed back in the 37.degree. C. incubator for four
hours. The plate(s) are then read at 530 nm excitation and 590 nm
emission using the CytoFluor fluorescence reader. Direct output is
recorded in relative fluorescence units.
[1790] Alamar blue is an oxidation-reduction indicator that both
fluoresces and changes color in response to chemical reduction of
growth medium resulting from cell growth. As cells grow in culture,
innate metabolic activity results in a chemical reduction of the
immediate surrounding environment. Reduction related to growth
causes the indicator to change from oxidized (non-fluorescent blue)
form to reduced (fluorescent red) form (i.e., stimulated
proliferation will produce a stronger signal and inhibited
proliferation will produce a weaker signal and the total signal is
proportional to the total number of cells as well as their
metabolic activity). The background level of activity is observed
with the starvation medium alone. This is compared to the output
observed from the positive control samples (bFGF in growth medium)
and protein dilutions.
Example 73
Detection of Inhibition of a Mixed Lymphocyte Reaction
[1791] This assay can be used to detect and evaluate inhibition of
a Mixed Lymphocyte Reaction (MLR) by fusion proteins of the
invention. Inhibition of a MLR may be due to a direct effect on
cell proliferation and viability, modulation of costimulatory
molecules on interacting cells, modulation of adhesiveness between
lymphocytes and accessory cells, or modulation of cytokine
production by accessory cells. Multiple cells may be targeted by
the albumin fusion proteins that inhibit MLR since the peripheral
blood mononuclear fraction used in this assay includes T, B and
natural killer lymphocytes, as well as monocytes and dendritic
cells.
[1792] Albumin fusion proteins of the invention found to inhibit
the MLR may find application in diseases associated with lymphocyte
and monocyte activation or proliferation. These include, but are
not limited to, diseases such as asthma, arthritis, diabetes,
inflammatory skin conditions, psoriasis, eczema, systemic lupus
erythematosus, multiple sclerosis, glomerulonephritis, inflammatory
bowel disease, crohn's disease, ulcerative colitis,
arteriosclerosis, cirrhosis, graft vs. host disease, host vs. graft
disease, hepatitis, leukemia and lymphoma.
[1793] Briefly, PBMCs from human donors are purified by density
gradient centrifugation using Lymphocyte Separation Medium
(LSM.RTM., density 1.0770 g/ml, Organon Teknika Corporation, West
Chester, Pa.). PBMCs from two donors are adjusted to
2.times.10.sup.6 cells/ml in RPMI-1640 (Life Technologies, Grand
Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs
from a third donor is adjusted to 2.times.10.sup.5 cells/ml. Fifty
microliters of PBMCs from each donor is added to wells of a 96-well
round bottom microliter plate. Dilutions of the fusion protein test
material (50 .mu.l) is added in triplicate to microtiter wells.
Test samples (of the protein of interest) are added for final
dilution of 1:4; rhuIL-2 (R&D Systems, Minneapolis, Minn.,
catalog number 202-IL) is added to a final concentration of 1
.mu.g/ml; anti-CD4 mAb (R&D Systems, clone 34930.11, catalog
number MAB379) is added to a final concentration of 10 .mu.g/ml.
Cells are cultured for 7-8 days at 37.degree. C. in 5% CO.sub.2,
and 1 .mu.C of [.sup.3H] thymidine is added to wells for the last
16 hrs of culture. Cells are harvested and thymidine incorporation
determined using a Packard TopCount. Data is expressed as the mean
and standard deviation of triplicate determinations.
[1794] Samples of the fusion protein of interest are screened in
separate experiments and compared to the negative control
treatment, anti-CD4 mAb, which inhibits proliferation of
lymphocytes and the positive control treatment, IL-2 (either as
recombinant material or supernatant), which enhances proliferation
of lymphocytes.
Example 74
Assays for Protease Activity
[1795] The following assay may be used to assess protease activity
of an albumin fusion protein of the invention.
[1796] Gelatin and casein zymography are performed essentially as
described (Heusen et al., Anal. Biochem., 102:196-202 (1980);
Wilson et al., Journal of Urology, 149:653-658 (1993)). Samples are
run on 10% polyacryamide/0.1% SDS gels containing 1% gelain
orcasein, soaked in 2.5% triton at room temperature for 1 hour, and
in 0.1M glycine, pH 8.3 at 37.degree. C. 5 to 16 hours. After
staining in amido black areas of proteolysis apear as clear areas
agains the blue-black background. Trypsin (Sigma T8642) is used as
a positive control.
[1797] Protease activity is also determined by monitoring the
cleavage of n-a-benzoyl-arginine ethyl ester (BAEE) (Sigma B-4500,
Reactions are set up in (25 mM NaPO.sub.4, 1 mM EDTA, and 1 mM
BAEE), pH 7.5. Samples are added and the change in absorbance at
260 nm is monitored on the Beckman DU-6 spectrophotometer in the
time-drive mode. Trypsin is used as a positive control.
[1798] Additional assays based upon the release of acid-soluble
peptides from casein or hemoglobin measured as absorbance at 280 nm
or colorimetrically using the Folin method are performed as
described in Bergmeyer, et al., Methods of Enzymatic Analysis, 5
(1984). Other assays involve the solubilization of chromogenic
substrates (Ward, Applied Science, 251-317 (1983)).
Example 75
Identifying Serine Protease Substrate Specificity
[1799] Methods known in the aft or described herein may be used to
determine the substrate specificity of the albumin fusion proteins
of the present invention having serine protease activity. A
preferred method of determining substrate specificity is by the use
of positional scanning synthetic combinatorial libraries as
described in GB 2 324 529 (incorporated herein in its
entirety).
Example 76
Ligand Binding Assays
[1800] The following assay may be used to assess ligand binding
activity of an albumin fusion protein of the invention.
[1801] Ligand binding assays provide a direct method for
ascertaining receptor pharmacology and are adaptable to a high
throughput format. The purified ligand for an albumin fusion
protein of the invention is radiolabeled to high specific activity
(50-2000 Ci/mmol) for binding studies. A determination is then made
that the process of radiolabeling does not diminish the activity of
the ligand towards the fusion protein. Assay conditions for
butters, ions, pH and other modulators such as nucleotides are
optimized to establish a workable signal to noise ratio for both
membrane and whole cell polypeptide sources. For these assays,
specific polypeptide binding is defined as total associated
radioactivity minus the radioactivity measured in the presence of
an excess of unlabeled competing ligand. Where possible, more than
one competing ligand is used to define residual nonspecific
binding.
Example 77
Functional Assay in Xenopus Oocytes
[1802] Capped RNA transcripts from linearized plasmid templates
encoding an albumin fusion protein of the invention is synthesized
in vitro with RNA polymerases in accordance with standard
procedures. In vitro transcripts are suspended in water at a final
concentration of 0.2 mg/ml. Ovarian lobes are removed from adult
female toads, Stage V defolliculated oocytes are obtained, and RNA
transcripts (10 ng/oocyte) are injected in a 50 nl bolus using a
microinjection apparatus. Two electrode voltage clamps are used to
measure the currents from individual Xenopus oocytes in response
fusion protein and polypeptide agonist exposure. Recordings are
made in Ca2+ free Barth's medium at room temperature. The Xenopus
system can be used to screen known ligands and tissue/cell extracts
for activating ligands.
Example 78
Microphysiometric Assays
[1803] Activation of a wide variety of secondary messenger systems
results in extrusion of small amounts of acid from a cell. The acid
formed is largely as a result of the increased metabolic activity
required to fuel the intracellular signaling process. The pH
changes in the media surrounding the cell are very small but are
detectable by the CYTOSENSOR microphysiometer (Molecular Devices
Ltd., Menlo Park, Calif.). The CYTOSENSOR is thus capable of
detecting the ability of an albumin fusion protein of the invention
to activate secondary messengers that are coupled to an energy
utilizing intracellular signaling pathway.
Example 79
Extract/Cell Supernatant Screening
[1804] A large number of mammalian receptors exist for which there
remains, as yet, no cognate activating ligand (agonist). Thus,
active ligands for these receptors may not be included within the
ligands banks as identified to date. Accordingly, the albumin
fusion proteins of the invention can also be functionally screened
(using calcium, cAMP, microphysiometer, oocyte electrophysiology,
etc., functional screens) against tissue extracts to identify
natural ligands for the Therapeutic protein portion and/or albumin
protein portion of an albumin fusion protein of the invention.
Extracts that produce positive functional responses can be
sequentially subtractionated until an activating ligand is isolated
and identified.
Example 80
ATP-Binding Assay
[1805] The following assay may be used to assess ATP-binding
activity of fusion proteins of the invention.
[1806] ATP-binding activity of an albumin fusion protein of the
invention may be detected using the ATP-binding assay described in
U.S. Pat. No. 5,858,719, which is herein incorporated by reference
in its entirety. Briefly, ATP-binding to an albumin fusion protein
of the invention is measured via photoaffinity labeling with
8-azido-ATP in a competition assay. Reaction mixtures containing 1
mg/ml of ABC transport protein are incubated with varying
concentrations of ATP, or the non-hydrolyzable ATP analog
adenyl-5'-imidodiphosphate for 10 minutes at 4.degree. C. A mixture
of 8-azido-ATP (Sigma Chem. Corp., St. Louis, Mo.) plus 8-azido-ATP
(.sup.32P-ATP) (5 mCi/.mu.mol, ICN, Irvine Calif.) is added to a
final concentration of 100 .mu.M and 0.5 ml aliquots are placed in
the wells of a porcelain spot plate on ice. The plate is irradiated
using a short wave 254 nm UV lamp at a distance of 2.5 cm from the
plate for two one-minute intervals with a one-minute cooling
interval in between. The reaction is stopped by addition of
dithiothreitol to a final concentration of 2 mM. The incubations
are subjected to SDS-PAGE electrophoresis, dried, and
autoradiographed. Protein bands corresponding to the albumin fusion
proteins of the invention are excised, and the radioactivity
quantified. A decrease in radioactivity with increasing ATP or
adenly-5' imidodiphosphate provides a measure of ATP affinity to
the fusion protein.
Example 81
Phosphorylation Assay
[1807] hi order to assay for phosphorylation activity of an albumin
fusion protein of the invention, a phosphorylation assay as
described in U.S. Pat. No. 5,958,405 (which is herein incorporated
by reference) is utilized. Briefly, phosphorylation activity may be
measured by phosphorylation, of a protein substrate using
gamma-labeled .sup.32P-ATP and quantitation of the incorporated
radioactivity using a gamma radioisotope counter. The fusion
protein of the invention is incubated with the protein substrate,
.sup.32P-ATP, and a kinase buffer. The .sup.32P incorporated into
the substrate is then separated from free .sup.32P-ATP by
electrophoresis, and the incorporated .sup.32P is counted and
compared to a negative control. Radioactivity counts above the
negative control are indicative of phosphorylation activity of the
fusion protein.
Example 82
Detection of Phosphorylation Activity (Activation) of an Albumin
Fusion Protein of the Invention in the Presence of Polypeptide
Ligands
[1808] Methods known in the art or described herein may be used to
determine the phosphorylation activity of an albumin fusion protein
of the invention. A preferred method of determining phosphorylation
activity is by the use of the tyrosine phosphorylation assay as
described in U.S. Pat. No. 5,817,471 (incorporated herein by
reference).
Example 83
Identification of Signal Transduction Proteins that Interact with
an Albumin Fusion Protein of the Present Invention
[1809] Albumin fusion proteins of the invention may serve as
research tools for the identification, characterization and
purification of signal transduction pathway proteins or receptor
proteins. Briefly, a labeled fusion protein of the invention is
useful as a reagent for the purification of molecules with which it
interacts. In one embodiment of affinity purification, an albumin
fusion protein of the invention is covalently coupled to a
chromatography column. Cell-free extract derived from putative
target cells, such as carcinoma tissues, is passed over the column,
and molecules with appropriate affinity bind to the albumin fusion
protein. The protein complex is recovered from the column,
dissociated, and the recovered molecule subjected to N-terminal
protein sequencing. This amino acid sequence is then used to
identify the captured molecule or to design degenerate
oligonucleotide probes for cloning the relevant gene from an
appropriate cDNA library.
Example 84
IL-6 Bioassay
[1810] A variety of assays are known in the art for testing the
proliferative effects of an albumin fusion protein of the
invention. For example, one such assay is the IL-6 Bioassay as
described by Marz et al. (Proc. Natl. Acad. Sci. USA., 95:3251-56
(1998), which is herein incorporated by reference). After 68 hrs.
at 37.degree. C., the number of viable cells is measured by adding
the tetrazolium salt thiazolyl blue (MTT) and incubating for a
further 4 hrs. at 37.degree. C. B9 cells are lysed by SDS and
optical density is measured at 570 nm. Controls containing IL-6
(positive) and no cytokine (negative) are Briefly, IL-6 dependent
B9 murine cells are washed three times in IL-6 free medium and
plated at a concentration of 5,000 cells per well in 50 .mu.l, and
50 .mu.l of fusion protein of the invention is added. Enhanced
proliferation in the test sample(s) (containing an albumin fusion
protein of the invention) relative to the negative control is
indicative of proliferative effects mediated by the fusion
protein.
Example 85
Support of Chicken Embryo Neuron Survival
[1811] To test whether sympathetic neuronal cell viability is
supported by an albumin fusion protein of the invention, the
chicken embryo neuronal survival assay of Senaldi et al may be
utilized (Proc. Natl. Acad. Sci., U.S.A., 96:11458-63 (1998), which
is herein incorporated by reference). Briefly, motor and
sympathetic neurons are isolated from chicken embryos, resuspended
in L15 medium (with 10% FCS, glucose, sodium selenite,
progesterone, conalbumin, putrescine, and insulin; Life
Technologies. Rockville, Md.) and Dulbecco's modified Eagles medium
[with 10% FCS, glutamine, penicillin, and 25 mM Hepes buffer (pH
7.2); Life Technologies, Rockville, Md.], respectively, and
incubated at 37.degree. C. in 5% CO.sub.2 in the presence of
different concentrations of the purified fusion protein of the
invention, as well as a negative control lacking any cytokine.
After 3 days, neuron survival is determined by evaluation of
cellular morphology, and through the use of the colorimetric assay
of Mosmann (Mosmann, T., J. Immunol. Methods, 65:55-63 (1983)).
Enhanced neuronal cell viability as compared to the controls
lacking cytokine is indicative of the ability of the albumin fusion
protein to enhance the survival of neuronal cells.
Example 86
Assay for Phosphatase Activity
[1812] The following assay may be used to assess serine/threonine
phosphatase (PTPase) activity of an albumin fusion protein of the
invention.
[1813] In order to assay for serine/threonine phosphatase (PTPase)
activity, assays can be utilized which are widely known to those
skilled in the art. For example, the serine/threonine phosphatase
(PSPase) activity of an albumin fusion protein of the invention may
be measured using a PSPase assay kit from New England Biolabs, Inc.
Myelin basic protein (MyBP), a substrate for PSPase, is
phosphorylated on serine and threonine residues with cAMP-dependent
Protein Kinase in the presence of [.sup.32P]ATP. Protein
serine/threonine phosphatase activity is then determined by
measuring the release of inorganic phosphate from 32P-labeled
MyBP.
Example 87
Interaction of Serine/Threonine Phosphatases with Other
Proteins
[1814] Fusion protein of the invention having serine/threonine
phosphatase activity (e.g., as determined in Example 86) are
useful, for example, as research tools for the identification,
characterization and purification of additional interacting
proteins or receptor proteins, or other signal transduction pathway
proteins. Briefly, a labeled fusion protein of the invention is
useful as a reagent for the purification of molecules with which it
interacts. In one embodiment of affinity purification, an albumin
fusion protein of the invention is covalently coupled to a
chromatography column. Cell-free extract derived from putative
target cells, such as neural or liver cells, is passed over the
column, and molecules with appropriate affinity bind to the fusion
protein. The fusion protein complex is recovered from the column,
dissociated, and the recovered molecule subjected to N-terminal
protein sequencing. This amino acid sequence is then used to
identify the captured molecule or to design degenerate
oligonucleotide probes for cloning the relevant gene from an
appropriate cDNA library.
Example 88
Assaying for Heparanase Activity
[1815] There a numerous assays known in the art that may be
employed to assay for heparanase activity of an albumin fusion
protein of the invention. In one example, heparanase activity of an
albumin fusion protein of the invention, is assayed as described by
Vlodaysky et al., (Vlodaysky et al., Nat. Med., 5:793-802 (1999)).
Briefly, cell lysates, conditioned media, intact cells
(1.times.10.sup.6 cells per 35-mm dish), cell culture supernatant,
or purified fusion protein are incubated for 18 hrs at 37.degree.
C., pH 6.2-6.6, with .sup.35S-labeled ECM or soluble ECM derived
peak I proteoglycans. The incubation medium is centrifuged and the
supernatant is analyzed by gel Libation on a Sepharose CL-6B column
(0.9.times.30 cm). Fractions are eluted with PBS and their
radioactivity is measured. Degradation fragments of heparan sulfate
side chains are eluted from Sepharose 6B at 0.5<K.sub.av<0.8
(peak II). Each experiment is done at least three times.
Degradation fragments corresponding to "peak II," as described by
Vlodaysky et al., is indicative of the activity of an albumin
fusion protein of the invention in cleaving heparan sulfate.
Example 89
Immobilization of Biomolecules
[1816] This example provides a method for the stabilization of an
albumin fusion protein of the invention in non-host cell lipid
bilayer constricts (see, e.g., Bieri et al., Nature Biotech
17:1105-1108 (1999), hereby incorporated by reference in its
entirety herein) which can be adapted for the study of fusion
proteins of the invention in the various functional assays
described above. Briefly, carbohydrate-specific chemistry for
biotinylation is used to confine a biotin tag to an albumin fusion
protein of the invention, thus allowing uniform orientation upon
immobilization. A 50 uM solution of an albumin fusion protein of
the invention in washed membranes is incubated with 20 mM NaIO4 and
1.5 mg/ml (4 mM) BACH or 2 mg/ml (7.5 mM) biotin-hydrazide for 1 hr
at room temperature (reaction volume, 150 ul). Then the sample is
dialyzed (Pierce Slidealizer Gossett, 10 kDa cutoff; Pierce
Chemical Co., Rockford Ill.) at 4 C first for 5 h, exchanging the
buffer after each hour, and finally for 12 h against 500 ml buffer
R (0.15 M NaCl, 1 mM MgCl2, 10 mM sodium phosphate, pH7). Just
before addition into a cuvette, the sample is diluted 1:5 in buffer
ROG50 (Buffer R supplemented with 50 mM octylglucoside).
Example 90
Assays for Metalloproteinase Activity
[1817] Metalloproteinases are peptide hydrolases which use metal
ions, such as Zn.sup.+2, as the catalytic mechanism.
Metalloproteinase activity of an albumin fusion protein of the
present invention can be assayed according to methods known in the
art. The following exemplary methods are provided:
[1818] Proteolysis of alpha-2-macroglobulin
[1819] To confirm protease activity, a purified fusion protein of
the invention is mixed with the substrate alpha-2-macroglobulin
(0.2 unit/ml; Boehringer Mannheim, Germany) in 1.times. assay
buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl.sub.2, 25 .mu.M
ZnCl.sub.2 and 0.05% Brij-35) and incubated at 37.degree. C. for
1-5 days. Trypsin is used as positive control. Negative controls
contain only alpha-2-macroglobulin in assay buffer. The samples are
collected and boiled in SDS-PAGE sample buffer containing 5%
2-mercaptoethanol for 5-min, then loaded onto 8% SDS-polyacrylamide
gel. After electrophoresis the proteins are visualized by silver
staining. Proteolysis is evident by the appearance of lower
molecular weight bands as compared to the negative control.
[1820] Inhibition of Alpha-2-Macroglobulin Proteolysis by
Inhibitors of Metalloproteinases
[1821] Known metalloproteinase inhibitors (metal chelators (EDTA,
EGTA, AND HgCl.sub.2), peptide metalloproteinase inhibitors (TIMP-1
and TIMP-2), and commercial small molecule MMP inhibitors) may also
be used to characterize the proteolytic activity of an albumin
fusion protein of the invention. Three synthetic MMP inhibitors
that may be used are: MMP inhibitor I, [IC.sub.50=1.0 .mu.M against
MMP-1 and MMP-8; IC.sub.50=30 .mu.M against MMP-9; IC.sub.50=150
.mu.M against MMP-3]; MMP-3 (stromelysin-1) inhibitor I
[IC.sub.50=5 .mu.M against MMP-3], and MMP-3 inhibitor II
[K.sub.i=130 nM against MMP-3]; inhibitors available through
Calbiochem, catalog #444250, 444218, and 444225, respectively).
Briefly, different concentrations of the small molecule MMP
inhibitors are mixed with a purified fusion protein of the
invention (50 .mu.g/ml) in 22.9 .mu.l of 1.times. HEPES buffer (50
mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl.sub.2, 25 .mu.M ZnCl.sub.2
and 0.05% Brij-35) and incubated at room temperature (24.degree.
C.) for 2-hr, then 7.1 .mu.l of substrate alpha-2-macroglobulin
(0.2 unit/ml) is added and incubated at 37.degree. C. for 20-hr.
The reactions are stopped by adding 4.times. sample buffer and
boiled immediately for 5 minutes. After SDS-PAGE, the protein bands
are visualized by silver stain.
[1822] Synthetic Fluorogenic Peptide Substrates Cleavage Assay
[1823] The substrate specificity for fusion proteins of the
invention with demonstrated metalloproteinase activity may be
determined using techniques known in the art, such as using
synthetic fluorogenic peptide substrates (purchased from SACHEM
Bioscience Inc). Test substrates include, M-1985, M-2225, M-2105,
M-2110, and M-2255. The first four are MMP substrates and the last
one is a substrate of tumor necrosis factor-.alpha. (TNF-.alpha.)
converting enzyme (TACE). These substrates are preferably prepared
in 1:1 dimethyl sulfoxide (DMSO) and water. The stock solutions are
50-500 .mu.M. Fluorescent assays are performed by using a Perkin
Elmer LS 50B luminescence spectrometer equipped with a constant
temperature water bath. The excitation .lamda. is 328 nm and the
emission .lamda. is 393 nm. Briefly, the assay is carried out by
incubating 176 .mu.l 1.times. HEPES buffer (0.2 M NaCl, 10 mM
CaCl.sub.2, 0.05% Brij-35 and 50 mM HEPES, pH 7.5) with 4 .mu.l of
substrate solution (50 .mu.M) at 25.degree. C. for 15 minutes, and
then adding 20 .mu.l of a purified fusion protein of the invention
into the assay cuvett. The final concentration of substrate is 1
.mu.M. Initial hydrolysis rates are monitored for 30-min.
Example 91
Identification and Cloning of VH and VL Domains
[1824] One method to identify and clone VH and VL domains from cell
lines expressing a particular antibody is to perform PCR with VH
and VL specific primers on cDNA made from the antibody expressing
cell lines. Briefly, RNA is isolated from the cell lines and used
as a template for RT-PCR designed to amplify the VH and VL domains
of the antibodies expressed by the EBV cell lines. Cells may be
lysed in the TRIzol.RTM. reagent (Life Technologies, Rockville,
Md.) and extracted with one fifth volume of chloroform. After
addition of chloroform, the solution is allowed to incubate at room
temperature for 10 minutes, and the centrifuged at 14,000 rpm for
15 minutes at 4.degree. C. in a tabletop centrifuge. The
supernatant is collected and RNA is precipitated using an equal
volume of isopropanol. Precipitated RNA is pelleted by centrifuging
at 14,000 rpm for 15 minutes at 4.degree. C. in a tabletop
centrifuge. Following centrifugation, the supernatant is discarded
and washed with 75% ethanol. Following washing, the RNA is
centrifuged again at 800 rpm for 5 minutes at 4.degree. C. The
supernatant is discarded and the pellet allowed to air dry. RNA is
the dissolved in DEPC water and heated to 60.degree. C. for 10
minutes. Quantities of RNA can determined using optical density
measurements.
[1825] cDNA may be synthesized, according to methods well-known in
the art, from 1.5-2.5 micrograms of RNA using reverse transcriptase
and random hexamer primers. cDNA is then used as a template for PCR
amplification of VH and VL domains. Primers used to amplify VH and
VL genes are shown in Table 6. Typically a PCR reaction makes use
of a single 5' primer and a single 3' primer. Sometimes, when the
amount of available RNA template is limiting, or for greater
efficiency, groups of 5' and/or 3' primers may be used. For
example, sometimes all five VH-5' primers and all JH3' primers are
used in a single PCR reaction. The PCR reaction is carried out in a
50 microliter volume containing 1.times.PCR buffer, 2 mM of each
dNTP, 0.7 units of High Fidelity Taq polymerse, 5' primer mix, 3'
primer mix and 7.5 microliters of cDNA. The 5' and 3' primer mix of
both VH and VL can be made by pooling together 22 pmole and 28
pmole, respectively, of each of the individual primers. PCR
conditions are: 94.degree. C. for 5 minutes; followed by 25 cycles
of 94.degree. C. for 1 minute, 50.degree. C. for 1 minute, and
72.degree. C. for 1 minute; followed by an extension cycle of
72.degree. C. for 10 minutes. After the reaction is completed,
sample tubes are stored 4.degree. C.
TABLE-US-00032 TABLE 6 Primer Sequences Used to Amplify VII and VL
domains. SEQ Primer name ID NO Primer Sequence (5'-3') VH Primers
Hu VH1-5' 342 CAGGTGCAGCTGGTGCAGTCTGG Hu VH2-5' 343
CAGGTCAACTTAAGGGAGTCTGG Hu VH3-5' 344 GAGGTGCAGCTGGTGGAGTCTGG Hu
VH4-5' 345 CAGGTGCAGCTGCAGGAGTCGGG Hu VH5-5' 346
GAGGTGCAGCTGTTGCAGTCTGC Hu VH6-5' 347 CAGGTACAGCTGCAGCAGTCAGG Hu
JH1,2-5' 348 TGAGGAGACGGTGACCAGGGTGCC Hu JH3-5' 349
TGAAGAGACGGTGACCATTGTCCC Hu JH4,5-5' 350 TGAGGAGACGGTGACCAGGGTTCC
Hu JH6-5' 351 TGAGGAGACGGTGACCGTGGTCCC VL Primers Hu Vkappa1-5' 352
GACATCCAGATGACCCAGTCTCC Hu Vkappa2a-5' 353 GATGTTGTGATGACTCAGTCTCC
Hu Vkappa2b-5' 354 GATATTGTGATGACTCAGTCTCC Hu Vkappa3-5' 355
GAAATTGTGTTGACGCAGTCTCC Hu Vkappa4-5' 356 GACATCGTGATGACCCAGTCTCC
Hu Vkappa5-5' 357 GAAACGACACTCACGCAGTCTCC Hu Vkappa6-5' 358
GAAATTGTGCTGACTCAGTCTCC Hu Vlambda1-5' 359 CAGTCTGTGTTGACGCAGCCGCC
Hu Vlambda2-5' 360 CAGTCTGCCCTGACTCAGCCTGC Hu Vlambda3-5' 361
TCCTATGTGCTGACTCAGCCACC Hu Vlambda3b-5' 362 TCTTCTGAGCTGACTCAGGACCC
Hu Vlambda4-5' 363 CACGTTATACTGACTCAACCGCC Hu Vlambda5-5' 364
CAGGCTGTGCTCACTCAGCCGTC Hu Vlambda6-5' 365 AATTTTATGCTGACTCAGCCCCA
Hu Jkappa1-5' 366 ACGTTTGATTTCCACCTTGGTCCC Hu Jkappa2-5' 367
ACGTTTGATCTCCAGCTTGGTCCC Hu Jkappa3-5' 368 ACGTTTGATATCCACTTTGGTCCC
Hu Jkappa4-5' 369 ACGTTTGATCTCCACCTTGGTCCC Hu Jkappa5-5' 370
ACGTTTAATCTCCAGTCGTGTCCC Hu Jlambda1-5' 371 CAGTCTGTGTTGACGCAGCCGCC
Hu Jlambda2-5' 372 CAGTCTGCCCTGACTCAGCCTGC Hu Jlambda3-5' 373
TCCTATGTGCTGACTCAGCCACC Hu Jlambda3b-5' 374 TCTTCTGAGCTGACTCAGGACCC
Hu Jlambda4-5' 375 CACGTTATACTGACTCAACCGCC Hu Jlambda5-5' 376
CAGGCTGTGCTCACTCAGCCGTC Hu Jlambda6-5' 377
AATTTTATGCTGACTCAGCCCCA
[1826] PCR samples are then electrophoresed on a 1.3% agarose gel.
DNA bands of the expected sizes (.about.506 base pairs for VH
domains, and 344 base pairs for VL domains) can be cut out of the
gel and purified using methods well known in the art. Purified PCR
products can be ligated into a PCR cloning vector (TA vector from
Invitrogen Inc, Carlsbad, Calif.). Individual cloned PCR products
can be isolated after transfection of E. coli and blue/white color
selection. Cloned PCR products may then be sequenced using methods
commonly known in the art.
[1827] The PCR bands containing the VH domain and the VL domains
can also be used to create full-length Ig expression vectors. VH
and VL domains can be cloned into vectors containing the nucleotide
sequences of a heavy (e.g., human IgG1 or human IgG4) or light
chain (human kappa or human lambda) constant regions such that a
complete heavy or light chain molecule could be expressed from
these vectors when transfected into an appropriate host cell.
Further, when cloned heavy and light chains are both expressed in
one cell line (from either one or two vectors), they can assemble
into a complete functional antibody molecule that is secreted into
the cell culture medium. Methods using polynucleotides encoding VH
and VL antibody domain to generate expression vectors that encode
complete antibody molecules are well known within the an.
Example 92
Construct ID 2249, IFNa2-HSA, Generation
[1828] Construct ID 2249, pSAC35:IFNa2.HSA, comprises DNA encoding
an IFNa2 albumin fusion protein which has the HSA chimeric leader
sequence, followed by the mature form of IFNa2 protein, i.e.,
C1-E165, fused to the amino-terminus of the mature form of USA in
the yeast S. cerevisiae expression vector pSAC35.
[1829] Cloning of IFNa2 cDNA
[1830] The polynucleotide encoding IFNa2 was PCR amplified using
primers IFNa2-1 and IFNa2-2, described below. The PCR amplimer was
cut with Sal 1/Cla I, and ligated into Xho I/Cla I cut pScCHSA.
Construct ID #2249 encodes an albumin fusion protein containing the
chimeric leader sequence of HSA, the mature form of IFNa2, followed
by the mature HSA protein.
[1831] Two oligonucleotides suitable for PCR amplification of the
polynucleotide encoding the mature form of IFNa2, IFNa2-1 and
IFNa2-2, were synthesized:
TABLE-US-00033 IFNa2-1: (SEQ ID NO: 838)
5'-CGCGCGCGTCGACAAAAGATGTGATCTGCCTCAAACCCACA-3' IFNa2-2: (SEQ ID
NO: 839) 5'-GCGCGCATCGATGAGCAACCTCACTCTTGTGTGCATCTTCC TTAC
TTCTTAAACTTTCT-3'
[1832] The IFNa2-1 primer incorporates a Sal I cloning site (shown
underlined), nucleotides encoding the last three amino acid
residues of the chimeric HSA leader sequence, as well as 22
nucleotides (shown in bold) encoding the first 7 amino acid
residues of the mature form of IFNa2. In IFNa2-2, the Cla I site
(shown underlined) and the DNA following it are the reverse
complement of DNA encoding the first 10 amino acids of the mature
HSA protein and the last 22 nucleotides (shown in bold) are the
reverse complement of DNA encoding the last 7 amino acid residues
of IFNa2 (see Example 2). A PCR autotimer of IFNa2-HSA was
generated using these primers, purified, digested with Sal I and
Cla I restriction enzymes, and cloned into the Xho 1 and Cla I
sites of the pScCHSA vector. After the sequence was confirmed, the
expression cassette encoding this IFNa2 albumin fusion protein was
subcloned into Not I digested pSAC35.
[1833] Further, analysis of the N-terminus of the expressed albumin
fusion protein by amino acid sequencing can confirm the presence of
the expected IFNa2 sequence (see below).
[1834] Other IFNa2 albumin fusion proteins using different leader
sequences have been constructed by methods known in the art (see
Example 2). Examples of the various leader sequences include, but
are not limited to, invertase "INV" (constructs 2343 and 2410) and
mating alpha factor "MAF" (construct 2366). These IFNa2 albumin
fusion proteins can be subcloned into mammalian expression vectors
such as pC4 (constructs 2382) and pEE12.1 as described previously
(see Example 5). IFNa2 albumin fusion proteins with the therapeutic
portion fused C-terminus to HSA can also be constructed (construct
2381).
[1835] IFNa2 albumin fusion proteins of the invention preferably
comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to
either the N- or C-terminus of the mature form of IFNa2, i.e.,
Cys-1 to Glu-165. In one embodiment of the invention, IFNa2 albumin
fusion proteins of the invention further comprise a signal sequence
which directs the nascent fusion polypeptide in the secretory
pathways of the host used for expression. In a further preferred
embodiment, the signal peptide encoded by the signal sequence is
removed, and the mature IFNa2 albumin fusion protein is secreted
directly into the culture medium. IFNa2 albumin fusion proteins of
the invention may comprise heterologous signal sequences including,
but not limited to, MAF, INV, Ig, Fibulin B, Clusterin,
Insulin-Like Growth Factor Binding Protein 4, variant HSA leader
sequences including, but not limited to, a chimeric HSA/MAF leader
sequence, or other heterologous signal sequences known in the art.
In a preferred embodiment, IFNa2 albumin fusion proteins of the
invention comprise the native IFNa2. In further preferred
embodiments, the IFNa2 albumin fusion proteins of the invention
further comprise an N-terminal methionine residue. Polynucleotides
encoding these polypeptides, including fragments and/or variants,
are also encompassed by the invention.
[1836] Expression and Purification of Construct ID 2249.
[1837] Expression in Yeast S. cerevisiae.
[1838] Transformation of construct 2249 inn) yeast S. cerevisiae
strain BXP10 was carried out by methods known in the an (see
Example 3). Cells can be collected at stationary phase after 72
hours of growth. Supernatants are collected by clarifying cells at
3000 g for 10 min. Expression levels are examined by immunoblot
detection with anti-HSA serum (Kent Laboratories) or as the primary
antibody. The IFNa2 albumin fusion protein of approximate molecular
weight of 88.5 ala can be obtained.
Purification from Yeast S. cerevisiae Cell Supernatant.
[1839] The cell supernatant containing IFNa2 albumin fusion protein
expressed from construct ID #2249 in yeast S. cerevisiae cells can
be purified either small scale over a Dyax peptide affinity column
(see Example 4) or large scale by following 5 steps: diafiltration,
anion exchange chromatography using DEAE-Sepharose Fast Flow
column, hydrophobic interaction chromatography (HIC) using Butyl
650S column, cation exchange chromatography using an SP-Sepharose
Fast Flow column or a Blue Sepharose chromatography, and high
performance chromatography using Q-sepharose high performance
column chromatography (see Example 4). The IFNa2 albumin fusion
protein may elute flour the DEAE-Sepharose Fast Flow column with
100-250 in M NaCl, from the SP-Sepharose Fast Flow column with
150-250 in M NaCl, and from the Q-Sepharose High Performance column
at 5-7.5 mS/cm. N-terminal sequencing should yield the sequence
CDLPQ (SEQ ID NO:849) which corresponds to the mature form of
IFNa2.
[1840] The Activity of IFNa2 can be Assayed Using an In Vitro
ISRE-SEAP Assay.
[1841] Method
[1842] The IFNa2 albumin fusion protein encoded by construct ID
#2249 can be tested for activity in the ISRE-SEAP assay as
previously described in Example 19. Briefly, conditioned yeast
supernatants were tested at a 1:1000 dilution for their ability to
direct ISRE signal transduction on the ISRE-SEAP/293F reporter
cell-line. The ISRE-SEAP/293F reporter cells were plated at
3.times.10.sup.4 cell/well in 96-well, poly-D-lysine coated,
plates, one day prior to treatment. The reporter cells were then
incubated for 18 or 24 hours prior to removing 40 .mu.L for use in
the SEAP Reporter Gene Chemiluminescent Assay (Roche catalog
#1779842). Recombinant human Interferon beta, "rhIFNb" (Biogen),
was used as a positive control.
[1843] Result
[1844] The purified preparation of IFNa2-HSA demonstrated a
relatively linear increase in the ISRE-SEAP assay over
concentrations ranging from 10.sup.-1 to 10.sup.1 ng/mL or
10.sup.-10 to 10.sup.-4 ng/ml, (see FIG. 9).
[1845] In Vivo Induction of OAS by Interferon Alpha Fusion Encoded
by Construct ID 2249.
[1846] Method
[1847] The OAS enzyme, 2'-5'-OligoAdenylate Synthetase, is
activated at the transcriptional level by interferon in response to
antiviral infection. The effect of interferon constructs can be
measured by obtaining blood samples from treated monkeys and
analyzing these samples for transcriptional activation of two OAS
mRNA, p41 and p69. A volume of 0.5 mL of whole blood was obtained
from 4 animals per group at 7 different time points, day 0, day 1,
day 2, day 4, day 8, day 10, and day 14 per animal. The various
groups include vehicle control, intravenous injection of 30
.mu.g/kg HSA-IFN on day 1, subcutaneous injection of 30 .mu.g/kg of
HSA-IFN on day 1, subcutaneous injection of 300 .mu.g/kg of HSA-IFN
on day 1, and subcutaneous injection of 40 .mu.g/kg of Interferon
alpha (Schering-Plough) as a positive Control on days 1, 3, and 5.
The levels of the p41 and the p69 mRNA transcripts were determined
by real-time quantitative PCR (Taqman) using probes specific for
p41-OAS and p69-GAS. OAS mRNA levels were quantitated relative to
18S ribosomal RNA endogenous control. The albumin fusion encoded by
construct 2249 can be subjected to similar experimentation.
[1848] Results
[1849] A significant increase in mRNA transcript levels for both
p41 and p69 OAS was observed in HSA-interferon treated monkeys in
contrast to IFNa treated monkeys (see FIG. 10 for p41 data). The
effect lasted nearly 10 days.
Example 93
Indications for IFNa2 Albumin Fusion Proteins
[1850] IFN alpha albumin fusion protein (including, but not limited
to, those encoded by constructs 2249, 2343, 2410, 2366, 2382, and
2381) can be used to treat, prevent, ameliorate, and/or detect
multiple sclerosis. Other indications include, but are not limited
to, Hepatitis C, oncology uses, cancer, hepatitis, human papilloma
virus, fibromyalgia, Sjogren's syndrome, hairy cell leukemia,
chronic myelogeonus leukemia, AIDS-related Kaposi's sarcoma,
chronic hepatitis B, malignant melanoma, non-Hodgkin's lymphoma,
external condylomata acuminata, HIV infection, small cell lung
cancer, hematological malignancies, herpes simplex virus
infections, multiple sclerosis, viral hemorrhagic fevers, solid
tumors, renal cancer, bone marrow disorders, bone disorders,
bladder cancer, gastric cancer, hepatitis D, multiple myeloma, type
I diabetes mellitus, viral infections, cutaneous lymphoma, cervical
dysplasia, chronic fatigue syndrome, and renal cancer.
[1851] Preferably, the IFN.alpha.-albumin fusion protein or IFN
hybrid fusion protein is administered in combination with a CCR5
antagonist, further in association with at least one of ribavirin,
IL-2, IL-12, pentafuside alone or in combination with an anti-HIV
drug therapy, e.g., HAART, for preparation of a medicament for the
treatment of HIV-1 infections, HCV, or HIV-1 and HCV co-infections
in treatment-naive as well as treatment-experienced adult and
pediatric patients.
Example 94
Preparation of HA-IFN Fusion Protein (Such as IFN.alpha.)
[1852] The cDNA for the interferon of interest such as IFN.alpha.
can be isolated by a variety of means including but not
exclusively, from cDNA libraries, by RT-PCR and by PCR using a
series of overlapping synthetic oligonucleotide primers, all using
standard methods. The nucleotide sequences for interferons, such as
IFN.alpha. are known and available, for instance, in U.S. Pat. Nos.
5,326,859 and 4,588,585, in EP 32 134, as well as in public
databases such as GenBank. The cDNA can be tailored at the 5' and
3' ends to generate restriction sites, such that oligonucleotide
linkers can be used to clone the cDNA into a vector containing the
cDNA for HA. This can be at the N or C-terminus of the HA sequence,
with or without the use of a spacer sequence. The IFN.alpha. (or
other interferon) cDNA is cloned into a vector such as pPPC0005
(FIG. 3), pScCHSA, pScNHSA, or pC4:HSA from which the complete
expression cassette is then excised and inserted into the plasmid
pSAC35 to allow the expression of the albumin fusion protein in
yeast. The albumin fusion protein secreted from the yeast can then
be collected and purified from the media and tested for its
biological activity. For expression in mammalian cell lines a
similar procedure is adopted except that the expression cassette
used employs a mammalian promoter, leader sequence and terminator
(See Example 1). This expression cassette is then excised and
inserted into a plasmid suitable for the transfection of mammalian
cell lines.
[1853] Maximum Protein Recovery from Vials
[1854] The albumin fusion proteins of the invention have a high
degree of stability even when they are packaged at low
concentrations. In addition, in spite of the low protein
concentration, good fusion-protein recovery is observed even when
the aqueous solution includes no other protein added to minimize
binding to the vial walls. The recovery of vial-stored HA-IFN
solutions was compared with a stock solution. 6 or 30 .mu.g/ml
HA-IFN solutions were placed in vials and stored at 4.degree. C.
After 48 or 72 hrs a volume originally equivalent to 10 ng of
sample was removed and measured in an IFN sandwich ELISA. The
estimated values were compared to that of a high concentration
stock solution. As shown, there is essentially no loss of the
sample in these vials, indicating that addition of exogenous
material such as albumin is not necessary to prevent sample loss to
the wall of the vials
[1855] In Vivo Stability and Bioavailability of HA-.alpha.-IFN
Fusions
[1856] To determine the in vivo stability and bioavailability of a
HA-.alpha.-IFN fusion molecule, the purified fusion molecule (from
yeast) was administered to monkeys. Pharmaceutical compositions
formulated from HA-.alpha.-IFN fusions may account for the extended
serum half-life and bioavailability. Accordingly, pharmaceutical
compositions may be formulated to contain lower dosages of
alpha-interferon activity compared to the native alpha-interferon
molecule.
[1857] Pharmaceutical compositions containing HA-.alpha.-IFN
fusions may be used to treat or prevent disease in patients with
any disease or disease state that can be modulated by the
administration of .alpha.-IFN. Such diseases include, but are not
limited to, hairy cell leukemia, Kaposi's sarcoma, genital and anal
warts, chronic hepatitis B, chronic non-A, non-B hepatitis, in
particular hepatitis C, hepatitis D, chronic myelogenous leukemia,
renal cell carcinoma, bladder carcinoma, ovarian and cervical
carcinoma, skin cancers, recurrent respirator papillomatosis,
non-Hodgkin's and cutaneous T-cell lymphomas, melanoma, multiple
myeloma, AIDS, multiple sclerosis, glioblastoma, etc. (see
Interferon Alpha, In: AHFS Drug Information, 1997.
[1858] Accordingly, the invention includes pharmaceutical
compositions containing a HA-.alpha.-IFN fusion protein,
polypeptide or peptide formulated with the proper dosage for human
administration. The invention also includes methods of treating
patients in need of such treatment comprising at least the step of
administering a pharmaceutical composition containing at least one
HA-.alpha.-IFN fusion protein, polypeptide or peptide.
[1859] Bifunctional HA-.alpha.-IFN Fusions
[1860] A HA-.alpha.-IFN expression vector may be modified to
include an insertion for the expression of bifunctional
HA-.alpha.-IFN fusion proteins. For instance, the cDNA for a second
protein of interest may be inserted in frame downstream of the
"rHA-IFN" sequence after the doable stop cotton has been removed or
shifted downstream of the coding sequence.
[1861] In one version of a bifunctional HA-.alpha.-IFN fusion
protein, an antibody or fragment against B-lymphocyte stimulator
protein (GenBank Acc 4455139) or polypeptide may be fused to one
end of the HA component of the fusion molecule. This bifunctional
protein is useful for modulating any immune response generated by
the .alpha.-IFN component of the fusion.
Example 95
Expression of an Albumin Fusion Protein in Mammalian Cells
[1862] The albumin fusion proteins of the present invention can be
expressed in a mammalian cell. A typical mammalian expression
vector contains a promoter element, which mediates the initiation
of transcription of mRNA, a protein coding sequence, and signals
required for the termination of transcription and polyadenylation
of the transcript. Additional elements include enhancers, Kozak
sequences and intervening sequences flanked by donor and acceptor
sites for RNA splicing. Highly efficient transcription is achieved
with the early and late promoters from SV40, the long terminal
repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the
early promoter of the cytomegalovirus (CMV). However, cellular
elements can also be used (e.g., the human actin promoter).
[1863] Suitable expression vectors for use in practicing the
present invention include, for example, vectors such as, pSVL and
pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr
(ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport
3.0. Mammalian host cells that could be used include, but are not
limited to human I Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and
C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells
and Chinese hamster ovary (CHO) cells.
[1864] Alternatively, the albumin fusion protein can be expressed
in stable cell lines containing the polynucleotide encoding the
albumin fusion protein integrated into a chromosome. The
co-transfection with a selectable marker such as DHFR, gpt,
neomycin, or hygromycin allows the identification and isolation of
the transfected cells.
[1865] The transfected polynucleotide encoding the fusion protein
can also be amplified to express large amounts of the encoded
fusion protein. The DHFR (dihydrofolate reductase) marker is useful
in developing cell lines that carry several hundred or even several
thousand copies of the gene of interest (See, e.g., Ali et al., J.
Biol. Chem. 253:1357-1370 (1978); Hamlin et al., Biochem. et
Biophys. Acta. 1097:107-143 (1990); Page et al., Biotechnology
9:64-68 (1991)). Another useful selection marker is the enzyme
glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279
(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using
these markers, the mammalian cells are grown in selective medium
and the cells with the highest resistance are selected. These cell
lines contain the amplified gene(s) integrated into a chromosome.
Chinese hamster ovary (CHO) and NSO cells are often used for the
production of proteins.
[1866] Derivatives of the plasmid pSV2-dhfr (ATCC Accession No.
37146), the expression vectors pC4 (ATCC Accession No. 209646) and
pC6 (ATCC Accession No. 209647) contain the strong promoter (LTR)
of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular
Biology, 438447 (March. 1985)) plus a fragment of the CMV-enhancer
(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites BamHI, XbaI and
Asp718, facilitate the cloning of the gene of interest. The vectors
also contain the 3' intron, the polyadenylation and termination
signal of the rat preproinsulin gene, and the mouse DHFR gene under
control of the SV40 early promoter.
[1867] Specifically, the plasmid pC6, for example, is digested with
appropriate restriction enzymes and then dephosphorylated using
calf intestinal phosphates by procedures known in the art. The
vector is then isolated from a 1% agarose gel.
[1868] A polynucleotide encoding an albumin fusion protein of the
present invention is generated using techniques known in the art
and this polynucleotide is amplified using PCR technology known in
the art. If a naturally occurring signal sequence is used to
produce the fusion protein of the present invention, the vector
does not need a second signal peptide. Alternatively, if a
naturally occurring signal sequence is not used, the vector can be
modified to include a heterologous signal sequence. (See, e.g.,
International Publication No. WO 96/34891.)
[1869] The amplified fragment encoding the fusion protein of the
invention is isolated from a 1% agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Calif.). The
fragment then is digested with appropriate restriction enzymes and
again purified on a 1% agarose gel.
[1870] The amplified fragment encoding the albumin fusion protein
of the invention is then digested with the same restriction enzyme
and purified on a 1% agarose gel. The isolated fragment and the
dephosphorylated vector are then ligated with T4 DNA ligase. E.
coli HB101 or XL-1 Blue cells are then transformed and bacteria are
identified that contain the fragment inserted into plasmid pC6
using, for instance, restriction enzyme analysis.
[1871] Chinese hamster ovary cells lacking an active DHFR gene is
used for transfection. Five .mu.g of the expression plasmid pC6 or
pC4 is cotransfected with 0.5 .mu.g of the plasmid pSVneo using
lipofectin (Feigner et al., supra). The plasmid pSV2-neo contains a
dominant selectable marker, the neo gene from Tn5 encoding an
enzyme that confers resistance to a group of antibiotics including
G418. The cells are seeded in alpha minus MEM supplemented with 1
mg/ml G418. After 2 days, the cells are trypsinized and seeded in
hybridoma cloning plates (Greiner, Germany) in alpha minus MEM
supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml
G418. After about 10-14 days single clones are trypsinized and then
seeded in 6-well petri dishes or 10 ml flasks using different
concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800
nM). Clones growing at the highest concentrations of methotrexate
are then transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 .mu.M, 2 .mu.M, 5 .mu.M, 10 nM,
20 mM). The same procedure is repeated until clones are obtained
which grow at a concentration of 100-200 .mu.M. Expression of the
desired fusion protein is analyzed, for instance, by SDS-PAGE and
Western blot or by reversed phase HPLC analysis.
Example 96
Construct ID 2876, HSA-IFN.alpha. Hybrid
[1872] Construct ID 2876,
pSAC35:HSA.IFN.alpha.A(Cl-Q91)/D(L93-E166) R23K,A113V comprises DNA
encoding an IFN.alpha. hybrid albumin fusion protein which has
mature HSA fused downstream of the HSA/kex2 leader sequence and
upstream of an IFN.alpha. A/D hybrid amino acid sequence, in the
yeast S. cerevisiae expression vector pSAC35. Regarding the
composition of the hybrid IFN, the first 91 amino acids are from
the subtype IFN.alpha.2 (also called IFN.alpha.A) and the remaining
75 as are from IFN.alpha.1 (IFN.alpha.D). We incorporated two point
mutations (R23K, A113V). The fusion was generated by PCR and fused
downstream of HSA within the yeast expression vector pSAC35.
[1873] Results
[1874] CID 2876 Expression and Purification
[1875] The yeast strain BXP-10 was transformed with pSAC35:CID 2876
and a transformant selected for fermentation. A 5-liter
fermentation was performed and analysis of supernatant demonstrated
high expression (approximately 500 mg/l). A small proportion of the
supernatant was processed to pilot purification. Approximately 1 mg
of CID 2876 protein (greater than 95% pure based on N-terminal
sequence) was obtained following a purification through
Blue-sepharose, followed by gel filtration, followed by Q-anion
exchange. The remaining fermentation starting material is available
for further purification if needed.
[1876] ISRE Activity
[1877] All type I IFNs mediate their activities through engagement
of a common IFN receptor complex and activation of the ISRE signal
transduction pathway. Activation of gene transcription through this
pathway leads to the cellular responses associated with IFNs
including anti-proliferation, antiviral and immune modulation.
Using a reporter based strategy, the ability of CID 2876 to
activate the ISRE signal transduction pathway was determined. CID
2876 was found to be a potent activator of the ISRE pathway,
demonstrating an EC.sub.50 of 2.7 ng/ml (data not shown). This
compares favorably with the potency of CID 3165 in this assay
system.
[1878] Anti-Viral Activity
[1879] A hallmark activity of IFNs is their ability to mediate
cellular protection against viral infection. While most human type
I IFNs display antiviral activity in a species restricted manner,
the hybrid IFN employed in this study has been demonstrated to be
active on murine cells. Thus the antiviral activity of CID 2876 was
evaluated on the marine cell line L929 infected with EMCV. Results
indicate that CID 2876 does demonstrate antiviral activity in a
cross species manner (data not shown).
[1880] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
[1881] The entire disclosure of each document cited (including
patents, patent applications, patent publications, journal
articles, abstracts, laboratory manuals, books, or other
disclosures) as well as information available through Identifiers
specific to databases such as GenBank, GeneSeq, or the CAS
Registry, referred to in this application are herein incorporated
by reference in their entirety.
[1882] Furthermore, the specification and sequence listing of each
of the following U.S. applications are herein incorporated by
reference in their entirety: U.S. Application No. 60/341,811, filed
Dec. 21, 2001; U.S. Application No. 60/360,000, filed Feb. 28,
2002; U.S. Application No. 60/378,950, filed May 10, 2002; U.S.
Application No. 60/398,008, filed Jul. 24, 2002; U.S. Application
No. 60/411,355, filed Sep. 18, 2002; U.S. Application No.
60/414,984, filed Oct. 2, 2002; U.S. Application No. 60/417,611,
filed Oct. 11, 2002; U.S. Application No. 60/420,246, filed Oct.
23, 2002; U.S. Application No. 60/423,623, filed Nov. 5, 2002; U.S.
Application No. 60/350,358, filed Jan. 24, 2002; U.S. Application
No. 60/359,370, filed Feb. 26, 2002; U.S. Application No.
60/367,500, filed Mar. 27, 2002; U.S. Application No. 60/402,131,
filed Aug. 9, 2002; U.S. Application No. 60/402,708, filed Aug. 13,
2002; U.S. Application No. 60/370,227, filed Apr. 8, 2002;
International Application No. PCT/US02/40892, filed Dec. 23, 2002;
International Application No. PCT/US02/40891, filed Dec. 23, 2002
and U.S. application Ser. No. 10/775,180 filed Feb. 11, 2004.
Furthermore, the specification and the sequence listing of related
U.S. application Ser. No. 10/775,204, filed concurrently with U.S.
application Ser. No. 10/775,180 on Feb. 11, 2004, is hereby
incorporated by reference in its entirety.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140328794A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140328794A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References