U.S. patent application number 10/515430 was filed with the patent office on 2007-03-22 for modified transferrin fusion proteins.
Invention is credited to Char-Huei Lai, Christopher P. Prior, Homayoun Sadeghi, Andrew J. Turner.
Application Number | 20070066813 10/515430 |
Document ID | / |
Family ID | 31981207 |
Filed Date | 2007-03-22 |
United States Patent
Application |
20070066813 |
Kind Code |
A1 |
Prior; Christopher P. ; et
al. |
March 22, 2007 |
Modified transferrin fusion proteins
Abstract
Modified fusion proteins of transferrin and therapeutic proteins
or peptides including soluble toxin receptors, with increased serum
half-life or serum stability are disclosed. Preferred fusion
proteins include those modified so that the transferrin moiety
exhibits no or reduced glycosylation, binding to iron and/or
binding to the transferrin receptor.
Inventors: |
Prior; Christopher P.; (King
of Prussia, PA) ; Lai; Char-Huei; (Chester Springs,
PA) ; Sadeghi; Homayoun; (King of Prussia, PA)
; Turner; Andrew J.; (King of Prussia, PA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Family ID: |
31981207 |
Appl. No.: |
10/515430 |
Filed: |
August 28, 2003 |
PCT Filed: |
August 28, 2003 |
PCT NO: |
PCT/US03/26818 |
371 Date: |
October 11, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10378094 |
Mar 4, 2003 |
|
|
|
10515430 |
Oct 11, 2005 |
|
|
|
10231494 |
Aug 30, 2002 |
|
|
|
10378094 |
Mar 4, 2003 |
|
|
|
60315745 |
Aug 30, 2001 |
|
|
|
60334059 |
Nov 30, 2001 |
|
|
|
60406977 |
Aug 30, 2002 |
|
|
|
Current U.S.
Class: |
530/400 |
Current CPC
Class: |
C07K 14/47 20130101;
C07K 2319/00 20130101; A61K 38/00 20130101; A61P 35/00 20180101;
C07K 14/79 20130101; A61P 3/10 20180101; C07K 7/06 20130101; A61P
31/18 20180101; C12N 2740/16122 20130101; A01K 2217/05 20130101;
C07K 14/005 20130101; A61K 47/644 20170801; A61P 7/06 20180101;
C07K 14/565 20130101; C07K 2319/035 20130101; C07K 7/08
20130101 |
Class at
Publication: |
530/400 |
International
Class: |
C07K 14/79 20060101
C07K014/79 |
Claims
1. A fusion protein comprising a modified transferrin (Tf) protein
fused to a therapeutic protein or peptide, wherein the Tf protein
exhibits reduced glycosylation, reduced metal binding, or reduced
receptor binding.
2. A fusion protein of claim 1, wherein the therapeutic protein or
peptide is selected from the group comprising .beta.-interferon
(IFN), glucagon-like peptide (GLP-1), erythropoietin mimetic
peptide (EMP1), T-20, insulin, and soluble toxin receptor.
3. A fusion protein of claim 2, wherein the soluble toxin receptor
is synaptotagmin I.
4. A fusion protein of claim 1, wherein the therapeutic protein or
peptide or the soluble toxin receptor is fused to the C-Terminal
end of Tf.
5. A fusion protein of claim 1, wherein the therapeutic protein or
peptide or the soluble toxin receptor is fused to the N-terminal
end of Tf.
6. A fusion protein of claim 1, wherein the therapeutic protein or
peptide or the soluble toxin receptor is inserted into at least one
loop of the Tf.
7. A fusion protein of claim 1, wherein the Tf protein has reduced
affinity for a transferrin receptor (TfR).
8. A fusion protein of claim 1, wherein the Tf protein is
lactoferrin.
9. A fusion protein of claim 7, wherein the Tf protein does not
bind a TfR.
10. A fusion protein of claim 1, wherein the Tf protein has reduced
affinity for iron.
11. A fusion protein of claim 10, wherein the Tf protein does not
bind iron.
12. A fusion protein of claim 1, wherein said Tf protein comprises
at least one mutation that prevents glycosylation.
13. A fusion protein of claim 12, wherein the Tf protein is
lactoferrin.
14. A fusion protein of claim 1, which is expressed in the presence
of tunicamycin.
15. A fusion protein of claim 1, wherein said Tf protein comprises
a portion of the N domain of a Tf protein, a bridging peptide and a
portion of the C domain of a Tf protein.
16. A fusion protein of claim 15, wherein the bridging peptide
links the therapeutic protein or peptide to Tf
17. A fusion protein of claim 15, wherein the therapeutic protein
or peptide is inserted between an N and a C domain of Tf
protein.
18. A fusion protein of claim 1, wherein the Tf protein comprises
at least one amino acid substitution, deletion or addition in the
hinge region.
19. A fusion protein of claim 18, wherein said hinge region is
selected from the group consisting of about residue 94 to about
residue 96 of SEQ ID NO: 3, about residue 245 to about residue 247
of SEQ ID NO: 3, about residue 316 to about residue 318 of SEQ ID
NO: 3, about residue 425 to about residue 427 of SEQ ID NO: 3,
about residue 581 to about residue 582 of SEQ ID NO: 3, and about
residue 652 to about residue 658 of SEQ ID NO: 3.
20. A fusion protein of claim 1, wherein said Tf protein has at
least one amino acid substitution, deletion or addition at a
position in SEQ ID NO: 3 selected from the group consisting of Asp
63, Gly 65, Tyr 95, Tyr 188, Lys 206, His 207, His 249, Asp 392,
Tyr 426, Tyr 514, Tyr 517, His 585, Thr 120, Arg 124, Ala 126, Gly
127, Thr 452, Arg 456, Ala 458 and Gly 459.
21. A fusion protein of claim 6, wherein the therapeutic protein or
peptide replaces at least one loop of Tf.
22. A fusion protein of claim 12, wherein the glycosylation site is
selected from the group consisting of an amino acid residue
corresponding to amino acids N413 of SEQ ID NO: 3 and N611 of SEQ
ID NO: 3.
23. A fusion protein of claim 7 or 9, wherein the Tf comprises at
least one amino acid substitution, deletion or addition at an amino
acid residue corresponding to an amino acid in SEQ D NO: 3 selected
from the group consisting of Asp 63, Gly 65, Tyr 95, Tyr 188, Lys
206, His 207, His 249, Asp 392, Tyr 426, Tyr 514, Tyr 517, His 585,
Thr 120, Arg 124, Ala 126, Gly 127, Thr 452, Arg 456, Ala 458 and
Gly 459.
24. A fusion protein of claim 4, wherein the Tf C-terminal proline
residue is deleted.
25. A fusion protein of claim 4, wherein the Tf C-terminal cysteine
loop is deleted.
26. A fusion protein of claim 1, wherein the serum half-life of the
therapeutic protein or peptide is increased over the serum
half-life of the therapeutic protein or peptide or soluble toxin
receptor in an unfused state.
27. A fusion protein of claim 1, wherein the Tf protein does not
bind a TfR.
28. A fusion protein of claim 1, wherein said Tf protein exhibits
reduced or no glycosylation.
29. A fusion protein of claim 28, comprising at least one mutation
that prevents glycosylation.
30. A nucleic acid molecule encoding a fusion protein of either
claim 1.
31. A vector comprising a nucleic acid molecule of claim 30.
32. A host cell comprising a vector of claim 31.
33. A host cell comprising a nucleic acid molecule of claim 30.
34. A method of expressing a Tf fusion protein comprising culturing
a host cell of claim 32 under conditions which express the encoded
fusion protein.
35. A method of expressing a Tf fusion protein comprising culturing
a host cell of claim 33 under conditions which express the encoded
fusion protein.
36. A host cell of claim 32, wherein the cell is prokaryotic or
eukaryotic.
37. A host cell of claim 33, wherein the cell is prokaryotic or
eukaryotic.
38. A host cell of claim 36, wherein the cell is a yeast cell.
39. A host cell of claim 37, wherein the cell is a yeast cell.
40. A transgenic animal comprising a nucleic acid molecule of
30.
41. A method of producing a Tf fusion protein comprising isolating
a fusion protein from a transgenic animal of claim 40.
42. A method of claim 41, wherein the Tf fusion protein comprises
lactoferrin.
43. A method of claim 42, wherein the fusion protein is isolated
from a biological fluid from the transgenic animal.
44. A method of claim 42, wherein the fluid is serum or milk.
45. A method of treating a disease or disease symptom in a patient,
comprising the step of administering a fusion protein of claim
1.
46. The fusion protein of claim 1, wherein the Tf protein has a
N-terminal domain at each end of the protein.
47. The fusion protein of claim 46, wherein the therapeutic protein
or peptide or the soluble toxin receptor is fused to each
N-terminal domain of the Tf protein.
48. The fusion protein of claim 2, wherein the soluble toxin
receptor binds specifically to a toxin.
49. A fusion protein of claim 2, wherein the therapeutic protein or
peptide is an analog of .beta.-IFN, GLP-1, erythropoietin mimetic
peptide (EMP1), T-20, insulin, or soluble toxin receptor wherein
the analog is effective in treating, preventing, or ameliorating a
disease, condition or disorder.
50. A pharmaceutical composition comprising the fusion protein of
claims 1, 2 or 3, and a carrier.
51. A method of treating a subject comprising administering to the
subject a therapeutically effective amount of a fusion protein of
claim 1.
52. A method of claim 51, wherein the subject is suffering from
multiple sclerosis, brain tumor, skin cancer, hepatitis B, or
hepatitis C, and wherein the fusion protein comprises .beta.-IFN or
an analog thereof.
53. A method of claim 52, wherein the subject is suffering from
multiple sclerosis.
54. A method of claim 51, wherein the subject is suffering from
elevated level of glucose as compared to a healthy subject and
wherein the fusion protein comprises GLP-1 or an analog
thereof.
55. A method of claim 54, wherein the elevated level of glucose is
associated with diabetes.
56. A method of claim 55, wherein the diabetes is Type II
diabetes.
57. A method of claim 51, wherein the subject is suffering from low
or defective red blood cell production as compared to a healthy
subject and wherein the fusion protein comprises EMP1 or an analog
thereof.
58. A method of claim 57, wherein the low or defective red blood
cell production is associated with anemia, .beta.-thalassemia,
pregnancy or menstrual disorders, rheumatoid arthritis, AIDS, and
cancer.
59. A method of claim 51, wherein the subject is suffering from a
disease caused by the transmission of a retrovirus and wherein the
therapeutic peptide is an inhibitor of virus entry.
60. A method of claim 59, wherein the retrovirus is a human
retrovirus.
61. A method of claim 60, wherein the human retrovirus is selected
from the group consisting of HIV-1, HIV-2, and the human
T-lymphocyte virus I (HTLV-1), HTLV-II, and HTLV-III.
62. A method of claim 59, wherein the retrovirus is a non-human
retrovirus.
63. A method of claim 62, wherein the non-human retrovirus is
selected from the group consisting of bovine leukosis virus, feline
sarcoma and leukemia viruses, simian sarcoma and leukemia viruses,
and sheep progress pneumonia viruses.
64. A method of claim 59, wherein the inhibitor is T-20, T-1249 or
an analog thereof.
65. A method of claim 59, wherein the disease is AIDS.
66. A method of preventing or treating a disease or condition
associated with a toxin comprising administering to the subject a
therapeutically effective amount of a fusion protein comprising a
modified Tf protein fused to a soluble toxin receptor, wherein the
modified Tf protein exhibits reduced glycosylation, reduced metal
binding, or reduced receptor binding.
67. A method of claim 66, wherein the soluble toxin receptor is
selected from the group consisting of anthrax toxin receptor,
botulinum toxin receptor, and diptheria toxin receptor.
68. A method of claim 67, wherein the soluble botulinum toxin
receptor is amino acids 1-53 (SEQ ID NO: 4) of synaptotagmin.
69. A fusion protein of claim 1, wherein a therapeutic protein or
peptide is inserted in one or more of the transferrin loops.
70. A fusion protein of claim 69, wherein a therapeutic protein is
inserted in each of the 5 transferrin loops.
71. A fusion protein of claim 2, wherein the GLP-1 further
comprises an additional amino acid at the N-terminus.
72. The fusion protein of claim 71, wherein the amino acid is
Gly.
73. The fusion protein of claim 71, wherein the GLP-1 analog is
fused to modified transferrin protein at the N-terminal end.
74. A method of regulating glucose level in a subject comprising
administering to the subject a therapeutically effective amount of
a fusion protein comprising a modified Tf protein fused to GLP-1 or
analog thereof, wherein the modified Tf protein exhibits reduced
glycosylation, reduced metal binding, or reduced receptor
binding.
75. A fusion protein of claim 1, wherein the therapeutic protein or
peptide is inserted into the N-domain of mTf at one or more of the
sites in SEQ ID NO: 3 selected from the group consisting of Asp33,
Asn55, Asn 75, Asp9O, Gly257, Lys280, His289, Ser298, Ser105,
Glu141, Asp166, Gln184, asp197, Lys217, Thr231, and Cys241.
76. A fusion protein of claim 1, wherein the therapeutic protein or
peptide is inserted into the C-domain of mTf at one or more sites
in SEQ ID NO: 3 corresponding to Asp33, Asn55, Asn 75, Asp90,
Gly257, Lys280, His289, Ser298, Ser105, Glu141, Asp166, Gln184,
asp197, Lys217, Thr231, or Cys241.
77. A fusion protein of claim 75, wherein the therapeutic protein
or peptide is further inserted into the mTf at one or more sites in
SEQ ID NO: 3 corresponding to Asp33, Asn55, Asn 75, Asp90, Gly257,
Lys280, His289, Ser298, Ser105, Glu141, Asp166, Gln184, asp197,
Lys217, Thr231, or Cys241,
78. A fusion protein of claim 2, wherein the therapeutic peptide is
EMP1 and wherein EMP1 is inserted into the N-domain of mTf at one
or more sites selected from the group consisting of His289 of SEQ
ID NO: 3 and Asp166 of SEQ ID NO: 3.
79. A fusion protein of claim 2, wherein the therapeutic peptide is
EMP1 and wherein EMP1 is inserted into the C-domain of mTf at one
or more sites corresponding to His289 of SEQ ID NO: 3 or Asp166 of
SEQ ID NO: 3.
80. A fusion protein of claim 78, wherein the therapeutic peptide
is further inserted into the C-domain of mTf at one or more sites
corresponding to His289 of SEQ ID NO: 3 or Asp166 of SEQ ID NO:
3.
81. A fusion protein of claim 1, wherein mTf is further modified by
deletion of the C-terminus Pro.
82. A fusion protein of claim 81, wherein mTf is further modified
by deletion of Arg-Arg adjacent to the C-terminus Pro.
83. A fusion protein of claim 1, wherein the mTf is further
modified by removing the disulfide bond between Cys402 and Cys674
of SEQ ID NO: 3.
84. A fusion protein of claim 83, wherein the disulfide bond is
removed by mutating Cys402 and Cys674 of SEQ ID NO: 3 into Gly
residues.
85. A fusion protein of claim 82, wherein the mTf is further
modified by mutating Cys402 and Cys674 of SEQ ID NO: 3 into Gly
residues.
86. A fusion protein of claim 1, wherein the therapeutic peptide or
protein is inserted into one or more of the loops in SEQ ID NO: 3
selected from the group consisting of N.sub.1(286-292),
N.sub.2(162-170), C.sub.1(489-495), and C.sub.2(623-628).
87. A fusion protein of claim 1, wherein the modified Tf protein
comprises a single N-terminus domain.
88. A method of claim 51, wherein the subject is suffering from
elevated level of glucose as compared to a healthy subject and
wherein the fusion protein comprises insulin or an analog
thereof.
89. A method of claim 88, wherein the elevated level of glucose is
associated with diabetes.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. application Ser.
No. 10/378,094, filed Mar. 4, 2003, and to U.S. Provisional
Application 60/406,977, filed Aug. 30, 2002, which are incorporated
by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to therapeutic proteins or
peptides and soluble toxin receptor fragments with extended serum
stability or in vivo circulatory half-life fused to or inserted in
a transferrin molecule modified to reduce or inhibit glycosylation,
iron binding and/or transferrin receptor binding. Specifically, the
present invention includes IFN-.beta., GLP-1, EMP1, and T-20 fused
to or inserted in a transferrin molecule or a modified transferrin
molecule. The present invention also includes an anti-toxin fusion
protein comprising a fragment of synaptotagmin 1 fused to or
inserted in a transferrin molecule or a modified transferrin
molecule.
BACKGROUND OF THE INVENTION
[0003] Therapeutic proteins or peptides in their native state or
when recombinantly produced are typically labile molecules
exhibiting short periods of serum stability or short in vivo
circulatory half-lives. In addition, these molecules are often
extremely labile when formulated, particularly when formulated in
aqueous solutions for diagnostic and therapeutic purposes.
[0004] Few practical solutions exist to extend or promote the
stability in vivo or in vitro of proteinaceous therapeutic
molecules. Polyethylene glycol (PEG) is a substance that can be
attached to a protein, resulting in longer-acting, sustained
activity of the protein. If the activity of a protein is prolonged
by the attachment to PEG, the frequency that the protein needs to
be administered may be decreased. PEG attachment, however, often
decreases or destroys the protein's therapeutic activity. While in
some instance PEG attachment can reduce immunogenicity of the
protein, in other instances it may increase immunogenicity.
[0005] Therapeutic proteins or peptides have also been stabilized
by fusion to a protein capable of extending the in vivo circulatory
half-life of the therapeutic protein. For instance, therapeutic
proteins fused to albumin or to antibody fragments may exhibit
extended in vivo circulatory half-life when compared to the
therapeutic protein in the unfused state. See U.S. Pat. Nos.
5,876,969 and 5,766,883.
[0006] Another serum protein, glycosylated human transferrin (Tf)
has also been used to make fusions with therapeutic proteins to
target delivery to the interior of cells or to carry agents across
the blood-brain barrier. These fusion proteins comprising
glycosylated human Tf have been used to target nerve growth factor
(NGF) or ciliary neurotrophic factor (CNTF) across the blood-brain
barrier by fusing full-length Tf to the agent. See U.S. Pat. Nos.
5,672,683 and 5,977,307. In these fusion proteins, the Tf portion
of the molecule is glycosylated and binds to two atoms of iron,
which is required for Tf binding to its receptor on a cell and,
according to the inventors of these patents, to target delivery of
the NGF or CNTF moiety across the blood-brain barrier. Transferrin
fusion proteins have also been produced by inserting an HIV-1
protease target sequence into surface exposed loops of glycosylated
transferrin to investigate the ability to produce another form of
Tf fusion for targeted delivery to the inside of a cell via the Tf
receptor (Ali et al. (1999) J. Biol. Chem.
274(34):24066-24073).
[0007] Serum transferrin (Tf) is a monomeric glycoprotein with a
molecular weight of 80,000 daltons that binds iron in the
circulation and transports it to various tissues via the
transferrin receptor (TfR) (Aisen et al. (1980) Ann. Rev. Biochem.
49: 357-393; MacGillivray et al. (1981) J. Biol. Chem. 258:
3543-3553, U.S. Pat. No. 5,026,651). Tf is one of the most common
serum molecules, comprising up to about 5-10% of total serum
proteins. Carbohydrate deficient transferrin occurs in elevated
levels in the blood of alcoholic individuals and exhibits a longer
half life (approximately 14-17 days) than that of glycosylated
transferrin (approximately 7-10 days). See van Eijk et al. (1983)
Clin. Chim. Acta 132:167-171, Stibler (1991) Clin. Chem.
37:2029-2037 (1991), Arndt (2001) Clin. Chem. 47(1):13-27 and
Stibler et al. in "Carbohydrate-deficient consumption", Advances in
the Biosciences, (Ed Nordmann et al.), Pergamon, 1988, Vol. 71,
pages 353-357).
[0008] The structure of Tf has been well characterized and the
mechanism of receptor binding, iron binding and release and
carbonate ion binding have been elucidated (U.S. Pat. Nos.
5,026,651, 5,986,067 and MacGillivray et al. (1983) J. Biol. Chem.
258(6):3543-3546).
[0009] Transferrin and antibodies that bind the transferrin
receptor have also been used to deliver or carry toxic agents to
tumor cells as cancer therapy (Baselga and Mendelsohn, 1994), and
transferrin has been used as a non-viral gene therapy vector to
deliver DNA to cells (Frank et al., 1994; Wagner et al., 1992). The
ability to deliver proteins to the central nervous system (CNS)
using the transferrin receptor as the entry point has been
demonstrated with several proteins and peptides including CD4
(Walus et al., 1996), brain derived neurotrophic factor (Pardridge
et al., 1994), glial derived neurotrophic factor (Albeck et al.), a
vasointestinal peptide analogue (Bickel et al., 1993), a
beta-amyloid peptide (Saito et al., 1995), and an antisense
oligonucleotide (Pardridge et al., 1995).
[0010] Transferrin fusion proteins have not, however, been modified
or engineered to extend the in vivo circulatory half-life of a
therapeutic protein nor peptide or to increase bioavailability by
reducing or inhibiting glycosylation of the Tf moiety nor to reduce
or prevent iron and/or Tf receptor binding.
SUMMARY OF THE INVENTION
[0011] As described in more detail below, the present invention
includes modified Tf fusion proteins comprising at least one
therapeutic protein, polypeptide or peptide entity, wherein the Tf
portion is engineered to extend the in vivo circulatory half-life
or bioavailability of the molecule. The invention also includes
pharmaceutical formulations and compositions comprising the fusion
proteins, methods of extending the serum stability, in vivo
circulatory half-life and bioavailability of a therapeutic protein
by fusion to modified transferrin, nucleic acid molecules encoding
the modified Tf fusion proteins, and the like. Another aspect of
the present invention relates to methods of treating a patient with
a modified Tf fusion protein.
[0012] Preferably, the modified Tf fusion proteins comprise a human
transferrin Tf moiety that has been modified to reduce or prevent
glycosylation and/or iron and receptor binding.
[0013] The present invention provides fusion proteins comprising
therapeutic proteins fused to or inserted into the transferrin or
modified transferrin molecules. Preferably, the therapeutic
proteins of the present invention include .beta.-interferon
(.beta.-IFN), glucagon-like peptide (GLP-1), EPO (erythropoietin)
mimetic peptide (EMP1), and T-20.
[0014] The present invention also provides fusion proteins
comprising a soluble toxin receptor fragment that bins a toxin
fused to or inserted into the transferrin or modified transferrin
molecules. The soluble toxin receptor may be synaptotagmin 1 and
the soluble fragment is amino acids 1-53 (SEQ ID NO: 4).
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows an alignment of the N and C Domains of Human
(Hu) transferrin (Tf) (SEQ ID NO: 3) with similarities and
identities highlighted.
[0016] FIGS. 2A-2B show an alignment of transferrin sequences from
different species. Light shading: Similarity; Dark shading:
Identity (SEQ ID NOS: 84-90).
[0017] FIG. 3 shows the location of a number of Tf surface exposed
insertion sites for therapeutic proteins, polypeptides or
peptides.
[0018] FIG. 4 shows the pharmacokinetics of mTf-T20 in two
rabbits.
DETAILED DESCRIPTION
General Description
[0019] The present invention is based in part on the finding by the
inventors that therapeutic proteins can be stabilized to extend
their serum half-life and/or activity in vivo by genetically fusing
the therapeutic proteins to transferrin, modified transferrin, or a
portion of transferrin or modified transferrin sufficient to extend
the half-life of the therapeutic protein in serum. The modified
transferrin fusion proteins include a transferrin protein or domain
covalently linked to a therapeutic protein or peptide, wherein the
transferrin portion is modified to contain one or more amino acid
substitutions, insertions or deletions compared to a wild-type
transferrin sequence. In one embodiment, Tf fusion proteins are
engineered to reduce or prevent glycosylation within the Tf or a Tf
domain. In other embodiments, the Tf protein or Tf domain(s) is
modified to exhibit reduced or no binding to iron or carbonate ion,
or to have a reduced affinity or not bind to a Tf receptor
(TfR).
[0020] The therapeutic proteins contemplated by the present
invention include, but are not limited to polypeptides, antibodies,
peptides, or fragments or variants thereof Preferably, the
therapeutic proteins of the present invention include
.beta.-interferon, glucagon-like peptide-1 (GLP-1), EPO mimetic
peptide (EMP1), and T-20.
[0021] The present invention also contemplates anti-toxin fusion
proteins comprising a soluble toxin receptor fragment fused or
inserted into transferrin or modified transferrin. Preferably, the
soluble toxin receptor fragment binds a specific toxin. In one
embodiment, the soluble toxin receptor fragment is amino acids 1-53
(SEQ ID NO: 4) of synaptotagmin 1.
[0022] The present invention therefore includes transferrin fusion
proteins, therapeutic compositions comprising the fusion proteins,
and methods of treating, preventing, or ameliorating diseases or
disorders by administering the fusion proteins. A transferrin
fusion protein of the invention includes at least a fragment or
variant of a therapeutic protein and at least a fragment or variant
of modified transferrin, which are associated with one another,
preferably by genetic fusion (i.e., the transferrin 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 modified transferrin) or chemical conjugation to one another.
The therapeutic protein and transferrin protein, once part of the
transferrin fusion protein, may be referred to as a "portion",
"region" or "moiety" of the transferrin fusion protein (e.g., a
"therapeutic protein portion` or a "transferrin protein
portion").
[0023] In one embodiment, the invention provides a transferrin
fusion protein comprising, or alternatively consisting of, a
therapeutic protein and a modified serum transferrin protein. In
other embodiments, the invention provides a transferrin fusion
protein comprising, or alternatively consisting of, a biologically
active and/or therapeutically active fragment of a therapeutic
protein and a modified transferrin protein. In other embodiments,
the invention provides a transferrin fusion protein comprising, or
alternatively consisting of, a biologically active and/or
therapeutically active variant of a therapeutic protein and
modified transferrin protein. In further embodiments, the invention
provides a transferrin fusion protein comprising a therapeutic
protein, and a biologically active and/or therapeutically active
fragment of modified transferrin. In another embodiment, the
therapeutic protein portion of the transferrin fusion protein is
the active form of the therapeutic protein.
[0024] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
Definitions
[0025] As used herein, an "amino acid corresponding to" or an
"equivalent amino acid" in a transferrin sequence is identified by
alignment to maximize the identity or similarity between a first
transferrin sequence and at least a second transferrin sequence.
The number used to identify an equivalent amino acid in a second
transferrin sequence is based on the number used to identify the
corresponding amino acid in the first transferrin sequence. In
certain cases, these phrases may be used to describe the amino acid
residues in human transferrin compared to certain residues in
rabbit serum transferrin.
[0026] As used herein, the term "biological activity" refers to a
function or set of activities performed by a therapeutic molecule,
protein or peptide in a biological context (i.e., in an organism or
an in vitro facsimile thereof). Biological activities may include
but are not limited to the functions of the therapeutic molecule
portion of the claimed fusion proteins, such as, but not limited
to, the induction of extracellular matrix secretion from responsive
cell lines, the induction of hormone secretion, the induction of
chemotaxis, the induction of mitogenesis, the induction of
differentiation, or the inhibition of cell division of responsive
cells. A fusion protein or peptide of the invention is considered
to be biologically active if it exhibits one or more biological
activities of its therapeutic protein's native counterpart.
[0027] As used herein, "binders" are agents used to impart cohesive
qualities to the powdered material. Binders, or "granulators" as
they are sometimes known, impart a cohesiveness to the tablet
formulation, which insures the tablet remaining intact after
compression, as well as improving the free-flowing qualities by the
formulation of granules of desired hardness and size. Materials
commonly used as binders include starch; gelatin; sugars, such as
sucrose, glucose, dextrose, molasses, and lactose; natural and
synthetic gums, such as acacia, sodium alginate, extract of Irish
moss, panwar gum, ghatti gum, mucilage of isapol husks,
carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone,
Veegum, microcrystalline cellulose, microcrystalline dextrose,
amylose, and larch arabogalactan, and the like.
[0028] As used herein, the term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which a composition 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.
[0029] As used herein, "coloring agents" are agents that give
tablets a more pleasing appearance, and in addition help the
manufacturer to control the product during its preparation and help
the user to identify the product. Any of the approved certified
water-soluble FD&C dyes, mixtures thereof, or their
corresponding lakes may be used to color tablets. A color lake is
the combination by adsorption of a water-soluble dye to a hydrous
oxide of a heavy metal, resulting in an insoluble form of the
dye.
[0030] As used herein, "diluents" are inert substances added to
increase the bulk of the formulation to make the tablet a practical
size for compression. Commonly used diluents include calcium
phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium
chloride, dry starch, powdered sugar, silica, and the like.
[0031] As used herein, "disintegrators" or "disintegrants" are
substances that facilitate the breakup or disintegration of tablets
after administration. Materials serving as disintegrants have been
chemically classified as starches, clays, celluloses, algins, or
gums. Other disintegrators include Veegum HV, methylcellulose,
agar, bentonite, cellulose and wood products, natural sponge,
cation-exchange resins, alginic acid, guar gum, citrus pulp,
cross-linked polyvinylpyrrolidone, carboxymethylcellulose, and the
like.
[0032] The term "dispersibility" or "dispersible" means a dry
powder having a moisture content of less than about 10% by weight
(% w) water, usually below about 5% w and preferably less than
about 3% w; a particle size of about 1.0-5.0 .mu.m mass median
diameter (MMD), usually 1.0-4.0 .mu.m MMD, and preferably 1.0-3.0
.mu.m MMD; a delivered dose of about >30%, usually >40%,
preferably >50%, and most preferred >60%; and an aerosol
particle size distribution of 1.0-5.0 .mu.m mass median aerodynamic
diameter (MMAD), usually 1.5-4.5 .mu.m MMAD, and preferably 1.5-4.0
.mu.m MMAD.
[0033] The term "dry" means that the composition has a moisture
content such that the particles are readily dispersible in an
inhalation device to form an aerosol. This moisture content is
generally below about 10% by weight (% w) water, usually below
about 5% w and preferably less than about 3% w.
[0034] As used herein, "effective amount" means an amount of a drug
or pharmacologically active agent that is sufficient to provide the
desired local or systemic effect and performance at a reasonable
benefit/risk ratio attending any medical treatment.
[0035] As used herein, "flavoring agents" vary considerably in
their chemical structure, ranging from simple esters, alcohols, and
aldehydes to carbohydrates and complex volatile oils. Synthetic
flavors of almost any desired type are now available.
[0036] As used herein, the terms "fragment of a Tf protein" or "Tf
protein," or "portion of a Tf protein" refer to an amino acid
sequence comprising at least about 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a naturally
occurring Tf protein or mutant thereof.
[0037] As used herein, the term "gene" refers to any segment of DNA
associated with a biological function. Thus, genes include, but are
not limited to, coding sequences and/or the regulatory sequences.
required for their expression. Genes can also include non-expressed
DNA segments that, for example, form recognition sequences for
other proteins. Genes can be obtained from a variety of sources,
including cloning from a source of interest or synthesizing from
known or predicted sequence information, and may include sequences
designed to have desired parameters.
[0038] As used herein, a "heterologous polynucleotide" or a
"heterologous nucleic acid" or a "heterologous gene" or a
"heterologous sequence" or an "exogenous DNA segment" refers to a
polynucleotide, nucleic acid or DNA segment that originates from a
source foreign to the particular host cell, or, if from the same
source, is modified from its original form. A heterologous gene in
a host cell includes a gene that is endogenous to the particular
host cell, but has been modified. Thus, the terms refer to a DNA
segment which is foreign or heterologous to the cell, or homologous
to the cell but in a position within the host cell nucleic acid in
which the element is not ordinarily found. As an example, a signal
sequence native to a yeast cell but attached to a human Tf sequence
is heterologous.
[0039] As used herein, an "isolated" nucleic acid sequence refers
to a nucleic acid sequence which is essentially free of other
nucleic acid sequences, e.g., at least about 20% pure, preferably
at least about 40% pure, more preferably about 60% pure, even more
preferably about 80% pure, most preferably about 90% pure, and even
most preferably about 95% pure, as determined by agarose gel
electrophoresis. For example, an isolated nucleic acid sequence can
be obtained by standard cloning procedures used in genetic
engineering to relocate the nucleic acid sequence from its natural
location to a different site where it will be reproduced. The
cloning procedures may involve excision and isolation of a desired
nucleic acid fragment comprising the nucleic acid sequence encoding
the polypeptide, insertion of the fragment into a vector molecule,
and incorporation of the recombinant vector into a host cell where
multiple copies or clones of the nucleic acid sequence will be
replicated. The nucleic acid sequence may be of genomic, cDNA, RNA,
semi-synthetic, synthetic origin, or any combinations thereof.
[0040] As used herein, two or more DNA coding sequences are said to
be "joined" or "fused" when, as a result of in-frame fusions
between the DNA coding sequences, the DNA coding sequences are
translated into a fusion polypeptide. The term "fusion" in
reference to Tf fusions includes, but is not limited to, attachment
of at least one therapeutic protein, polypeptide or peptide to the
N-terminal end of Tf, attachment to the C-terminal end of Tf,
and/or insertion between any two amino acids within Tf.
[0041] As used herein, "lubricants" are materials that perform a
number of functions in tablet manufacture, such as improving the
rate of flow of the tablet granulation, preventing adhesion of the
tablet material to the surface of the dies and punches, reducing
interparticle friction, and facilitating the ejection of the
tablets from the die cavity. Commonly used lubricants include talc,
magnesium stearate, calcium stearate, stearic acid, and
hydrogenated vegetable oils. Typical amounts of lubricants range
from about 0.1% by weight to about 5% by weight.
[0042] As used herein, "Modified transferrin" as used herein refers
to a transferrin molecule that exhibits at least one modification
of its amino acid sequence, compared to wild-type transferrin.
[0043] As used herein, "Modified transferrin fusion protein" as
used herein refers to a protein formed by the fusion of at least
one molecule of modified transferrin (or a fragment or variant
thereof) to at least one molecule of a therapeutic protein (or
fragment or variant thereof).
[0044] As used herein, the terms "nucleic acid" or "polynucleotide"
refer to deoxyribonucleotides or ribonucleotides and polymers
thereof in either single- or double-stranded form. Unless
specifically limited, the terms encompass nucleic acids containing
analogues of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.
degenerate codon substitutions) and complementary sequences as well
as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J.
Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al.
(1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used
interchangeably with gene, cDNA, and mRNA encoded by a gene.
[0045] As used herein, a DNA segment is referred to as "operably
linked" when it is placed into a functional relationship with
another DNA segment. For example, DNA for a signal sequence is
operably linked to DNA encoding a fusion protein of the invention
if it is expressed as a preprotein that participates in the
secretion of the fusion protein; a promoter or enhancer is operably
linked to a coding sequence if it stimulates the transcription of
the sequence. Generally, DNA sequences that are operably linked are
contiguous, and in the case of a signal sequence or fusion protein
both contiguous and in reading phase. However, enhancers need not
be contiguous with the coding sequences whose transcription they
control. Linking, in this context, is accomplished by ligation at
convenient restriction sites or at adapters or linkers inserted in
lieu thereof.
[0046] As used herein, "pharmaceutically acceptable" refers to
materials and compositions that are physiologically tolerable and
do not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human. Typically, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans.
[0047] As used herein, "physiologically effective amount" is that
amount delivered to a subject to give the desired palliative or
curative effect. This amount is specific for each drug and its
ultimate approved dosage level.
[0048] As used herein, the term "powder" means a composition that
consists of finely dispersed solid particles that are free flowing
and capable of being readily dispersed in an inhalation device and
subsequently inhaled by a subject so that the particles reach the
lungs to permit penetration into the alveoli. Thus, the powder is
said to be "respirable." Preferably the average particle size is
less than about 10 microns (.mu.m) in diameter with a relatively
uniform spheroidal shape distribution. More preferably the diameter
is less than about 7.5 .mu.m and most preferably less than about
5.0 .mu.m. Usually the particle size distribution is between about
0.1 .mu.m and about 5 .mu.m in diameter, particularly about 0.3
.mu.m to about 5 .mu.m.
[0049] As used herein, the term "promoter" refers to a region of
DNA involved in binding RNA polymerase to initiate
transcription.
[0050] As used herein, the term "recombinant" refers to a cell,
tissue or organism that has undergone transformation with a new
combination of genes or DNA.
[0051] As used herein, the term "subject" can be a human, a mammal,
or an animal. The subject being treated is a patient in need of
treatment.
[0052] As used herein, a targeting entity, protein, polypeptide or
peptide refers to a molecule that binds specifically to a
particular cell type [normal (e.g., lymphocytes) or abnormal e.g.,
(cancer cell)] and therefore may be used to target a Tf fusion
protein or compound (drug, or cytotoxic agent) to that cell type
specifically.
[0053] As used herein, "tablets" are solid pharmaceutical dosage
forms containing drug substances with or without suitable diluents
and prepared either by compression or molding methods well known in
the art. Tablets have been in widespread use since the latter part
of the 19.sup.th century and their popularity continues. Tablets
remain popular as a dosage form because of the advantages afforded
both to the manufacturer (e.g., simplicity and economy of
preparation, stability, and convenience in packaging, shipping, and
dispensing) and the patient (e.g., accuracy of dosage, compactness,
portability, blandness of taste, and ease of administration).
Although tablets are most frequently discoid in shape, they may
also be round, oval, oblong, cylindrical, or triangular. They may
differ greatly in size and weight depending on the amount of drug
substance present and the intended method of administration. They
are divided into two general classes, (1) compressed tablets, and
(2) molded tablets or tablet triturates. In addition to the active
or therapeutic ingredient or ingredients, tablets contain a number
or inert materials or additives. A first group of such additives
includes those materials that help to impart satisfactory
compression characteristics to the formulation, including diluents,
binders, and lubricants. A second group of such additives helps to
give additional desirable physical characteristics to the finished
tablet, such as disintegrators, colors, flavors, and sweetening
agents.
[0054] As used herein, the term "therapeutically effective amount"
refers to that amount of the transferrin fusion protein comprising
a therapeutic molecule which, when administered to a subject in
need thereof, is sufficient to effect treatment. The amount of
transferrin fusion protein which constitutes a "therapeutically
effective amount" will vary depending on the therapeutic protein
used, the severity of the condition or disease, and the age and
body weight of the subject to be treated, but can be determined
routinely by one or ordinary skill in the art having regard to
his/her own knowledge and to this disclosure.
[0055] As used herein, "therapeutic protein" refers to proteins,
polypeptides, 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. Additionally, the term "therapeutic protein" may refer to
the endogenous or naturally occurring correlate of a therapeutic
protein. 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. As a non-limiting example, a "therapeutic
protein" is a protein that is useful to treat, prevent or
ameliorate a disease, condition or disorder. Such a disease,
condition or disorder may be in humans or in a non-human animal,
e.g., veterinary use.
[0056] As used herein, the term "toxin" refers to a poisonous
substance of biological origin.
[0057] As used herein, the term "transformation" refers to the
transfer of nucleic acid (i.e., a nucleotide polymer) into a cell.
As used herein, the term "genetic transformation" refers to the
transfer and incorporation of DNA, especially recombinant DNA, into
a cell.
[0058] As used herein, the term "transformant" refers to a cell,
tissue or organism that has undergone transformation.
[0059] As used herein, the term "transgene" refers to a nucleic
acid that is inserted into an organism, host cell or vector in a
manner that ensures its function.
[0060] As used herein, the term "transgenic" refers to cells, cell
cultures, organisms, bacteria, fungi, animals, plants, and progeny
of any of the preceding, which have received a foreign or modified
gene and in particular a gene encoding a modified Tf fusion protein
by one of the various methods of transformation, wherein the
foreign or modified gene is from the same or different species than
the species of the organism receiving the foreign or modified
gene.
[0061] "Variants or 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. As used herein, "variant"
refers to a therapeutic protein portion of a transferrin fusion
protein of the invention, differing in sequence from a native
therapeutic protein but retaining at least one functional and/or
therapeutic property thereof as described elsewhere herein or
otherwise known in the art.
[0062] As used herein, the term "vector" refers broadly to any
plasmid, phagemid or virus encoding an exogenous nucleic acid. The
term is also be construed to include non-plasmid, non-phagemid and
non-viral compounds which facilitate the transfer of nucleic acid
into virions or cells, such as, for example, polylysine compounds
and the like. The vector may be a viral vector that is suitable as
a delivery vehicle for delivery of the nucleic acid, or mutant
thereof, to a cell, or the vector may be a non-viral vector which
is suitable for the same purpose. Examples of viral and non-viral
vectors for delivery of DNA to cells and tissues are well known in
the art and are described, for example, in Ma et al. (1997, Proc.
Natl. Acad. Sci. U.S.A. 94:12744-12746). Examples of viral vectors
include, but are not limited to, a recombinant vaccinia virus, a
recombinant adenovirus, a recombinant retrovirus, a recombinant
adeno-associated virus, a recombinant avian pox virus, and the like
(Cranage et al., 1986, EMBO J. 5:3057-3063; International Patent
Application No. WO 94/17810, published Aug. 18, 1994; International
Patent Application No. WO 94/23744, published Oct. 27, 1994).
Examples of non-viral vectors include, but are not limited to,
liposomes, polyamine derivatives of DNA, and the like.
[0063] As used herein, the term "wild type" refers to a
polynucleotide or polypeptide sequence that is naturally
occurring.
[0064] Transferrin and Transferrin Modifications
[0065] The present invention provides fusion proteins comprising
therapeutic protein or soluble toxin receptor fragment and
transferrin or modified transferrin. Preferably, the therapeutic
proteins provided by the present invention include .beta.-IFN,
GLP-1, EMP1, and T-20. Preferably, the soluble toxin receptor
fragment is amino acids 1-53 (SEQ ID NO: 4) of synaptotagmin 1. Any
transferrin may be used to make modified Tf fusion proteins of the
invention.
[0066] Any transferrin may be used to make modified Tf fusion
proteins of the invention. As an example, the wild-type human Tf
(Tf) is a 679 amino acid protein of approximately 75 kDa (not
accounting for glycosylation), with two main domains, N (about 330
amino acids) and C (about 340 amino acids), which appear to
originate from a gene duplication. See GenBank accession numbers
NM.sub.--001063, XM.sub.--002793, M12530, XM.sub.--039845,
XM.sub.--039847 and S95936 (www.ncbi.nlm.nih.gov/), all of which
are herein incorporated by reference in their entirety, as well as
SEQ ID NOS 1, 2 and 3. The two domains have diverged over time but
retain a large degree of identity/similarity (FIG. 1).
[0067] Each of the N and C domains is further divided into two
subdomains, N1 and N2, C1 and C2. The function of Tf is to
transport iron to the cells of the body. This process is mediated
by the Tf receptor (TfR), which is expressed on all cells,
particularly actively growing cells. TfR recognizes the iron bound
form of Tf (two molecules of which are bound per receptor),
endocytosis then occurs whereby the TfR/Tf complex is transported
to the endosome, at which point the localized drop in pH results in
release of bound iron and the recycling of the TfR/Tf complex to
the cell surface and release of Tf (known as apoTf in its
iron-unbound form). Receptor binding is through the C domain of Tf.
The two glycosylation sites in the C domain do not appear to be
involved in receptor binding as unglycosylated iron bound Tf does
bind the receptor.
[0068] Each Tf molecule can carry two iron ions (Fe.sup.3+). These
are complexed in the space between the N1 and N2, C1 and C2 sub
domains resulting in a conformational change in the molecule. Tf
crosses the blood brain barrier (BBB) via the Tf receptor.
[0069] In human transferrin, the iron binding sites comprise at
least amino acids Asp 63 (Asp 82 of SEQ ID NO: 2 which includes the
native Tf signal sequence), Asp 392 (Asp 411 of SEQ ID NO: 2), Tyr
95 (Tyr 114 of SEQ ID NO: 2), Tyr 426 (Tyr 445 of SEQ ID NO: 2),
Tyr 188 (Tyr 207 of SEQ ID NO: 2), Tyr 514 or 517 (Tyr 533 or Tyr
536 SEQ ID NO: 2), His 249 (His 268 of SEQ ID NO: 2), and His 585
(His 604 of SEQ ID NO: 2) of SEQ ID NO: 3. The hinge regions
comprise at least N domain amino acid residues 94-96, 245-247
and/or 316-318 as well as C domain amino acid residues 425-427,
581-582 and/or 652-658 of SEQ ID NO: 3. The carbonate binding sites
comprise at least amino acids Thr 120 (Thr 139 of SEQ ID NO: 2),
Thr 452 (Thr 471 of SEQ ID NO: 2), Arg 124 (Arg 143 of SEQ ID NO:
2), Arg 456 (Arg 475 of SEQ ID NO: 2), Ala 126 (Ala 145 of SEQ ID
NO: 2), Ala 458 (Ala 477 of SEQ ID NO: 2), Gly 127 (Gly 146 of SEQ
ID NO: 2), and Gly 459 (Gly 478 of SEQ ID NO: 2) of SEQ ID NO:
3.
[0070] In one embodiment of the invention, the modified transferrin
fusion protein includes a modified human transferrin, although any
animal Tf molecule may be used to produce the fusion proteins of
the invention, including human Tf variants, cow, pig, sheep, dog,
rabbit, rat, mouse, hamster, echnida, platypus, chicken, frog,
hornworm, monkey, as well as other bovine, canine and avian
species. All of these Tf sequences are readily available in GenBank
and other public databases. The human Tf nucleotide sequence is
available (see SEQ ID NOS 1, 2 and 3 and the accession numbers
described above and available at www.ncbi.nlm.nih.gov/) and can be
used to make genetic fusions between Tf or a domain of Tf and the
therapeutic molecule of choice. Fusions may also be made from
related molecules such as lacto transferrin (lactoferrin) GenBank
Acc: NM.sub.--002343) or melanotransferrin (GenBank Acc.
NM.sub.--013900, murine melanotransferrin).
[0071] Melanotransferrin is a glycosylated protein found at high
levels in malignant melanoma cells and was originally named human
melanoma antigen p97 (Brown et al., 1982, Nature, 296: 171-173). It
possesses high sequence homology with human serum transferrin,
human lactoferrin, and chicken transferrin (Brown et al., 1982,
Nature, 296: 171-173; Rose et al., Proc. Natl. Acad. Sci. USA,
1986, 83: 1261-1265). However, unlike these receptors, no cellular
receptor has been identified for melanotransferrin.
Melanotransferrin reversibly binds iron and it exists in two forms,
one of which is bound to cell membranes by a glycosyl
phosphatidylinositol anchor while the other form is both soluble
and actively secreted (Baker et al., 1992, FEBS Lett, 298: 215-218;
Alemany et al., 1993, J. Cell Sci., 104: 1155-1162; Food et al.,
1994, J. Biol. Chem. 274: 7011-7017).
[0072] Lactoferrin (Lf), a natural defense iron-binding protein,
has been found to possess antibacterial, antimycotic, antiviral,
antineoplastic and anti-inflammatory activity. The protein is
present in exocrine secretions that are commonly exposed to normal
flora: milk, tears, nasal exudate, saliva, bronchial mucus,
gastrointestinal fluids, cervico-vaginal mucus and seminal fluid.
Additionally, Lf is a major constituent of the secondary specific
granules of circulating polymorphonuclear neutrophils (PMNs). The
apoprotein is released on degranulation of the PMNs in septic
areas. A principal function of Lf is that of scavenging free iron
in fluids and inflamed areas so as to suppress free
radical-mediated damage and decrease the availability of the metal
to invading microbial and neoplastic cells. In a study that
examined the turnover rate of .sup.125I Lf in adults, it was shown
that Lf is rapidly taken up by the liver and spleen, and the
radioactivity persisted for several weeks in the liver and spleen
(Bennett et al. (1979), Clin. Sci. (Lond.) 57: 453-460).
[0073] In one embodiment, the transferrin portion of the
transferrin fusion protein of the invention includes a transferrin
splice variant. In one example, a transferrin splice variant can be
a splice variant of human transferrin. In one specific embodiment,
the human transferrin splice variant can be that of Genbank
Accession AAA61140.
[0074] In another embodiment, the transferrin portion of the
transferrin fusion protein of the invention includes a lactoferrin
splice variant. In one example, a human serum lactoferrin splice
variant can be a novel splice variant of a neutrophil lactoferrin.
In one specific embodiment, the neutrophil lactoferrin splice
variant can be that of Genbank Accession AAA59479. In another
specific embodiment, the neutrophil lactoferrin splice variant can
comprise the following amino acid sequence EDCIALKGEADA (SEQ ID NO:
8), which includes the novel region of splice-variance.
[0075] In another embodiment, the transferrin portion of the
transferrin fusion protein of the invention includes a
melanotransferrin variant.
[0076] Modified Tf fusions may be made with any Tf protein,
fragment, domain, or engineered domain. For instance, fusion
proteins may be produced using the full-length Tf sequence, with or
without the native Tf signal sequence. Tf fusion proteins may also
be made using a single Tf domain, such as an individual N or C
domain or a modified form of Tf comprising 2N or 2C domains (see
U.S. Provisional Application 60/406,977, filed Aug. 30, 2002, which
is herein incorporated by reference in its entirety). In some
embodiments, fusions of a therapeutic protein to a single C domain
may be produced, wherein the C domain is altered to reduce, inhibit
or prevent glycosylation. In other embodiments, the use of a single
N domain is advantageous as the Tf glycosylation sites reside in
the C domain and the N domain, on its own. A preferred embodiment
is the Tf fusion protein having a single N domain which is
expressed at a high level.
[0077] As used herein, a C terminal domain or lobe modified to
function as an N-like domain is modified to exhibit glycosylation
patterns or iron binding properties substantially like that of a
native or wild-type N domain or lobe. In a preferred embodiment,
the C domain or lobe is modified so that it is not glycosylated and
does not bind iron by substitution of the relevant C domain regions
or amino acids to those present in the corresponding regions or
sites of a native or wild-type N domain.
[0078] As used herein, a Tf moiety comprising "two N domains or
lobes" includes a Tf molecule that is modified to replace the
native C domain or lobe with a native or wild-type N domain or lobe
or a modified N domain or lobe or contains a C domain that has been
modified to function substantially like a wild-type or modified N
domain.
[0079] Analysis of the two domains by overlay of the two domains
(Swiss PDB Viewer 3.7b2, Iterative Magic Fit) and by direct amino
acid alignment (ClustalW multiple alignment) reveals that the two
domains have diverged over time. Amino acid alignment shows 42%
identity and 59% similarity between the two domains. However,
approximately 80% of the N domain matches the C domain for
structural equivalence. The C domain also has several extra
disulfide bonds compared to the N domain.
[0080] Alignment of molecular models for the N and C domain reveals
the following structural equivalents: TABLE-US-00001 N 4- 36- 94-
138- 149- 168- 178- 219- 259- 263- 271- 279- 283- 309- domain 24 72
136 139 164 173 198 255 260 268 275 280 288 327 (1-330) 75- 200-
290- 88 214 304 C 340- 365- 425- 470- 475- 492- 507- 555- 593- 597-
605- 614- 620- 645- domain 361 415 437 471 490 497 542 591 594 602
609 615 640 663 (340-679) 439- 468
[0081] The disulfide bonds for the two domains align as follows:
TABLE-US-00002 N C C339-C596 C9-C48 C345-C377 C19-C39 C355-C368
C402-C674 C418-C637 C118-C194 C450-C523 C137-C331 C474-C665
C158-C174 C484-C498 C161-C179 C171-C177 C495-C506 C227-C241
C563-C577 C615-C620 Bold aligned disulfide bonds Italics bridging
peptide
[0082] In one embodiment, the transferrin portion of the
transferrin fusion protein includes at least two N terminal lobes
of transferrin. In further embodiments, the transferrin portion of
the transferrin fusion protein includes at least two N terminal
lobes of transferrin derived from human serum transferrin.
[0083] In another embodiment, the transferrin portion of the
transferrin fusion protein includes, comprises, or consists of at
least two N terminal lobes of transferrin having a mutation in at
least one amino acid residue selected from the group consisting of
Asp63, Gly65, Tyr95, Tyr188, and His249 of SEQ ID NO: 3.
[0084] In another embodiment, the transferrin portion of the
modified transferrin fusion protein includes a recombinant human
serum transferrin N-terminal lobe mutant having a mutation at
Lys206 or His207 of SEQ ID NO: 3.
[0085] In another embodiment, the transferrin portion of the
transferrin fusion protein includes, comprises, or consists of at
least two C terminal lobes of transferrin. In further embodiments,
the transferrin portion of the transferrin fusion protein includes
at least two C terminal lobes of transferrin derived from human
serum transferrin.
[0086] In a further embodiment, the C terminal lobe mutant further
includes a mutation of at least one of Asn413 and Asn611 of SEQ ID
NO: 3 which does not allow glycosylation.
[0087] In another embodiment, the transferrin portion of the
transferrin fusion protein includes at least two C terminal lobes
of transferrin having a mutation in at least one amino acid residue
selected from the group consisting of Asp392, Tyr426, Tyr514,
Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant retains the
ability to bind metal. In an alternate embodiment, the transferrin
portion of the transferrin fusion protein includes at least two C
terminal lobes of transferrin having a mutation in at least one
amino acid residue selected from the group consisting of Tyr426,
Tyr514, Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant has a
reduced ability to bind metal. In another embodiment, the
transferrin portion of the transferrin fusion protein includes at
least two C terminal lobes of transferrin having a mutation in at
least one amino acid residue selected from the group consisting of
Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO:3, wherein the
mutant does not retain the ability to bind metal and functions
substantially like an N domain.
[0088] In some embodiments, the Tf or Tf portion will be of
sufficient length to increase the in vivo circulatory half-life,
serum stability, in vitro solution stability or bioavailability of
the therapeutic protein or peptide or soluble toxin receptor
compared to the in vivo circulatory half-life, serum stability, in
vitro solution stability or bioavailability of the therapeutic
protein or peptide or soluble toxin receptor in an unfused state.
Such an increase in stability, serum half-life or bioavailability
may be about a 30%, 50%, 70%, 80%, 90% or more increase over the
unfused therapeutic protein or peptide or soluble toxin receptor.
In some cases, the transferrin fusion proteins comprising modified
transferrin exhibit a serum half-life of about 10-20 or more days,
about 12-18 days or about 14-17 days.
[0089] When the C domain of Tf is part of the fusion protein, the
two N-linked glycosylation sites, amino acid residues corresponding
to N413 and N611 of SEQ ID NO: 3 may be mutated for expression in a
yeast system to prevent glycosylation or hypermannosylationn and
extend the serum half-life of the fusion protein and/or therapeutic
protein (to produce asialo-, or in some instances, monosialo-Tf or
disialo-Tf). In addition to Tf amino acids corresponding to N413
and N611, mutations may be to the adjacent residues within the
N--X--S/T glycosylation site to prevent or substantially reduce
glycosylation. See U.S. Pat. No. 5,986,067 of Funk et al. It has
also been reported that the N domain of Tf expressed in Pichia
pastoris becomes O-linked glycosylated with a single hexose at S32
which also may be mutated or modified to prevent such
glycosylation.
[0090] Accordingly, in one embodiment of the invention, the
transferrin fusion protein includes a modified transferrin molecule
wherein the transferrin exhibits reduced glycosylation, including
but not limited to asialo- monosialo- and disialo-forms of Tf. In
another embodiment, the transferrin portion of the transferrin
fusion protein includes a recombinant transferrin mutant that is
mutated to prevent glycosylation. In another embodiment, the
transferrin portion of the transferrin fusion protein includes a
recombinant transferrin mutant that is fully glycosylated. In a
further embodiment, the transferrin portion of the transferrin
fusion protein includes a recombinant human serum transferrin
mutant that is mutated to prevent glycosylation, wherein at least
one of Asn413 and Asn611 of SEQ ID NO: 3 are mutated to an amino
acid which does not allow glycosylation. In another embodiment, the
transferrin portion of the transferrin fusion protein includes a
recombinant human serum transferrin mutant that is mutated to
prevent or substantially reduce glycosylation, wherein mutations
may be to the adjacent residues within the N--X--S/T glycosylation
site. Moreover, glycosylation may be reduced or prevented by
mutating the serine or threonine residue. Further, changing the X
to proline is known to inhibit glycosylation.
[0091] As discussed below in more detail, modified Tf fusion
proteins of the invention may also be engineered to not bind iron
and/or bind the Tf receptor. In other embodiments of the invention,
the iron binding is retained and the iron binding ability of Tf may
be used to deliver a therapeutic protein or peptide(s) to the
inside of a cell, across an epithelial or endothelial cell membrane
and/or across the BBB. These embodiments that bind iron and/or the
Tf receptor will often be engineered to reduce or prevent
glycosylation to extend the serum half-life of the therapeutic
protein. The N domain alone will not bind to TfR when loaded with
iron, and the iron bound C domain will bind TfR but not with the
same affinity as the whole molecule.
[0092] In another embodiment, the transferrin portion of the
transferrin fusion protein includes a recombinant transferrin
mutant having a mutation wherein the mutant does not retain the
ability to bind metal ions. In an alternate embodiment, the
transferrin portion of the transferrin fusion protein includes a
recombinant transferrin mutant having a mutation wherein the mutant
has a weaker binding avidity for metal ions than wild-type serum
transferrin. In an alternate embodiment, the transferrin portion of
the transferrin fusion protein includes a recombinant transferrin
mutant having a mutation wherein the mutant has a stronger binding
avidity for metal ions than wild-type serum transferrin.
[0093] In another embodiment, the transferrin portion of the
transferrin fusion protein includes a recombinant transferrin
mutant having a mutation wherein the mutant does not retain the
ability to bind to the transferrin receptor. In an alternate
embodiment, the transferrin portion of the transferrin fusion
protein includes a recombinant transferrin mutant having a mutation
wherein the mutant has a weaker binding avidity for the transferrin
receptor than wild-type serum transferrin. In an alternate
embodiment, the transferrin portion of the transferrin fusion
protein includes a recombinant transferrin mutant having a mutation
wherein the mutant has a stronger binding avidity for the
transferrin receptor than wild-type serum transferrin.
[0094] In another embodiment, the transferrin portion of the
transferrin fusion protein includes a recombinant transferrin
mutant having a mutation wherein the mutant does not retain the
ability to bind to carbonate ions. In an alternate embodiment, the
transferrin portion of the transferrin fusion protein includes a
recombinant transferrin mutant having a mutation wherein the mutant
has a weaker binding avidity for carbonate ions than wild-type
serum transferrin. In an alternate embodiment, the transferrin
portion of the transferrin fusion protein includes a recombinant
transferrin mutant having a mutation wherein the mutant has a
stronger binding avidity for carbonate ions than wild-type serum
transferrin.
[0095] In another embodiment, the transferrin portion of the
transferrin fusion protein includes a recombinant human serum
transferrin mutant having a mutation in at least one amino acid
residue selected from the group consisting of Asp63, Gly65, Tyr95,
Tyr188, His249, Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID
NO: 3, wherein the mutant retains the ability to bind metal ions.
In an alternate embodiment, a recombinant human serum transferrin
mutant having a mutation in at least one amino acid residue
selected from the group consisting of Asp63, Gly65, Tyr95, Tyr188,
His249, Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3,
wherein the mutant has a reduced ability to bind metal ions. In
another embodiment, a recombinant human serum transferrin mutant
having a mutation in at least one amino acid residue selected from
the group consisting of Asp63, Gly65, Tyr95, Tyr188, His249,
Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO: 3, wherein the
mutant does not retain the ability to bind metal ions.
[0096] In another embodiment, the transferrin portion of the
transferrin fusion protein includes a recombinant human serum
transferrin mutant having a mutation at Lys206 or His207 of SEQ ID
NO:3, wherein the mutant has a stronger binding avidity for metal
ions than wild-type human serum transferrin (see U.S. Pat. No.
5,986,067, which is herein incorporated by reference in its
entirety). In an alternate embodiment, the transferrin portion of
the transferrin fusion protein includes a recombinant human serum
transferrin mutant having a mutation at Lys206 or His207 of SEQ ID
NO:3, wherein the mutant has a weaker binding avidity for metal
ions than wild-type human serum transferrin. In a further
embodiment, the transferrin portion of the transferrin fusion
protein includes a recombinant human serum transferrin mutant
having a mutation at Lys206 or His207 of SEQ ID NO:3, wherein the
mutant does not bind metal ions.
[0097] Any available technique may be used to produce the
transferrin fusion proteins of the invention, including but not
limited to molecular techniques commonly available, for instance,
those disclosed in Sambrook et al. Molecular Cloning: A Laboratory
Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989. When
carrying out nucleotide substitutions using techniques for
accomplishing site-specific mutagenesis that are well known in the
art, the encoded amino acid changes are preferably of a minor
nature, that is, conservative amino acid substitutions, although
other, non-conservative, substitutions are contemplated as well,
particularly when producing a modified transferrin portion of a Tf
fusion protein, e.g., a modified Tf protein exhibiting reduced
glycosylation, reduced iron binding and the like. Specifically
contemplated are amino acid substitutions, small deletions or
insertions, typically of one to about 30 amino acids; insertions
between transferrin domains; small amino- or carboxyl-terminal
extensions, such as an amino-terminal methionine residue, or small
linker peptides of less than 50, 40, 30, 20 or 10 residues between
transferrin domains or linking a transferrin protein and
therapeutic protein or peptide or soluble toxin receptor or a small
extension that facilitates purification, such as a poly-histidine
tract, an antigenic epitope or a binding domain.
[0098] Examples of conservative amino acid substitutions are
substitutions made within the same group such as within the group
of basic amino acids (such as arginine, lysine, histidine), acidic
amino acids (such as glutamic acid and aspartic acid), polar amino
acids (such as glutamine and asparagine), hydrophobic amino acids
(such as leucine, isoleucine, valine), aromatic amino acids (such
as phenylalanine, tryptophan, tyrosine) and small amino acids (such
as glycine, alanine, serine, threonine, methionine).
[0099] Non-conservative substitutions encompass substitutions of
amino acids in one group by amino acids in another group. For
example, a non-conservative substitution would include the
substitution of a polar amino acid for a hydrophobic amino acid.
For a general description of nucleotide substitution, see e.g. Ford
et al. (1991), Prot. Exp. Pur. 2: 95-107. Non-conservative
substitutions, deletions and insertions are particularly useful to
produce Tf fusion proteins of the invention that exhibit no or
reduced binding of iron, no or reduced binding of the fusion
protein to the Tf receptor and/or no or reduced glycosylation.
[0100] Iron binding and/or receptor binding may be reduced or
disrupted by mutation, including deletion, substitution or
insertion into, amino acid residues corresponding to one or more of
Tf N domain residues Asp63, Tyr95, Tyr188, His249 and/or C domain
residues Asp 392, Tyr 426, Tyr 514 and/or His 585 of SEQ ID NO: 3.
Iron binding may also be affected by mutation to amino acids
Lys206, His207 or Arg632 of SEQ ID NO: 3. Carbonate binding may be
reduced or disrupted by mutation, including deletion, substitution
or insertion into, amino acid residues corresponding to one or more
of Tf N domain residues Thr120, Arg124, Ala126, Gly 127 and/or C
domain residues Thr 452, Arg 456, Ala 458 and/or Gly 459 of SEQ ID
NO: 3. A reduction or disruption of carbonate binding may adversely
affect iron and/or receptor binding.
[0101] Binding to the Tf receptor may be reduced or disrupted by
mutation, including deletion, substitution or insertion into, amino
acid residues corresponding to one or more of Tf N domain residues
described above for iron binding.
[0102] As discussed above, glycosylation may be reduced or
prevented by mutation, including deletion, substitution or
insertion into, amino acid residues corresponding to one or more of
Tf C domain residues around the N--X--S/T sites corresponding to C
domain residues N413 and/or N611 (See U.S. Pat. No. 5,986,067). For
instance, the N413 and/or N611 may be mutated to Glu residues.
[0103] In instances where the Tf fusion proteins of the invention
are not modified to prevent glycosylation, iron binding, carbonate
binding and/or receptor binding, glycosylation, iron and/or
carbonate ions may be stripped from or cleaved off of the fusion
protein. For instance, available deglycosylases may be used to
cleave glycosylation residues from the fusion protein, in
particular the sugar residues attached to the Tf portion, yeast
deficient in glycosylation enzymes may be used to prevent
glycosylation and/or recombinant cells may be grown in the presence
of an agent that prevents glycosylation, e.g., tunicamycin.
[0104] The carbohydrates on the fusion protein may also be reduced
or completely removed enzymatically by treating the fusion protein
with deglycosylases. Deglycosylases are well known in the art.
Examples of deglycosylases include but are not limited to
galactosidase, PNGase A, PNGase F, glucosidase, mannosidase,
fucosidase, and Endo H deglycosylase.
[0105] Nevertheless, in certain circumstances, it may be preferable
for oral delivery that the Tf portion of the fusion protein be
fully glycosylated
[0106] Additional mutations may be made with Tf to alter the three
dimensional structure of Tf, such as modifications to the hinge
region to prevent the conformational change needed for iron biding
and Tf receptor recognition. For instance, mutations may be made in
or around N domain amino acid residues 94-96, 245-247 and/or
316-318 as well as C domain amino acid residues 425-427, 581-582
and/or 652-658. In addition, mutations may be made in or around the
flanking regions of these sites to alter Tf structure and
function.
[0107] In one aspect of the invention, the transferrin fusion
protein can function as a carrier protein to extend the half life
or bioavailability of the therapeutic protein as well as, in some
instances, delivering the therapeutic protein inside a cell and/or
across the blood brain barrier. In an alternate embodiment, the
transferrin fusion protein includes a modified transferrin molecule
wherein the transferrin does not retain the ability to cross the
blood brain barrier.
[0108] In another embodiment, the transferrin fusion protein
includes a modified transferrin molecule wherein the transferrin
molecule retains the ability to bind to the transferrin receptor
and transport the therapeutic peptide inside cells. In an alternate
embodiment, the transferrin fusion protein includes a modified
transferrin molecule wherein the transferrin molecule does not
retain the ability to bind to the transferrin receptor and
transport the therapeutic peptide inside cells.
[0109] In further embodiments, the transferrin fusion protein
includes a modified transferrin molecule wherein the transferrin
molecule retains the ability to bind to the transferrin receptor
and transport the therapeutic peptide inside cells and retains the
ability to cross the blood brain barrier. In an alternate
embodiment, the transferrin fusion protein includes a modified
transferrin molecule wherein the transferrin molecule retains the
ability to cross the blood brain barrier, but does not retain the
ability to bind to the transferrin receptor and transport the
therapeutic peptide inside cells.
[0110] Modified Transferrin Fusion Proteins
[0111] The fusion of proteins of the invention may contain one or
more copies of the therapeutic protein or polypeptide or soluble
toxin receptor attached to the N-terminus and/or the C-terminus of
the Tf protein. In some embodiments, the therapeutic protein or
polypeptide or soluble toxin receptor is attached to both the N-
and C-terminus of the Tf protein and the fusion protein may contain
one or more equivalents of the therapeutic protein or polypeptide
on either or both ends of Tf. In other embodiments, the therapeutic
protein or polypeptide is inserted into known domains of the Tf
protein, for instance, into one or more of the loops of Tf (see Ali
et al. (1999) J. Biol. Chem. 274(34):24066-24073). In fact, the
therapeutic protein or polypeptide may be inserted into all five
loops of transferrin to create a pentavalent molecule with
increased avidity for the antigen, receptor, or targeting molecule,
which the therapeutic protein binds. In other embodiments, the
therapeutic protein or polypeptide is inserted between the N and C
domains of Tf. Alternatively, the therapeutic protein or
polypeptide is inserted anywhere in the transferrin molecule.
[0112] Generally, the transferrin fusion protein of the invention
may have one modified transferrin-derived region and one
therapeutic protein region. Multiple regions of each protein,
however, may be used to make a transferrin fusion protein of the
invention. Similarly, more than one therapeutic protein may be used
to make a transferrin fusion protein of the invention, thereby
producing a multi-functional modified Tf fusion protein.
[0113] In one embodiment, the transferrin fusion protein of the
invention contains a therapeutic protein or polypeptide or portion
thereof or a soluble toxin receptor fused to a transferrin molecule
or portion thereof. In another embodiment, the transferrin fusion
protein of the inventions contains a therapeutic protein or
polypeptide fused to the N terminus of a transferrin molecule. In
an alternate embodiment, the transferrin fusion protein of the
invention contains a therapeutic protein or polypeptide fused to
the C terminus of a transferrin molecule. In a further embodiment,
the transferrin fusion protein of the invention contains a
transferrin molecule fused to the N terminus of a therapeutic
protein or polypeptide. In an alternate embodiment, the transferrin
fusion protein of the invention contains a transferrin molecule
fused to the C terminus of a therapeutic protein or
polypeptide.
[0114] In other embodiments, the transferrin fusion protein of the
inventions contains a therapeutic protein fused to both the
N-terminus and the C-terminus of modified transferrin. In another
embodiment, the therapeutic proteins fused at the N- and C-termini
bind the same therapeutic proteins. In an alternate embodiment, the
therapeutic proteins fused at the N- and C-termini are different
therapeutic proteins. In another alternate embodiment, the
therapeutic proteins fused to the N- and C-termini bind different
therapeutic proteins which may be used to treat or prevent the same
disease, disorder, or condition. In another embodiment, the
therapeutic proteins fused at the N- and C-termini are different
therapeutic proteins which may be used to treat or prevent diseases
or disorders which are known in the art to commonly occur in
patients simultaneously.
[0115] In addition to modified transferrin fusion protein of the
invention in which the modified transferrin portion is fused to the
N terminal and/or C-terminal of the therapeutic protein portion,
transferrin fusion protein of the invention may also be produced by
inserting the therapeutic protein or peptide of interest (e.g., a
therapeutic protein or peptide as disclosed herein, or a fragment
or variant thereof) into an internal region of the modified
transferrin. Internal regions of modified transferrin include, but
are not limited to, the iron binding sites, the hinge regions, the
bicarbonate binding sites, or the receptor binding domain.
[0116] Within the protein sequence of the modified transferrin
molecule a number of loops or turns exist, which are stabilized by
disulfide bonds. 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 generate a modified transferrin molecule
with specific biological activity.
[0117] When therapeutic proteins are inserted into or replace at
least one loop of a Tf molecule, insertions may be made within any
of the surface exposed loop regions, in addition to other areas of
Tf. For instance, insertions may be made within the loops
comprising Tf amino acids 32-33, 74-75, 256-257, 279-280 and
288-289 (Ali et al., supra) (See FIG. 3). As previously described,
insertions may also be made within other regions of Tf such as the
sites for iron and bicarbonate binding, hinge regions, and the
receptor binding domain as described in more detail below. The
loops in the Tf protein sequence that are amenable to
modification/replacement for the insertion of proteins or peptides
may also be used for the development of a screenable library of
random peptide inserts. Any procedures may be used to produce
nucleic acid inserts for the generation of peptide libraries,
including available phage and bacterial display systems, prior to
cloning into a Tf domain and/or fusion to the ends of Tf.
[0118] The N-terminus of Tf is free and points away from the body
of the molecule. Fusions of proteins or peptides on the N-terminus
may therefore be a preferred embodiment. Such fusions may include a
linker region, such as but not limited to a poly-glycine stretch,
to separate the therapeutic protein from Tf. Attention to the
junction between the leader sequence, the choice of leader
sequence, and the structure of the mRNA by codon
manipulation/optimization (no major stem loops to inhibit ribosome
progress) will increase secretion and can be readily accomplished
using standard recombinant protein techniques.
[0119] The C-terminus of Tf appears to be more buried and secured
by a disulfide bond 6 amino acids from the C-terminus. In human Tf,
the C-terminal amino acid is a proline which, depending on the way
that it is orientated, will either point a fusion away or into the
body of the molecule. A linker or spacer moiety at the C-terminus
may be used in some embodiments of the invention. There is also a
proline near the N-terminus. In one aspect of the invention, the
proline at the N- and/or the C-termini may be changed out. In
another aspect of the invention, the C-terminal disulfide bond may
be eliminated to untether the C-terminus.
[0120] In yet other embodiments, small molecule therapeutics may be
complexed with iron and loaded on a modified Tf protein fusion for
delivery to the inside of cells and across the BBB. The addition of
a targeting peptide or, for example, a single chain antibody (SCA)
can be used to target the payload to a particular cell type, e.g.,
a cancer cell.
[0121] Therapeutic Proteins and Peptides
[0122] Any therapeutic molecule may be used as the fusion partner
to Tf according to the methods and compositions of the present
invention. As used herein, a therapeutic molecule is typically a
protein or peptide capable of exerting a beneficial biological
effect in vitro or in vivo and includes proteins or peptides that
exert a beneficial effect in relation to normal homeostasis,
physiology or a disease state. Therapeutic molecules do not include
fusion partners commonly used as markers or protein purification
aids, such as bacterial galactosidases (see for example, U.S. Pat.
No. 5,986,067 and Aldred et al. (1984) Biochem. Biophys. Res.
Commun. 122: 960-965). For instance, a beneficial effect as related
to a disease state includes any effect that is advantageous to the
treated subject, including disease prevention, disease
stabilization, the lessening or alleviation of disease symptoms or
a modulation, alleviation or cure of the underlying defect to
produce an effect beneficial to the treated subject.
[0123] A modified transferrin fusion protein of the invention
includes at least a fragment or variant of a therapeutic protein
and at least a fragment or variant of modified serum transferrin,
which are associated with one another, preferably by genetic
fusion.
[0124] In one embodiment, the transferrin fusion protein includes a
modified transferrin molecule linked to a neuropharmaceutical
agent. In another embodiment, the modified transferrin fusion
protein includes transferrin at the carboxyl terminus linked to a
neuropharmaceutical agent at the amino terminus. In an alternate
embodiment, the modified transferrin fusion protein includes
transferrin at the amino terminus linked to a neuropharmaceutical
agent at the carboxy terminus. In specific embodiments, the
neuropharmaceutical agent is either nerve growth factor or ciliary
neurotrophic factor.
[0125] In further embodiments, a modified transferrin fusion
protein of the invention may contain at least a fragment or variant
of a therapeutic protein. In a further embodiment, the transferrin
fusion proteins can contain peptide fragments or peptide variants
of proteins or antibodies wherein the variant or fragment retains
at least one biological or therapeutic activity. The transferrin
fusion proteins can contain therapeutic proteins that can be
peptide fragments or peptide variants at least about 4, at least 5,
at least 6, 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 about
40, at least about 50, at least about 55, at least about 60 or at
least about 70 or more amino acids in length fused to the N and/or
C termini, inserted within, or inserted into a loop of a modified
transferrin.
[0126] The modified transferrin fusion proteins of the present
invention may contain one or more peptides. Increasing the number
of peptides enhances the function of the peptides fused to
transferrin and the function of the entire transferrin fusion
protein. The peptides may be used to make a bi- or multi-functional
fusion protein by including peptide or protein domains with
multiple functions. For instance, a multi-functional fusion protein
can be made with a therapeutic protein and a second protein to
target the fusion protein to a specific target. Other peptides may
be used to induce the immune response of a cellular system, or
induce an antiviral, antibacterial, or anti-pathogenic
response.
[0127] In another embodiment, the modified transferrin fusion
molecules contain a therapeutic protein portion that can be
fragments of a therapeutic protein that include the full length
protein as well as polypeptides having one or more residues deleted
from the amino terminus of the amino acid sequence.
[0128] In another embodiment, the modified transferrin fusion
molecules contain a therapeutic protein portion that can be
fragments of a therapeutic protein that include the full length
protein as well as polypeptides having one or more residues deleted
from the carboxy terminus of the amino acid sequence.
[0129] In another embodiment, the modified transferrin fusion
molecules contain a therapeutic protein portion that can have one
or more amino acids deleted from both the amino and the carboxy
termini.
[0130] In another embodiment, the modified transferrin fusion
molecules contain a therapeutic protein portion that is at least
about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a
reference therapeutic protein set forth herein, or fragments
thereof. In further embodiments, the transferrin fusion molecules
contain a therapeutic protein portion that is at least about 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.
[0131] In another embodiment, the modified transferrin fusion
molecules contain the therapeutic protein portion that is at least
about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to,
for example, the native or wild-type amino acid sequence of a
therapeutic protein. Fragments, of these polypeptides are also
provided.
[0132] The therapeutic proteins corresponding to a therapeutic
protein portion of a modified transferrin fusion protein of the
invention, such as cell surface and secretory proteins, can be
modified by the attachment of one or more oligosaccharide groups.
The modification referred to as glycosylation can significantly
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 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 an amino acid
except proline. 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. For
example, several types of human interferon are glycosylated.
[0133] Therapeutic proteins corresponding to a therapeutic protein
portion of a transferrin 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
glycosylation-deficient yeast. These approaches are known in the
art.
[0134] Therapeutic proteins and their nucleic acid sequences are
well known in the art and available in public databases such as
Chemical Abstracts Services Databases (e.g., the CAS Registry),
GenBank, and GenSeq. The Accession Numbers and sequences referred
to below are herein incorporated by reference in their
entirety.
[0135] In other embodiments, the transferrin fusion proteins of the
invention are capable of a therapeutic activity and/or biologic
activity, corresponding to the therapeutic activity and/or biologic
activity of the therapeutic protein described elsewhere in this
application. In further embodiments, the therapeutically active
protein portions of the transferrin fusion proteins of the
invention are fragments or variants of the reference sequences
cited herein.
[0136] The present invention is further directed to modified Tf
fusion proteins comprising fragments of the therapeutic proteins
herein described. 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 portion,
other therapeutic activities and/or functional activities (e.g.,
biological activities, 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 with less than the majority
of the residues of the complete polypeptide removed from the
N-terminus. Whether a particular polypeptide lacking N-terminal
residues of a complete polypeptide retains such immunologic
activities can be assayed by routine methods described herein and
otherwise known in the art. It is not unlikely that a mutant 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.
[0137] Also as mentioned above, even if deletion of one or more
amino acids from the N-terminus or C-terminus of a therapeutic
protein results in modification or loss of one or more biological
functions of the protein, other functional activities (e.g.,
biological activities, 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.
[0138] Peptide fragments of the therapeutic proteins can be
fragments comprising, or alternatively, consisting of, an amino
acid sequence that displays a therapeutic activity and/or
functional activity (e.g. biological activity) of the polypeptide
sequence of the therapeutic protein of which the amino acid
sequence is a fragment.
[0139] The peptide fragments of the therapeutic protein may
comprise only the N- and C-termini of the protein, i.e., the
central portion of the therapeutic protein has been deleted.
Alternatively, the peptide fragments may comprise non-adjacent
and/or adjacent portions of the central part of the therapeutic
protein.
[0140] Other polypeptide fragments are biologically active
fragments. Biologically active fragments are those exhibiting
activity similar, but not necessarily identical, to an activity of
a therapeutic protein used in the present invention. The biological
activity of the fragments may include an improved desired activity,
or a decreased undesirable activity.
[0141] Generally, variants of proteins 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 a transferrin fusion protein of the invention. Nucleic acids
encoding these variants are also encompassed by the invention.
[0142] Further therapeutic polypeptides that may be used in the
invention are polypeptides encoded by polynucleotides which
hybridize to the complement of a nucleic acid molecule encoding an
amino acid sequence of a therapeutic protein under stringent
hybridization conditions which are known to those of skill in the
art. (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). Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0143] By a polypeptide-having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, 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.
[0144] As a practical matter, whether any particular polypeptide is
at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical
to, for instance, the amino acid sequence of a transferrin fusion
protein of the invention or a fragment thereof (such, as the
therapeutic protein portion of the transferrin fusion protein or
the transferrin portion of the transferrin 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 Brufiag et al. (Comp. App. Biosci 245 (1990)).
[0145] The polynucleotide variants of the invention may contain
alterations in the coding regions, non-coding regions, or both.
Polynucleotide variants containing alterations which produce silent
substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide may be used to
produce modified Tf fusion proteins. Nucleotide variants produced
by silent substitutions due to the degeneracy of the genetic code
can be utilized. Moreover, polypeptide variants in which less than
about 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 can also be utilized.
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 host, such as,
yeast or E. coli as described above).
[0146] In other embodiments, the therapeutic protein moiety has
conservative substitutions compared to the wild-type sequence. By
"conservative substitutions" is intended swaps within groups such
as replacement of the aliphatic or hydrophobic amino acids Ala,
Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr;
replacement of the acidic residues Asp and Glu; replacement of the
amide residues Asn and Gln, replacement of the basic residues Lys,
Arg, and His; replacement of the aromatic residues Phe, Tyr, and
Trp, and replacement of the small-sized amino acids Ala, Ser, Thr,
Met, and Gly. Guidance concerning how to make phenotypically silent
amino acid substitutions is provided, for example, in Bowie et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990). In specific
embodiments, the polypeptides of the invention comprise, or
alternatively, consist of, fragments or variants of the amino acid
sequence of a therapeutic protein described herein and/or serum
transferrin, and/modified transferrin protein of the invention,
wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50
or 50-150 amino acid residue additions, substitutions, and/or
deletions when compared to the reference amino acid sequence. In
further embodiments, the amino acid substitutions are conservative.
Nucleic acids encoding these polypeptides are also encompassed by
the invention.
[0147] The modified fusion proteins of the present invention can be
composed of amino-acids joined to each other by peptide bonds or
modified peptide bonds and may contain amino acids other than the
20 gene-encoded amino acids. The polypeptides may be modified by
either natural processes, such as post-translational processing, or
by chemical modification techniques which are well known in the
art. Such modifications are well described in basic texts and in
more detailed monographs, as well as in a voluminous research
literature.
[0148] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxy termini. 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. Also, a given polypeptide may
contain many types of modifications. Polypeptides 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 heme 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, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristylation,
oxidation, pegylation, proteolytic processing, phosphorylation,
prenylation, racemization, 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. (1990) Meth. Enzymol. 182:626-646;
Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62.
[0149] The therapeutic proteins of the present invention include,
but are not limited to polypeptide, peptide, antibody, or fragments
and variants thereof. Preferably, the therapeutic proteins of the
present invention include .beta.-interferon (.beta.-IFN),
glucagon-like peptide-1 (GLP-1), EPO nimetic peptide (EMP-1), T-20,
and soluble toxin receptor, such as synaptotagmin I.
[0150] .beta.-Interferon
[0151] Most cytokines, including .beta.-IFN, have relatively short
circulation half-lives since they are produced in vivo to act
locally and transiently. To use .beta.-IFN as an effective systemic
therapeutic, one needs relatively large doses and frequent
administrations. Such frequent parenteral administrations are
inconvenient and painful. Further, toxic side effects are
associated with .beta.-IFN administration which are so severe that
some multiple sclerosis patients cannot tolerate the treatment.
These side effects are probably associated with administration of a
high dosage.
[0152] The present invention provides .beta.-IFN/transferrin fusion
proteins with increased half-lives and pharmaceutical compositions
comprising such fusion proteins with increased stability. Such
fusion proteins can be administered to patients at lower doses,
thus reducing the toxic side effects associated with .beta.-IFN.
The present invention contemplates the use of the
.beta.-IFN/transferrin fusion proteins to treat various diseases
and conditions associated with .beta.-IFN, such as but not limited
to multiple sclerosis, cancer including brain tumors and skin
cancer, and viral infections such as hepatitis B and C. Preferably,
the .beta.-IFN/transferrin fusion proteins are used to treat
subjects suffering from multiple sclerosis.
[0153] .beta.-interferon (.beta.-IFN) is a glycoprotein with an
apparent molecular weight (MW) of 23 kilodaltons. The gene encoding
.beta.-IFN is located on chromosome 9. Its amino acid sequence
containing 166 residues was determined by K. Hosoi et al. (J.
Interferon Res., 8, pp 375-384 (1988)), and its glucoside sequence
was reported by Y. Kagawa et al. (J. Biol. Chem., 263, pp
17508-17515 (1988)).
[0154] .beta.-IFN is secreted by fibroblasts in response to a viral
or bacterial infection, or exposure to foreign cells,
macromolecules, or RNA. In particular, .beta.-IFN inhibits the
proliferation of infected cells and stimulates the immune system.
The specific antiviral activity of homogeneous Hu-.beta.-IFN is
considered to be between 3.times.10.sup.8 and 1.times.10.sup.9
iu/mg (international units per milligram of total protein)
inclusive (see U.S. Pat. No. 4,289,689 and EP-A-94 672).
[0155] "Interferon-beta" (IFN-.beta.) or "beta-interferon"
(.beta.-IFN) includes native and recombinant Type I interferons
exhibiting the same or similar pharmaceutical characteristics as
the Type I interferons commonly known as IFN-.beta.-1a and
IFN-.beta.-1b.
[0156] Any .beta.-IFN sequence may be used to prepare Tf fusion
proteins of the present invention. For instance, U.S. Pat. No.
4,738,931 discloses the human .beta.-IFN gene derived from human
chromosomal DNA. A 1.8 kb EcoRI fragment, containing the nucleic
acid encoding the human .beta.-IFN, introduced into Escherichia
coli has been deposited with the American Type Culture Collection
in U.S.A. as Escherichia coli CI4 under accession number ATCC
31905. The GenBank accession number for the amino acid sequence of
Human .beta.-IFN amino acid sequence is AAA72588. The .beta.-IFN
could also be a mutein as described in U.S. Pat. No. 4,588,585, in
which the cysteine (Cys) normally occurring at position 17 of the
wild-type or native molecule has been replaced by a neutral amino
acid, such as serine or alanine. Mark et al. (Proc. Natl. Acad.
Sci. USA 81: 5662-5666 (1984)) showed that when Cys 17 was changed
for serine, the IFN exhibited the same spectrum of biological
activities as .beta.-IFN, such as anticellular and
antiproliferative activity, activation of NK cells and
neutralization of anti-human IFN antibodies. The mutein also
exhibited greater stability than natural human (Hu) .beta.-IFN when
incubated at 70.degree. C.
[0157] Because of its activity, .beta.-IFN is regarded as an active
principle not only in the treatment and prophylaxis of viral
diseases such as herpes, influenza etc, but also in the treatment
of tumoral conditions such as encephaloma and leukemia. .beta.-IFN
is used to treat multiple sclerosis, brain tumor, skin cancer and
hepatitis B and C. .beta.-IFN fusion proteins of the present
invention may be used to treat any of these diseases.
[0158] Human .beta.-IFN is also effective in treating coronary
restenosis in humans by selectively inhibiting the proliferation of
coronary smooth muscle cell at the site of vascular injury
following a surgical procedure while having no inhibitory effect on
the normal proliferation of coronary endothelial cells following
the procedure. U.S. Pat. No. 5,681,558 discloses a method of
treating restenosis comprising administering .beta.-IFN to the
patient. Accordingly, .beta.-IFN fusion proteins of the present
invention may be used to treat restenosis.
[0159] .beta.-IFN has an erythropoietic effect on the growth of
progenitor cells from individuals suffering from several diseases
with a very low production of red blood cells. Additionally,
.beta.-IFN increases burst formation as well as promotes a more
rapid maturation toward normoblasts and even late reticulocytes.
U.S. Pat. No. 5,104,653 discloses a method for the stimulation of
erythropoiesis in a patient suffering from a disorder characterized
by lack of maturation of progenitor blood cells to red blood cells
comprising administering to said patient an erythropoietic
effective amount of human .beta.-IFN. Therefore, .beta.-IFN fusion
proteins of the present invention may be used to stimulate
erythropoiesis.
[0160] .beta.-IFN, acting via STAT1 and STAT2, is known to
upregulate and downregulate a wide variety of genes, most of which
are involved in the antiviral immune response. Although most IFN
responses are induced by the presence of dsRNA, both DNA and RNA
viruses are sensitive to the effects of .beta.-IFN (Biron, Seminars
in Immunology, 10: 383-390 (1998)).
[0161] .beta.-IFN is generally produced in response to a viral
infection. Interferon .beta.-IFN exerts its biological effects by
binding to specific receptors on the surface of human cells. This
binding initiates a complex cascade of intracellular events that
leads to the expression of numerous interferon-induced gene
products and markers, for example, 2', 5'-oligoadenylate
synthetase, b.sub.2-microglobulin, and neopterin.
[0162] (2'-5')-Oligoadenylate synthetase and dsRNA dependent
protein kinase are the two best-known IFN-.beta.-induced proteins
(Biron,1998, supra). (2'-5')-oligoadenylate synthetase polymerizes
ATP in a unique 2'-5' fashion (Janeway et al., Immunobiology: The
Immune System in Health and Disease, 4th Edition, New York,
Elsevier Science/Garland Publishing pp 385-386(1999)); the
resultant oligomers activate RNase L, which cleaves mRNA (Biron,
1998, supra). dsRNA dependent protein kinase phosphorylates and
inactivates elF2, a transcriptional initiator. Both
(2'-5')-oligoadenylate synthetase and dsRNA dependent protein
kinase act only in the presence of dsRNA, i.e. in virally infected
cells. The net result of the action of these two proteins is to
inhibit protein translation, which will retard viral replication
(Biron, 1998, supra).
[0163] .beta.-IFN dependent upregulation of TAP (transporter
associated with antigen processing), Lmp2, Lmp7 serves to increase
presentation of viral peptides by MHC class I molecules in order to
facilitate CD8 T cell recognition and destruction of infected
cells. TAP is the molecule responsible for loading peptide
fragments onto MHC class I molecules in the ER; the Lmp proteins
are components of the proteasome which cleave proteins specifically
for MHC class I presentation (Janeway et al., 1999, supra).
[0164] .beta.-IFN is known to both activate and induce some
proliferation in natural killer (NK) cells (Janeway et al., 1999,
supra). However, interferons themselves are not mitogens. The
proliferation of NK cells is probably caused by an intermediary
cytokine which is induced by IFN-.beta. (Biron, 1998, supra). NK
cells can kill cells which exhibit atypical patterns of MHC class I
expression; such cells are generally virally infected (Janeway et
al, 1999, supra).
[0165] Although at the end of a successfully defeated infection, T
cells die by apoptosis as the immune system returns to a
homeostatic balance, some T cells must avoid apoptosis and enter a
G.sub.0/G.sub.1 memory state to preserve immunological memory.
These memory T cells are rescued from apoptosis by interacting with
stromal cells, which secrete .beta.-IFN and some IFN-.alpha.
(Pilling et al., European Journal of Immunology 29:1041-1050
(1999)). T cell apoptosis may be induced by either cytokine
deprivation or ligation of Fas on the cell surface, but .beta.-IFN
is able to block both apoptotic pathways. The former apoptotic
pathway is blocked by .beta.-IFN dependent upregulation of Bcl-x,
an apoptotic inhibitor. Fas ligation-induced apoptosis occurs much
too quickly to be blocked by upregulation of a gene, so .beta.-IFN
must block that apoptotic pathway by different means
(Scheel-Toellner et al., European Journal of Immunology
29:2603-2612 (1999)). The existence of a second blocking mechanism
is supported by the results of Marrack et al. (Journal of
Experimental Medicine 189:521-529(1999)), who found that .beta.-IFN
prevented T cell apoptosis without increased production of
Bcl-x.
[0166] Der et al. (Proc. Nat. Acad. Sci., USA 95: 15623-15628
(1998)) found that .beta.-IFN increased transcription of well over
100 proteins in human fibrosarcoma cells. Induced proteins ranged
in function from cytochromes and cell scaffolding proteins to
immunologically active proteins such as Complement components and
dsRNA adenosine deaminase. These results indicate that .beta.-IFN
has truly pleiotropic effects, many of which are not fully
understood.
[0167] Much clinical research on .beta.-IFN is currently focused on
its use as a treatment for multiple sclerosis (MS). MS is an
autoimmune disease in which T cells mount an immune response
against self myelin antigens in the glial cells of the central
nervous system (Goodkin, 1999. Multiple sclerosis: Treatment
options for patients with relapsing-remitting and secondary
progressive multiple sclerosis.
<http://www.msnews.org/goodkin1.sub.--99.htm>). In 1993, the
FDA approved subcutaneous injections of IFN-.beta.1b for treatment
of MS (Revelle M., 1993, FDA licenses interferon beta-1b.
(<http://www.fda.gov/bbs/topics/NEWS/NEW00424.html>).
.beta.-IFN 1b is a non-glycosylated form of IFN-.beta. produced in
E. coli (Arduini et al., Protein Science 8: 1867-1877 (1999)).
Adverse experiences associated with .beta.-IFN 1b therapy include:
injection site reactions (inflammation, pain, hypersensitivity and
necrosis), and a flu-like symptom complex (fever, chills, anxiety
and confusion). These adverse side effects may be, in fact, reduced
or alleviated by fusing .beta.-IFN 1b to transferrin as described
above.
[0168] Currently, .beta.-IFN 1a (a eukaryotic, glycosylated form)
is also available (Goodkin, 1999, supra). .beta.-IFN 1a is produced
by recombinant DNA technology. Interferon beta-1a is a 166 amino
acid glycoprotein with a predicted molecular weight of
approximately 22,500 daltons. It is produced by mammalian cells
(Chinese Hamster Ovary cells) into which the human IFN-.beta. gene
has been introduced. The amino acid sequence of .beta.-IFN 1a is
identical to that of natural human .beta.-IFN and may be used to
make Tf fusion proteins of the present invention.
[0169] .beta.-IFN/transferrin fusion proteins treatment may also
ameliorate autoimmune attacks by restoring suppressor T cell
function; cotreatment with all-trans-retinoic acid seems to
increase this restorative action for unknown reasons (Qu et al.,
1998. All-trans retinoic acid potentiates the ability of interferon
beta-1b.
<http://members.tripod.com/.about.ThJuland/ra-beta1b.html>).
.beta.-IFN may also inhibit the induction of inducible nitric oxide
synthase (INOS) expression by IL-1 and IFN-.gamma.. Production of
nitric oxide by INOS in astrocytes has been implicated as a factor
in the parthenogenesis of MS (Hua et al. 1998. Beta inteferon
prevents nitric oxide/peroxynitrate from damaging the central
nervous system.
(<http://members.tripod.com/.about.ThJuland/nitric-oxide_beta.html>-
).
[0170] In one aspect, the present invention includes the use of
.beta.-IFN analogs that are therapeutically effective for treating
various diseases associated with .beta.-IFN for generating
.beta.-IFN/transferrin fusion proteins.
[0171] In another aspect, the present invention includes the use of
the .beta.-IFN/transferrin fusion protein in the methods described
above to inhibit or stimulate various cellular processes and for
the treatment and prevention of the various disease and conditions
described above. In particular, the .beta.-IFN/transferrin fusion
protein may be used to treat multiple sclerosis, herpes, influenza,
brain tumor, and skin cancer.
[0172] The .beta.-IFN/transferrin fusion protein of the present
invention can be formulated into pharmaceutical compositions by
well known methods. See, e.g., Remington's Pharmaceutical Sciences
by E. W. Martin, hereby incorporated by reference, describes
suitable formulations. The pharmaceutical composition of the
.beta.-IFN/transferrin fusion protein of the present invention may
be formulated in a variety of forms, including liquid, gel,
lyophilized, or any other suitable form. The preferred form will
depend upon the particular indication being treated and will be
apparent to one of skill in the art.
[0173] The .beta.-IFN/transferrin fusion protein can be
administered in pure form or in an appropriate pharmaceutical
composition. Administration can be carried out via any of the
accepted modes. Thus, administration can be, for example, orally,
nasally, parenterally, topically, transdermally, or rectally, in
the form of solid, semi-solid, lyophilized powder, or liquid dosage
forms, such as for example, tablets, suppositories, pills, soft
elastic and hard gelatin capsules, powders, solutions, suspensions,
or aerosols, or the like, preferably in unit dosage forms suitable
for simple administration of precise dosages. The compositions will
include a conventional pharmaceutical carrier or excipient and the
.beta.-IFN/transferrin fusion protein as the active agent, and, in
addition, may include other medicinal agents, pharmaceutical
agents, carriers, adjuvants, etc.
[0174] Generally, depending on the intended mode of administration,
the pharmaceutically acceptable compositions will contain about 1%
to about 99% by weight of the .beta.-IFN/transferrin fusion
protein, and 99% to 1% by weight of a suitable pharmaceutical
excipient. The composition could be about 5% to 75% by weight of
the .beta.-IFN/transferrin fusion protein with the rest being
suitable pharmaceutical excipients.
[0175] The route of administration could be parenterally, using a
convenient daily dosage regimen which can be adjusted according to
the degree of severity of the disease, preferably multiple
sclerosis, to be treated. For such parenteral administration, a
pharmaceutically acceptable composition containing the
.beta.-IFN/transferrin fusion protein may be formed by the methods
disclosed in U.S. Pat. Nos. 4,462,940, 4,588,585 and 4,992,271.
[0176] Alternatively, the .beta.-IFN/transferrin fusion protein
pharmaceutical compositions may be administered orally,
intravenously, intramuscularly, intraperitoneally, intradermally or
subcutaneously or in any other acceptable manner. The preferred
mode of administration will depend upon the particular indication
being treated and will be apparent to one of skill in the art.
[0177] U.S. Pat. No. 6,333,032 describes effective methods of using
.beta.-IFN to treat diseases in warm-blooded vertebrates, such as
multiple sclerosis. Treatment of multiple sclerosis comprises
administering .beta.-IFN at a dosage of 0.01 to about 5 IU/lb per
day in a dosage form adapted to promote contact of said dosage of
interferon with the oral and pharyngeal mucosa of said animal. The
dosage of interferon could be from 0.1 to about 4.0 IU/lb per day,
or from 0.5 to about 1.5 IU/lb of body weight per day.
[0178] The present invention contemplates administering the
.beta.-IFN in a dosage form adapted to assure maximum contact of
the interferon in said dosage form with the oral and pharyngeal
mucosa of the human or animal undergoing treatment. Contact of
interferon with the mucosa can be enhanced by maximizing residence
time of the treatment solution in the oral or pharyngeal cavity.
Thus, best results seem to be achieved in human patients when the
patient is requested to hold said solution of interferon in the
mouth for a period of time. Contact of interferon with the oral and
pharyngeal mucosa and thereafter with the lymphatic system of the
treated human or animal is unquestionably the most efficient method
administering immunotherapeutic amounts of interferon.
[0179] Further, the present invention contemplates the use of the
.beta.-IFN/transferrin protein for the manufacture of a medicament
which is useful for the treatment of diseases associated with
.beta.-IFN. The diseases contemplated by the present invention
include but are not limited to those described above.
[0180] Glucagon-Like Peptide-1 (GLP-1)
[0181] Glucagon-Like Peptide-1 (GLP-1) is a gastrointestinal
hormone that regulates insulin secretion belonging to the so-called
enteroinsular axis. The enteroinsular axis designates a group of
hormones, released from the gastrointestinal mucosa in response to
the presence and absorption of nutrients in the gut, which promote
an early and potentiated release of insulin. The incretin effect
which is the enhancing effect on insulin secretion is probably
essential for a normal glucose tolerance. GLP-1 is a
physiologically important insulinotropic hormone because it is
responsible for the incretin effect.
[0182] GLP-1 is a product of proglucagon (Bell, et al., Nature,
1983, 304: 368-371). It is synthesized in intestinal endocrine
cells in two principal major molecular forms, as GLP-1(7-36)amide
and GLP-1(7-37). The peptide was first identified following the
cloning of cDNAs and genes for proglucagon in the early 1980s.
[0183] Initial studies done on the full length peptide GLP-1(1-37)
and GLP-1(1-36.sup.amide) concluded that the larger GLP-1 molecules
are devoid of biological activity. In 1987, three independent
research groups demonstrated that removal of the first six amino
acids resulted in a GLP-1 molecule with enhanced biological
activity.
[0184] The amino acid sequence of GLP-1 is disclosed by Schmidt et
al. (1985 Diabetologia 28 704-707). Human GLP-1 is a 37 amino acid
residue peptide originating from preproglucagon which is
synthesized in the L-cells in the distal ileum, in the pancreas,
and in the brain. Processing of preproglucagon to
GLP-1(7-36.sup.amide), GLP-1(7-37) and GLP-2 occurs mainly in the
L-cells. The amino acid sequence of GLP-1(7-36.sup.amide) and
GLP-1(7-37) is (SEQ ID NO: 6): TABLE-US-00003
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-
Tyr-Leu-Glu-Gly-Gln-Ala-Ala-
Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-X
wherein X is NH.sub.2 for GLP-1(7-36.sup.amide) and X is Gly for
GLP-1(7-37).
[0185] GLP-1 like molecules possesses anti-diabetic activity in
human subjects suffering from Type II (non-insulin-dependent
diabetes mellitus (NIDDM)) and, in some cases, even Type I
diabetes. Treatment with GLP-1 elicits activity, such as increased
insulin secretion and biosynthesis, reduced glucagon secretion,
delayed gastric emptying, only at elevated glucose levels, and thus
provides a potentially much safer therapy than insulin or
sulfonylureas. Post-prandial and glucose levels in patients can be
moved toward normal levels with proper GLP-1 therapy. There are
also reports suggesting GLP-1-like molecules possess the ability to
preserve and even restore pancreatic beta cell function in Type-II
patients.
[0186] Any GLP-1 sequence may be used to make Tf fusion proteins of
the present invention, including GLP-1(7-35), GLP-1(7-36), and
GLP-1(7-37). GLP-1 also has powerful actions on the
gastrointestinal tract. Infused in physiological amounts, GLP-1
potently inhibits pentagastrin-induced as well as meal-induced
gastric acid secretion (Schjoldager et al., Dig. Dis. Sci. 1989,
35:703-708; Wettergren et al., Dig Dis Sci 1993; 38:665-673). It
also inhibits gastric emptying rate and pancreatic enzyme secretion
(Wettergren et al., Dig Dis Sci 1993; 38:665-673). Similar
inhibitory effects on gastric and pancreatic secretion and motility
may be elicited in humans upon perfusion of the ileum with
carbohydrate- or lipid-containing solutions (Layer et al., Dig Dis
Sci 1995, 40:1074-1082; Layer et al., Digestion 1993, 54: 385-38).
Concomitantly, GLP-1 secretion is greatly stimulated, and it has
been speculated that GLP-1 may be at least partly responsible for
this so-called "ileal-brake" effect (Layer et al., Digestion 1993;
54: 385-38). In fact, recent studies suggest that, physiologically,
the ileal-brake effects of GLP-1 may be more important than its
effects on the pancreatic islets. Thus, in dose response studies
GLP-1 influences gastric emptying rate at infusion rates at least
as low as those required to influence islet secretion (Nauck et
al., Gut 1995; 37 (suppl. 2): A124).
[0187] GLP-1 seems to have an effect on food intake.
Intraventricular administration of GLP-1 profoundly inhibits food
intake in rats (Schick et al. in Ditschuneit et al. (eds.), Obesity
in Europe, John Libbey & Company ltd, 1994; pp. 363-367; Turton
et al., Nature 1996, 379: 69-72). This effect seems to be highly
specific. Thus, N-terminally extended GLP-1(PG 72-107) amide is
inactive and appropriate doses of the GLP-1 antagonist, exendin
9-39, abolish the effects of GLP-1(Tang-Christensen et al., Am. J.
Physiol., 1996, 271(4 Pt 2):R848-56). Acute, peripheral
administration of GLP-1 does not inhibit food intake acutely in
rats (Tang-Christensen et al., Am. J. Physiol., 1996, 271(4 Pt
2):R848-56; Turton et al., Nature 1996, 379: 69-72). However, it
remains possible that GLP-1 secreted from the intestinal L-cells
may also act as a satiety signal.
[0188] In diabetic patients, GLP's insulinotropic effects and the
effects of GLP-1 on the gastrointestinal tract are preserved
(Willms et al, Diabetologia 1994; 37, suppl.1: A118), which may
help curtail meal-induced glucose excursions, but, more
importantly, may also influence food intake. Administered
intravenously, continuously for one week, GLP-1 at 4 ng/kg/min has
been demonstrated to dramatically improve glycaemic control in
NIDDM patients without significant side effects (Larsen et al.,
Diabetes 1996; 45, suppl. 2: 233A.).
[0189] GLP-1/transferrin fusion proteins comprising at least one
analog of GLP-1 and fragments thereof are useful in the treatment
of Type 1 and Type 2 diabetes and obesity.
[0190] As used herein, the term "GLP-1 molecule" means GLP-1, a
GLP-1 analog, or GLP-1 derivative.
[0191] As used herein, the term "GLP-1 analog" is defined as a
molecule having one or more amino acid substitutions, deletions,
inversions, or additions compared with GLP-1. Many GLP-1 analogs
are known in the art and include, for example, GLP-1(7-34),
GLP-1(7-35), GLP-1(7-36), Val.sup.8-GLP-1(7-37),
Gly.sup.8-GLP-1(7-37), Ser.sup.8-GLP-1(7-37), Gln.sup.9-GLP1(7-37),
D-Gln.sup.9-GLP-1(7-37), Thr.sup.16-Lys.sup.18-GLP-1(7-37), and
Lys.sup.18-GLP-1(7-37). Other analogs include dipeptidyl-peptidase
resistant versions of GLP-1, wherein the N-terminal end of the
peptide is protected. Such analogs include, but are not limited to
GLP-1 with additional amino acids, such as histidine residue added
to the N-terminal end or substituted into the N-terminal amino
acids (amino acid 7 or 8 in GLP-1(7-36) or GLP-1(7-37). In these
analogs, the N-terminal end may comprise the residues His-His-Ala,
Gly-His-Ala, His-Gly-Glu, His-Ser-Glu, His-Ala-Glu, His-Gly-Glu,
His-Ser-Glu, His-His-Ala-Glu, His-His-Gly-Glu, His-His-Ser-Glu,
Gly-His-Ala-Glu, Gly-His-Gly-Glu, Gly-His-Ser-Glu, His-X-Ala-Glu,
His-X-Gly-Glu, His-X-Ser-Glu, wherein X is any amino acid. U.S.
Pat. No. 5,118,666 discloses examples of GLP-1 analogs such as
GLP-1(7-34) and GLP-1(7-35).
[0192] The term "GLP-1 derivative" is defined as a molecule having
the amino acid sequence of GLP-1 or a GLP-1 analog, but
additionally having chemical modification of one or more of its
amino acid side groups, .alpha.-carbon atoms, terminal amino group,
or terminal carboxylic acid group. A chemical modification
includes, but is not limited to, adding chemical moieties, creating
new bonds, and removing chemical moieties.
[0193] As used herein, the term "GLP-1 related compound" refers to
any compound falling within the GLP-1, GLP-1 analog, or GLP-1
derivative definition.
[0194] WO 91/11457 discloses analogs of the active GLP-1 peptides
7-34, 7-35, 7-36, and 7-37 which can also be useful as GLP-1
moieties.
[0195] EP 0708179-A2 (Eli Lilly & Co.) discloses GLP-1 analogs
and derivatives that include an N-terminal imidazole group and
optionally an unbranched C.sub.6-C.sub.10 acyl group in attached to
the lysine residue in position 34.
[0196] EP 0699686-A2 (Eli Lilly & Co.) discloses certain
N-terminal truncated fragments of GLP-1 that are reported to be
biologically active.
[0197] U.S. Pat. No. 5,545,618 discloses GLP-1 molecules consisting
essentially of GLP-1(7-34), GLP1(7-35), GLP-1(7-36), or
GLP-1(7-37), or the amide forms thereof, and
pharmaceutically-acceptable salts thereof, having at least one
modification selected from the group consisting of: (a)
substitution of glycine,serine, cysteine, threonine, asparagine,
glutamine, tyrosine, alanine, valine, isoleucine, leucine,
methionine, phenylalanine, arginine, or D-lysine for lysine at
position 26 and/or position 34; or substitution of glycine, serine,
cysteine, threonine, asparagine, glutamine, tyrosine, alanine,
valine, isoleucine, leucine, methionine, phenylalanine, lysine, or
a D-arginine for arginine at position 36; (b) substitution of an
oxidation-resistant amino acid for tryptophan at position 31; (c)
substitution of at least one of: tyrosine for valine at position
16; lysine for serine at position 18; aspartic acid for glutamic
acid at position 21; serine for glycine at position 22; arginine
for glutamine at position 23; arginine for alanine at position 24;
and glutamine for lysine at position 26; and (d) substitution of at
least one of: glycine, serine, or cysteine for alanine at position
8; aspartic acid, glycine, serine, cysteine, threonine, asparagine,
glutamine, tyrosine, alanine, valine, isoleucine, leucine,
methionine, or phenylalanine for glutamic acid at position 9;
serine, cysteine, threonine, asparagine, glutamine, tyrosine,
alanine, valine, isoleucine, leucine, methionine, or phenylalanine
for glycine at position 10; and glutamic acid for aspartic acid at
position 15; and (e) substitution of glycine, serine, cysteine,
threonine, asparagine, glutamine, tyrosine, alanine, valine,
isoleucine, leucine, methionine, or phenylalanine, or the D- or
N-acylated or alkylated form of histidine for histidine at position
7; wherein, in the substitutions is (a), (b), (d), and (e), the
substituted amino acids can optionally be in the D-form and the
amino acids substituted at position 7 can optionally be in the
N-acylated or N-alkylated form.
[0198] U.S. Pat. No. 5,118,666 discloses a GLP-1 molecule having
insulinotropic activity. Such molecule is selected from the group
consisting of a peptide having the amino acid sequence
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-A-
la-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys (SEQ ID NO: .7) or
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-A-
la-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (SEQ ID NO: 8); and a
derivative of said peptide and wherein said peptide is selected
from the group consisting of: a pharmaceutically-acceptable acid
addition salt of said peptide; a pharmaceutically-acceptable
carboxylate salt of said peptide; a pharmaceutically-acceptable
lower alkylester of said peptide; and a pharmaceutically-acceptable
amide of said peptide selected from the group consisting of amide,
lower alkyl amide, and lower dialkyl amide.
[0199] U.S. Pat. No. 6,277,819 teaches a method of reducing
mortality and morbidity after myocardial infarction comprising
administering GLP-1, GLP-1 analogs, and GLP-1 derivatives to the
patient. The GLP-1 analog being represented by the following
structural formula (SEQ ID NO: 9):
R.sub.1--X.sub.1-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-X.sub.2--
Gly-Gln-Ala-Ala-Lys-X.sub.3-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R.sub.2
and pharmaceutically-acceptable salts thereof, wherein: R.sub.1 is
selected from the group consisting of L-histidine, D-histidine,
desamino-histidine, 2-amino-histidine, .beta.-hydroxy-histidine,
homohistidine, alpha-fluoromethyl-histidine, and
alpha-methyl-histidine; X.sub.1 is selected from the group
consisting of Ala, Gly, Val, Thr, Ile, and alpha-methyl-Ala;
X.sub.2 is selected from the group consisting of Glu, Gin, Ala,
Thr, Ser, and Gly; X.sub.3 is selected from the group consisting of
Glu, Gln, Ala, Thr, Ser, and Gly; R.sub.2 is selected from the
group consisting of NH.sub.2, and Gly-OH; provided that the GLP-1
analog has an isoelectric point in the range from about 6.0 to
about 9.0 and further providing that when R.sub.1 is His, X.sub.1
is Ala, X.sub.2 is Glu, and X.sub.3 is Glu, R.sub.2 must be
NH.sub.2.
[0200] Ritzel et al. (Journal of Endocrinology, 1998, 159: 93-102)
disclose a GLP-1 analog, [Ser.sup.8]GLP-1, in which the N-terminal
second amino acid, alanine, is replaced with serine. The
modification did not impair the insulinotropic action of the
peptide but produced an analog with increased plasma stability as
compared to GLP-1.
[0201] U.S. Pat. No. 6,429,197 teaches that GLP-1 treatment after
acute stroke or hemorrhage, preferably intravenous administration,
can be an ideal treatment because it provides a means for
optimizing insulin secretion, increasing brain anabolism, enhancing
insulin effectiveness by suppressing glucagon, and maintaining
euglycemia or mild hypoglycemia with no risk of severe hypoglycemia
or other adverse side effects. The present invention provides a
method of treating the ischemic or reperfused brain with GLP-1 or
its biologically active analogues after acute stroke or hemorrhage
to optimize insulin secretion, to enhance insulin effectiveness by
suppressing glucagon antagonism, and to maintain euglycemia or mild
hypoglycemia with no risk of severe hypoglycemia.
[0202] U.S. Pat. No. 6,277,819 provides a method of reducing
mortality and morbidity after myocardial infarction, comprising
administering to a patient in need thereof, a compound selected
from the group consisting of GLP-1, GLP-1 analogs, GLP-1
derivatives and pharmaceutically-acceptable salts thereof, at a
dose effective to normalize blood glucose.
[0203] U.S. Pat. No. 6,191,102 discloses a method of reducing body
weight in a subject in need of body weight reduction by
administering to the subject a composition comprising a
glucagon-like peptide-1 (GLP-1), a glucagon-like peptide analog
(GLP-1 analog), a glucagon-like peptide derivative (GLP-1
derivative) or a pharmaceutically acceptable salt thereof in a dose
sufficient to cause reduction in body weight for a period of time
effective to produce weight loss, said time being at least 4
weeks.
[0204] GLP-1 is fully active after subcutaneous administration
(Ritzel et al., Diabetologia 1995; 38: 720-725), but is rapidly
degraded mainly due to degradation by dipeptidyl peptidase IV-like
enzymes (Deacon et al., J Clin Endocrinol Metab 1995, 80: 952-957;
Deacon et al.,1995, Diabetes 44: 1126-1131). Thus, unfortunately,
GLP-1 and many of its analogues have a short plasma half-life in
humans (Orskov et al., Diabetes 1993; 42:658-661). Accordingly, it
is an objective of the present invention to provide transferrin
fusion proteins comprising GLP-1 or analogues thereof which have a
protracted profile of action relative to GLP-1(7-37). It is a
further object of the invention to provide derivatives of GLP-1 and
analogues thereof which have a lower clearance than GLP-1(7-37).
Moreover, it is an object of the invention to provide
pharmaceutical compositions comprising GLP-1/transferrin fusion
proteins or GLP-1 analog/transferrin fusion proteins with improved
stability. Additionally, the present invention includes the use of
GLP-1/transferrin fusion proteins or GLP-1 analog/transferrin
fusion proteins to treat diseases associated with GLP-1 such as but
not limited to those described above.
[0205] In one aspect of the present invention, the pharmaceutical
compositions comprising the GLP-1 peptide/transferrin fusion
proteins and GLP-1 analog/transferrin fusion proteins may be
formulated by any of the established methods of formulating
pharmaceutical compositions, e.g. as described in Remington's
Pharmaceutical Sciences, 1985. The composition may be in a form
suited for systemic injection or infusion and may, as such, be
formulated with a suitable liquid vehicle such as sterile water or
an isotonic saline or glucose solution. The compositions may be
sterilized by conventional sterilization techniques which are well
known in the art. The resulting aqueous solutions may be packaged
for use or filtered under aseptic conditions and lyophilized, the
lyophilized preparation being combined with the sterile aqueous
solution prior to administration. The composition may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such as buffering agents,
tonicity adjusting agents and the like, for instance sodium
acetate, sodium lactate, sodium chloride, potassium chloride,
calcium chloride, etc.
[0206] The GLP-1/transferrin fusion proteins and GLP-1
analog/transferrin fusion proteins of the present invention may
also be adapted for nasal, transdermal, pulmonal or rectal
administration. The pharmaceutically acceptable carrier or diluent
employed in the composition may be any conventional solid carrier.
Examples of solid carriers are lactose, terra alba, sucrose, talc,
gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
Similarly, the carrier or diluent may include any sustained release
material known in the art, such as glyceryl monostearate or
glyceryl distearate, alone or mixed with a wax.
[0207] It may be of particular advantage to provide the composition
of the invention in the form of a sustained release formulation. As
such, the composition may be formulated as microcapsules or
microparticles containing the GLP-1/transferrin fusion protein or
GLP-1 analog/transferrin fusion protein encapsulated by or
dispersed in a suitable pharmaceutically acceptable biodegradable
polymer such as polylactic acid, polyglycolic acid or a lactic
acid/glycolic acid copolymer.
[0208] For nasal administration, the preparation may contain
GLP-1/transferrin fusion proteins or GLP-1 analog/transferrin
fusion proteins dissolved or suspended in a liquid carrier, in
particular an aqueous carrier, for aerosol application. The carrier
may contain additives such as solubilizing agents, e.g. propylene
glycol, surfactants, absorption enhancers such as lecithin
(phosphatidylcholine) or cyclodextrin, or preservatives such as
parabenes.
[0209] Generally, the compounds of the present invention are
dispensed in unit dosage form comprising 0.5-500 mg of the fusion
protein together with a pharmaceutically acceptable carrier per
unit dosage.
[0210] Moreover, the present invention contemplates the use of the
GLP-1/transferrin and GLP-1 analog/transferrin fusion proteins for
the manufacture of a medicinal product which can be used in the
treatment of diseases associated with elevated glucose level, such
as but not to limited to those described above. Specifically, the
present invention contemplates the use of GLP-1/transferrin fusion
protein for the treatment of diabetes including type II diabetes,
obesity, severe burns, and heart failure, including congestive
heart failure and acute coronary syndrome.
[0211] The N-terminus of GLP-1 is normally amidated. In yeast,
amidation does not occur. In one aspect of the invention, in order
to compensate for amidation on the N-terminus which does not occur
in yeast, an extra amino acid is added on the N-terminus of GLP-1.
The addition of an amino acid to the N-terminus of GLP-1 may
prevent dipeptidyl peptidase from cleaving at the second amino acid
of GLP-1 due to steric hindrance. Therefore, GLP-1 will remain
functionally active. Any one of the 20 amino acids may be added to
the N-terminus of GLP-1. In some instances, an uncharged or
positively charged amino acid may be used and preferably, a smaller
amino acid such as Glycine is added. The GLP-1 with the extra amino
acid is then fused to transferrin. Accordingly, the GLP-1 with the
added amino acid will be fused at the N-terminus of the
GLP-1/transferrin fusion protein.
[0212] In one embodiment of making the GLP-1(7-36) or GLP-1(7-37)
peptide more resistant to cleavage by dipeptidyl peptidase, a His
residue is added at the N-terminus of GLP-1 or is inserted after
the His residue at the N-terminus of GLP-1, so that the N-terminus
of GLP-1 begins with His-His.
[0213] In another embodiment of the invention, the second residue
from the N-terminus in the GLP-1(7-36) or GLP-1(7-37) peptide (SEQ
ID NO: 6) is substituted with another amino acid. For example, the
Ala residue at the second residue from the N-terminus in the
GLP-1(7-36) or GLP-1(7-37) peptide may be substituted with Ser,
Gly, Val, or another amino acid.
[0214] GLP-mTf Fusion Protein for Treating Type 2 Diabetes
[0215] As discussed above, GLP-1 activates and regulates important
endocrine hormone systems in the body and plays a critical
management role in the metabolism of glucose. Unlike all other
diabetic treatments on the market GLP-1 has the potential to be
restorative by acting as a growth factor for B-cells thus improving
the ability of the pancreas to secrete insulin and also, to make
the existing insulin levels act more efficiently by improving
sensitivity and better stabilizing glucose levels. This reduces the
burden on daily monitoring of glucose levels and potentially offers
a delay in the serious long term side effects caused by
fluctuations in blood glucose due to diabetes. Furthermore, GLP-1
can reduce appetite and reduce weight. Obesity is an inherent
consequence of poor control of glucose metabolism and this only
serves to aggravate the diabetic condition.
[0216] Clinical application of natural GLP-1 is limited because it
is rapidly degraded in the circulation (half-life is several
minutes). To maintain therapeutic levels in the circulation
requires constant administration of high doses using pumps or patch
devices which adds to the cost of treatment. This is inconvenient
for long term chronic use especially in conjunction with all the
other medications for treating diabetes and monitoring of glucose
levels. The GLP-1 fusion proteins retain the activity of GLP-1 but
have the long half-life (14-17 days), solubility, and
biodistribution properties of transferrin (mTf). These properties
could provide for a low cost, small volume, monthly s.c.
(subcutaneous) injection and this type of product is absolutely
needed for long term chronic use.
[0217] Insulin
[0218] Human insulin contains two peptide chains, known as the A
and B chains, which are 21 and 30 amino acids in length,
respectively, and which are connected by two cystine disulphide
bridges. This peptide has a molecular weight of approximately 6
kDa. The immediate precursor of insulin is proinsulin, a single
chain peptide composed of the B and A chains linked to a connecting
peptide of approximately 31 amino acids, known as the C-peptide, by
adjacent pairs of basic residues. The arrangement of these three
peptides in the proinsulin molecule, beginning with the
amino-terminal end, is as follows: B
chain-Arg-Arg-C-peptide-Lys-Arg-A chain. When translated into mRNA,
however, preproinsulin is produced, which contains proinsulin
joined at its amino-terminal end to a largely hydrophobic signal
peptide 24 amino acids in length.
[0219] Preproinsulin is synthesized in pancreatic beta cells
located within the islets of Langerhans, which are dispersed
throughout the pancreas. Removal of the signal peptide occurs in
the rough endoplasmic reticulum, and the resulting proinsulin is
then transported to the Golgi apparatus for packaging into
secretion granules. The folded proinsulin is stabilized by
disulfide bonds. During processing of the secretion granules, the
folded proinsulin molecule is cleaved by specific proteases at the
paired basic residues to liberate insulin and the C-peptide.
[0220] Diabetes mellitus is a disease that affects approximately 17
million people in the United States, or 6.2% of the population.
About one million people over the age of 20 are diagnosed with
diabetes annually, and diabetes is the sixth leading cause of death
in the United States
(http://www.niddk.nih.gov/health/diabetes/pubs/dmstats/dmstats.htm#7).
Approximately 90-95% of diabetes cases are Type 2, formerly called
adult-onset diabetes, which begins as insulin resistance (failure
of the body's cells to use insulin properly) as progresses to an
inability of the pancreas to produce insulin. Type 1 diabetes,
formerly called juvenile diabetes or insulin-dependent diabetes,
accounts for the remaining 5-10% of diabetes cases. In type 1
diabetes, the body's immune system destroys the beta cells in the
pancreas, which manufacture insulin.
[0221] Because human insulin contains only 51 amino acid residues,
it is readily made by recombinant techniques, and a large number of
insulin analogues and variants have been prepared. Any of these
analogues or variants can be used to make mTf fusion proteins of
the invention.
[0222] Diabetics typically require insulin replacement therapy,
which involves one or more doses of the drug per day by
subcutaneous injection. Treatment by injection, however, is both
psychologically and physically painful, as well as demanding of
technical expertise, and many diabetics require assistance in
administering injections. Oral formulations of insulin have not
been successful, however, because the peptide is rapidly degraded
in the acidic environment of the GI tract, particularly in the
stomach. Nevertheless, alternatives to injection, such as oral,
nasal and topical formulations have been attempted. U.S. Pat. No.
5,824,638, Burnside et al., describes oral emulsion preparations in
which insulin is dissolved in a hydrophilic phase, such as water,
saline or a water-miscible alcohol, and dispersed with a surfactant
in a hydrophobic phase, such as a long chain fatty acid or fatty
acid ester. Although an emulsion keeps insulin dispersed, it cannot
protect the peptide from the harsh conditions of the stomach. Nasal
preparations, which deliver insulin in an aerosol to the lungs, are
disclosed in U.S. Pat. No. 6,427,681, Gonda et al., while topical
preparations are disclosed in U.S. Pat. No. 6,399,566, Dardai et
al.
[0223] Modified insulins for injection, containing amino acid
substitutions or glycosylated residues, to enhance activity,
inhibit degradation or inhibit peptide aggregation have also been
developed (see U.S. Pat. No. 4,478,746, Kim et al., glycosylated
insulin derivatives; U.S. Pat. No. 4,992,418, Katsoyanis et al.,
Asp.sub.10-containing insulin (B chain) for increased activity;
U.S. Pat. No. 5,716,927, Balschmidt et al., Lys or Arg at position
28 in the B chain, or A18, A21 or B3 modified from Asn, or other
modifications at the C-terminal end of the B chain, to prevent
aggregation and reduced activity) Additional amino acid
substitutions that confer a longer active phase, because they can
be acylated, are disclosed in U.S. Pat. No. 5,750,497, Havelund et
al. A21 B3 and B30 can be replaced by any amino acid except Lys,
Arg or Cys. B1 may be deleted, and B30 may be replaced by a
lipophilic chain of 10-24 carbon atoms. Fusion proteins for
improved recombinant production of insulin (higher yields, soluble,
allowing correct folding) are described in U.S. Pat. No. 6,534,288,
Habermann et al. These peptides contain a fusion portion at the
amino terminal end of the B chain, followed by amino acids
RDVP-Y.sub.n-A chain, where Y is a peptide 2-50 amino acids in
length, terminating with a basic amino acid.
[0224] The present invention includes fusion proteins comprising
transferrin and an insulin protein or peptide. In one embodiment,
the fusion proteins are formulated for oral delivery. The present
invention, therefore, also includes methods of orally administering
insulin fusion proteins of the invention to a patient in need
thereof, in particular, a diabetic patient.
[0225] In one embodiment, the present invention includes
transferrin fusion protein comprising single chain insulin analog
(Lee et al., 2000, Nature, 408: 483). In another embodiment, the
insulin in the transferrin fusion protein may contain a protease
cleavage site specific to the gastrointestinal (GI) tract, or a
specific part of the gastrointestinal tract, such that the site
would be recognized by one or more enzymes in the GI tract. The
proinsulin could be activated in this manner. The cleavage site
could reside in the peptide linking the A and B chain.
[0226] EPO Mimetic Peptide (EMP)
[0227] Erythropoietin (EPO) is a glycoprotein hormone that is
synthesized in the kidneys of mammals for stimulating mitotic cell
division and differentiation of erythrocyte precursor cells.
Accordingly, EPO acts to stimulate and regulate the production of
erythrocytes. Because of its role in red blood cell formation, EPO
is useful in both the diagnosis and the treatment of blood
disorders characterized by low or defective red blood cell
production.
[0228] Studies have shown the efficacy of EPO therapy in a variety
of disease states, disorders, and states of hematologic
irregularity, for example, beta-thalassemia (Vedovato et al. (1984)
Acta. Haematol. 71:211-213); cystic fibrosis (Vichinsky et al.
(1984) J. Pediatric 105:15-21); pregnancy and menstrual disorders
(Cotes et al. (1983) Brit. J. Ostet. Gyneacol. 90:304-311); early
anemia of prematurity (Haga et al. (1983) Acta Pediatr. Scand.
72:827-831); spinal cord injury (Claus-Walker et al. (1984) Arch.
Phys. Med. Rehabil. 65:370-374); space flight (Dunn et al. (1984)
Eur. J. Appl. Physiol. 52:178-182); acute blood loss (Miller et al.
(1982) Brit. J. Haematol. 52:545-590); aging (Udupa et al. (1984)
J. Lab. Clin. Med. 103:574-588); various neoplastic disease states
accompanied by abnormal erythropoiesis (Dainiak et al. (1983)
Cancer 5:1101-1106); and renal insufficiency (Eschbach et al.
(1987) N. Eng. J. Med. 316:73-78). During the last fifteen years,
EPO has been used for the treatment of the anemia of renal failure,
anemia of chronic disease associated with rheumatoid arthritis,
inflammatory bowel disease, AIDS, and cancer, as well as for the
treatment of anemia in hematopoietic malignancies, post-bone marrow
transplantation, and autologous blood donation.
[0229] The activity of EPO is mediated by its receptor. The
EPO-receptor (EPO-R) belongs to the class of growth-factor-type
receptors which are activated by a ligand-induced protein
dimerization. Other hormones and cytokines such as human growth
hormone (hGH), granulocyte colony stimulating factor (G-CSF),
epidermal growth factor (EGF) and insulin can cross-link two
receptors resulting in juxtaposition of two cytoplasmic tails. Many
of these dimerization-activated receptors have protein kinase
domains within the cytoplasmic tails that phosphorylate the
neighboring tail upon dimerization. While some cytoplasmic tails
lack intrinsic kinase activity, these function by association with
protein kinases. The EPO receptor is of the latter type. In each
case, phosphorylation results in the activation of a signaling
pathway.
[0230] There has been an increasing interest in molecular mimicry
with EPO potency. For example, dimerization of the erythropoietin
receptor (EPOR) in the presence of either natural EPO or synthetic
EPO mimetic peptides (EMPs) is the extracellular event that leads
to activation of the receptor and downstream signal transduction
events. In general, there is an interest in obtaining mimetics with
equivalent potency to EPO.
[0231] Wrighton et al (1996, Science, 273:458-463) employed phage
display where random peptides are to be exposed on coat proteins of
filamentous phage. A library of random peptide-phage was allowed to
bind to and subsequently eluted from the extracellular domain of
EPO receptor in the screening system. They used weak-binding system
to first fish out EPO domain-weak-binding (Kd 10 mM) CRIGPITWVC
(SEQ ID NO: 10) as the consensus sequence. Consequently, a 20-amino
acid peptide, EMP1, (GGTYSCBFGPLTWVCKPQGG, SEQ ID NO: 11) with an
affinity (Kd) of 200 nM, compared to 200 pM for EPO was isolated,
the sequence of which does not actually exist in the native EPO.
The crystal structure at 2.8 .ANG. resolution of a complex of this
mimetic agonist peptide with the extracellular domain of EPO
receptor revealed that a peptide dimer induces an almost perfect
twofold dimerization of the receptor (Livnah et al., 1996 Science,
273 (274): 464-471). This 20-amino acid peptide has a b-sheet
structure and is stabilized by the C--C disulfide bond.
[0232] The biological activity of EMP1 indicates that EMP1 can act
as an EPO mimetic. For example, EMP1 competes with EPO in receptor
binding assays to cause cellular proliferation of cell lines
engineered to be responsive to EPO (Wrighton et al., 1996, Science,
273:458-463). Both EPO and EMP1 induce a similar cascade of
phosphorylation events and cell cycle progression in EPO responsive
cells (Wrighton et al., 1996, Science, 273:458-463). Further, EMP1
demonstrates significant erythropoietic effects in mice as
monitored by two different in vivo assays of nascent red blood cell
production (Wrighton et al., 1996, Science, 273:458-463).
[0233] Johnson et al. (1998, Biochemistry, 37:3699-3710) identified
the minimal peptide that retained activity in the assays for EPO
mimetic action. Using N- and C-terminal deletions, they found that
the minimal active peptide is EMP20 having the sequence,
YSCHFGPLTWVCK (SEQ ID NO: 12), namely amino acids 4 through 16 of
EMP1. They also found Tyr4 and Trp13 of EMP1 are critical for
mimetic action.
[0234] The present invention provides EMP1/transferrin fusion
proteins with increased half-life and pharmaceutical compositions
comprising such fusion proteins. Any EMP1 sequence may be used to
make EMP1/transferrin fusion proteins, including EMP1 sequences
wherein one or more C residues is deleted or replaced. These
sequences can then be inserted into a mTf loop to provide three
dimensional structure to the EMP1 region of the fusion protein. The
present invention contemplates the use of the fusion protein to
treat various diseases and conditions associated with EPO such as
but not limited to those described above.
[0235] In one embodiment of the present invention, the
pharmaceutical compositions comprising the EMP1/transferrin fusion
protein and may be formulated by any of the established methods of
formulating pharmaceutical compositions, e.g. as described in
Remington's Pharmaceutical Sciences, 1985. The composition may be
in a form suited for systemic injection or infusion and may, as
such, be formulated with a suitable liquid vehicle such as sterile
water or an isotonic saline or glucose solution. These
pharmaceutical compositions may contain buffers, salts and other
excipients to stabilize the composition or assist in the delivery
of the transferrin fusion proteins.
[0236] In a preferred embodiment, the present invention provides a
method for treating disorders associated with EPO. The method is
accomplished by administering a EMP1/transferrin fusion protein
provided herein for a time and under conditions sufficient to
alleviate the symptoms of the disorder, i.e. sufficient to effect
dimerization or biological activation of EPO receptors. In the case
of EPO such methodology is useful in the treatment of end-stage
renal failure/dialysis; anemia, especially associated with AIDS or
chronic inflammatory diseases such as rheumatoid arthritis and
chronic bowel inflammation; auto-immune disease; and for boosting
the red blood cell count of patient when necessary, e.g. prior to
surgery or as pretreatment to transfusion. The EMP1/transferrin
fusion protein of the present invention which behave as EPO
agonists can be used to activate megakaryocytes.
[0237] Since EPO has been shown to have a mitogenic and chemotactic
effect on vascular endothelial cells as well as an effect on
central cholinergic neurons (Amagnostou et al. (1990) Proc. Natl.
Acad. Sci. USA 87:597805982; Konishi et al. (1993) Brain Res.
609:29-35), the compounds of this invention can also be used to
treat a variety of vascular disorders, such as promoting wound
healing, growth of collateral coronary blood vessels (such as those
that may occur after myocardial infarction), trauma, and post
vascular graft treatment, and a variety of neurological disorders,
generally characterized by low absolute levels of acetyl choline or
low relative levels of acetyl choline as compared to other
neuroactive substances e.g., neurotransmitters.
[0238] Accordingly, the present invention includes pharmaceutical
compositions comprising, as an active ingredient, the
EMP1/transferrin fusion protein of the present invention in
association with a pharmaceutical carrier or diluent. The
EMP1/transferrin fusion protein of this invention can be
administered by oral, parenteral (intramuscular, intraperitoneal,
intravenous (IV) or subcutaneous injection), transdermal (either
passively or using iontophoresis or electroporation) or
transmucosal (nasal, vaginal, rectal, or sublingual) routes of
administration in dosage forms appropriate for each route of
administration.
[0239] Solid dosage forms for oral administration include capsules,
tablets, pill, powders, and granules. In such solid dosage forms,
the active compound is admixed with at least one inert
pharmaceutically acceptable carrier such as sucrose, lactose, or
starch. Such dosage forms can also comprise, as it normal practice,
additional substances other than inert diluents, e.g., lubricating,
agents such as magnesium stearate. In the case of capsules, tablets
and pills, the dosage forms may also comprise buffering, agents.
Tablets and pills can additionally be prepared with enteric
coatings.
[0240] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, with the elixirs containing inert diluents commonly used in
the art, such as water. Besides such inert diluents, compositions
can also include adjuvants, such as wetting agents, emulsifying and
suspending agents, and sweetening, flavoring and perfuming
agents.
[0241] Preparations according to this invention for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil and corn oil, gelatin, and injectable organic
esters such as ethyl oleate. Such dosage forms may also contain
adjuvants such as preserving, wetting, emulsifying, and dispersing
agents. They may be sterilized by, for example, filtration through
a bacteria retaining filter, by incorporating sterilizing agents
into the compositions, by irradiating the compositions, or by
heating the compositions. They can also be manufactured using
sterile water, or some other sterile injectable medium, immediately
before use.
[0242] Compositions for rectal or vaginal administration are
preferably suppositories which may contain, in addition to the
active substance, excipients such as cocoa butter or a suppository
wax. Compositions for nasal or sublingual administration are also
prepared with standard excipients well known in the art.
[0243] The dosage of active ingredient in the compositions of this
invention may be varied; however, it is necessary that the amount
of the active ingredient shall be such that a suitable dosage form
is obtained. The selected dosage depends upon the desired
therapeutic effect, on the route of administration, and on the
duration of the treatment desired. Generally dosage levels of
between 0.001 to 10 mg/kg of body weight daily are administered to
mammals.
[0244] Moreover, the present invention also contemplates the use of
the transferrin fusion protein comprising EMP1 or analogs thereof
for the manufacture of a medicinal product which can be used in the
treatment of diseases associated with low or defective red blood
cell production. Examples of such diseases are not limited to those
described above.
[0245] T-20 and T-1249
[0246] HIV infection is pandemic and HIV associated diseases
represent a major world health problem. Although considerable
effort is being put into the successful design of effective
therapeutics, currently no curative anti-retroviral drugs against
AIDS exist. In attempts to develop such drugs, several stages of
the HIV life cycle have been considered as targets for therapeutic
intervention (Mitsuya, H. et al., 1991, FASEB J. 5:2369-2381). For
example, virally encoded reverse transcriptase has been one focus
of drug development. A number of reverse-transcriptase-targeted
drugs, including 2',3'-dideoxynucleoside analogs such as AZT, ddI,
ddc, and d4T have been developed which have been shown to been
active against HIV (Mitsuya, H. et al., 1991, Science
249:1533-1544). While beneficial, these nucleoside analogs are not
curative, probably due to the rapid appearance of drug resistant
HIV mutants (Lander, B. et al., 1989, Science 243:1731-1734). In
addition, the drugs often exhibit toxic side effects, such as bone
marrow suppression, vomiting, and liver function abnormalities.
[0247] Entry inhibitors are distinct from the existing classes of
drugs that fight HIV. Other drugs work inside the infected cell.
Nucleoside reverse transcriptase inhibitors such as AZT and
abacavir and non-nucleoside reverse transcriptase inhibitors like
nevirapine and efavirenz all act by shutting down the reverse
transcriptase enzyme that HIV uses to replicate itself once it is
inside the cell. Protease inhibitors shut down the viral protease
enzyme HIV uses to package itself up for export. By contrast, entry
inhibitors are drugs that interfere with the processes involved in
the virus' initial assault on the cell's outer membrane.
[0248] T-20 is the most studied of all the entry inhibitors and is
the first member of the fusion inhibitor class. Unlike existing
AIDS drugs that work inside the cell and target viral enzymes
involved in the replication of the virus, T-20 inhibits fusion of
HIV with host cells before the virus enters the cell and begins its
replication process. T-20 binds to one of the two helical domains
of gp41. Gp41 is a spring-loaded HIV-1 protein that is activated
when CD4 binds to HIV gp-120. The fusion action of gp41 is
inhibited if its two helical domains cannot fold together. T-20
binds to gp41, effectively keeping the protein from functioning. It
has been shown in early, single-arm clinical studies to be about as
potent as a protease inhibitor by itself-giving greater than 10
fold reductions in viral load-and to be safe in combination with
other antiretrovirals.
[0249] U.S. Pat. No. 5,464,933 discloses T-20 (pentafuside, DP-178)
as a 36 amino acid synthetic peptide. Since this drug is a peptide,
it cannot be given orally because it is readily broken down by the
digestive system. When administered by subcutaneous injection, T-20
achieves sufficient levels in the blood to have anti-HIV activity.
It is administered by subcutaneous injection twice daily. However,
patients develop skin reactions at the injection site. The most
frequently reported treatment related adverse events were mild to
moderate local injection site reactions. These consist of mild
pain, temporary swelling and redness at the site of injection.
[0250] U.S. Pat. No. 6,479,055 discloses peptide analogs of the
DP-178 (peptides corresponding to amino acid residues 638 to 673 of
transmembrane protein gp41 of HIV-1.sub.LAI, which exhibit
anti-membrane fusion capability, antiviral activity, such as the
ability to inhibit HIV transmission to uninfected CD-4.sup.+ cells,
or an ability to modulate intracellular processes involving
coiled-coil peptide structures. Further, the patent relates to the
use of DP-178 and DP-178 portions and/or analogs as antifusogenic
or antiviral compounds or as inhibitors of intracellular events
involving coiled-coil peptide structures. Further, the patent
teaches the use of the peptides as diagnostic agents. For example,
a DP178 peptide may be used as an HV subtype-specific
diagnostic.
[0251] T-1249 is a sister compound of T-20. Like T-20, T-1249
targets the HIV glycoprotein known as gp41 which HIV uses to bind
onto CD4 cells. T-1249 has shown potent anti-HIV effects in animal
and laboratory studies. Preliminary safety, dosing and efficacy
studies in humans have provided support for ongoing research.
[0252] T-1249 is currently administered by subcutaneous (under the
skin) injection once or twice daily. The first safety study of
T-1249 conducted in humans found two serious adverse events:
hypersensitivity reaction (oral ulcers, maculopapular rash, fever)
and severe neutropenia. Forty percent of recipients developed
injection site reactions but these were deemed to be mild.
Dizziness, diarrhea, headache and fever have also been reported by
recipients. No dose-limiting toxicity was identified and
experiments with higher doses are likely.
[0253] T-1249 has completed phase I/II safety and dosing studies.
Initial results indicated that higher doses produced an average
viral load drop of 1.3 log.
[0254] Dose-dependent decreases in HIV RNA have been reported. In
the study of T-1249, the average reduction from baseline ranged
from 0.29 to 1.96 log copies/ml (Gulick 2002).
[0255] The present invention provides transferrin fusion proteins
comprising T-20, T-1249, or analogs thereof with increased
half-life and pharmaceutical compositions comprising such fusion
proteins. The present invention also provides pharmaceutical
compositions comprising these transferrin fusion proteins for
therapeutic purposes. The present invention contemplates the use of
such fusion proteins as inhibitors of human and non-human
retroviral, especially HIV, transmission to uninfected cells. The
human retroviruses whose transmission may be inhibited by the
peptides of the invention include, but are not limited to all
strains of HIV-1 and HIV-2 and the human T-lymphocyte viruses
(HTLV-I, II, III). The non-human retroviruses whose transmission
may be inhibited by the peptides of the invention include, but are
not limited to bovine leukosis virus, feline sarcoma and leukemia
viruses, simian sarcoma and leukemia viruses, and sheep progress
pneumonia viruses.
[0256] With respect to HIV, the transferrin fusion protein of the
present invention comprising T-20, T-1249 or analogs thereof may be
used as a therapeutic in the treatment of AIDS. These transferrin
fusion proteins may be administered using techniques well known to
those in the art. Preferably, the pharmaceutical compositions
comprising these transferrin fusion proteins are formulated and
administered systemically. Techniques for formulation and
administration may be found in "Remington's Pharmaceutical
Sciences" 18th ed., 1990 Mack Publishing Co., Easton, Pa. Suitable
routes may include oral, rectal, transmucosal, or intestinal
administration; parenteral delivery, including intramuscular,
subcutaneous, intramedullary injections, as well as intrathecal,
direct intraventricular, intravenous, intraperitoneal, intranasal,
or intraocular injections, just to name a few. Most preferably,
administration is intravenous. For injection, the transferrin
fusion proteins comprising T-20, T1249, or analogs thereof may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks' solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0257] In addition, the transferrin fusion protein comprising T-20,
T1249, or analogs thereof may be used as a prophylactic measure in
previously uninfected individuals after acute exposure to an HIV
virus. Examples of such prophylactic use of the peptides may
include, but are not limited to, prevention of virus transmission
from mother to infant and other settings where the likelihood of
HIV transmission exists, such as, for example, accidents in health
care settings wherein workers are exposed to HIV-containing blood
products. The transferrin fusion proteins of the present invention
comprising T-20, T-1249, or analogs thereof in such cases may serve
the role of a prophylactic vaccine, wherein the host raises
antibodies against the fusion proteins of the invention, which then
serve to neutralize HIV viruses by, for example, inhibiting further
HIV infection. Administration of the transferrin fusion proteins of
the invention as a prophylactic vaccine, therefore, would comprise
administering to a host a concentration of transferrin fusion
protein effective in raising an immune response which is sufficient
to neutralize HIV, by, for example, inhibiting HIV ability to
infect cells. The exact concentration will depend upon the specific
peptide in the transferrin fusion protein to be administered, but
may be determined by using standard techniques for assaying the
development of an immune response which are well known to those of
ordinary skill in the art. The transferrin fusions protein to be
used as vaccines are usually administered intramuscularly.
[0258] Effective dosages of the transferrin fusion proteins
comprising T-20, T-1249, or analogs thereof to be administered may
be determined through procedures well known to those in the art
which address such parameters as biological half-life,
bioavailability, and toxicity. Given the data presented below in
Section 6, DP-178, for example, may prove efficacious in vivo at
doses required achieve circulating levels of 10 ng per ml of
peptide.
[0259] Furthermore, the present invention contemplates the use of
the transferrin fusion proteins comprising T-20, T-1249, or analogs
thereof for the manufacture of a medicinal product for the
treatment of diseases associated with the transmission of a
virus.
[0260] Soluble Toxin Receptors
[0261] The present invention provides fusion proteins comprising
soluble toxin receptor and transferrin or modified transferrin. As
used herein, the term "toxin" refers to a poisonous substance of
biological origin. The fusion proteins comprising a soluble toxin
receptor may be used to treat patients suffering from diseases
associated with toxins. Such fusion proteins may also be used for
diagnostic purposes.
[0262] Examples of toxins include, but are not limited to,
Pseudomonas exotoxins (PE), Diphtheria toxins (DT), ricin toxin,
abrin toxin, anthrax toxins, shiga toxin, botulism toxin, tetanus
toxin, cholera toxin, maitotoxin, palytoxin, ciguatoxin,
textilotoxin, batrachotoxin, alpha conotoxin, taipoxin,
tetrodotoxin, alpha tityustoxin, saxitoxin, anatoxin, microcystin,
aconitine, exfoliatin toxins A and B, enterotoxins, toxic shock
syndrome toxin (TSST-1), Y. pestis toxin, gas gangrene toxin, and
others. Because of the seriousness of the diseases that some of
these toxins cause and the ease of obtaining some of them for
biological warfare, there is a need to develop methods to obtain
large quantities of potent anti-toxins at a low cost.
[0263] The present invention contemplates the use of soluble toxin
receptors as anti-toxins for treatment and prevention of diseases
associated with various toxins. Toxin receptors are molecules that
bind to a specific toxin. A soluble toxin receptor is one that is
capable of being dissolved. Usually peptides or fragments of a
receptor are soluble. The present invention is directed to soluble
peptides or fragments of a toxin receptor that bind a specific
toxin.
[0264] Similar to other peptides discussed above, these peptides
have a short half-life. The present invention provides fusion
proteins comprising a soluble peptide of a toxin receptor fused to
a transferrin or modified transferrin molecule. The resulting
fusion protein has an increased half-life as compared to the
soluble toxin receptor peptide. The fusion protein is also easy to
produce in large quantities by recombinant means. Since the binding
properties of the soluble peptide has not been altered, it will
bind the toxin in circulation and prevent the toxin from binding to
the target receptor, thus inactivating the toxin. Accordingly, the
fusion protein is a potent anti-toxin.
[0265] In one embodiment, the present invention provides
pharmaceutical composition comprising soluble toxin receptor fused
to transferrin or modified transferrin and a pharmaceutically
acceptable carrier. In another embodiment, the present invention
provides the use of transferrin fusion protein comprising soluble
toxin receptor for the manufacture of a medicament for the
treatment or prevention of diseases or conditions associated with a
toxin.
[0266] Unlike antibodies which are difficult and expensive to
produce in large quantities, the present transferrin/anti-toxin
fusion protein is highly potent and less costly to manufacture.
Additionally, immunization is not expected to maintain the antibody
titers required to protect in instances of mass exposure following
an act of bioterrorism. It is unrealistic for the population to be
immunized on a mass scale in anticipation of an exposure.
[0267] Bacillus Anthracis Toxin Receptor
[0268] Anthrax toxin is a well-known agent of biological warfare
derived from Bacillus anthracis. Bacillus anthracis produces three
proteins which when combined appropriately form two potent toxins,
collectively designated anthrax toxin. Protective antigen (PA,
82,684 Da (Dalton)) and edema factor (EF, 89,840 Da) combine to
form edema toxin (ET), while PA and lethal factor (LF, 90,237 Da)
combine to form lethal toxin (LT) (Leppla, S. H. Alouf, J. E. and
Freer, J. H., eds. Academic Press, London 277-302, 1991). ET and LT
each conform to the AB toxin model, with PA providing the target
cell binding (B) function and EF or LF acting as the effector or
catalytic (A) moieties. A unique feature of these toxins is that LF
and EF have no toxicity in the absence of PA, apparently because
they cannot gain access to the cytosol of eukaryotic cells.
[0269] Recently, two of the targets of Lethal factor (LF) were
identified in cells. LF is a metalloprotease that specifically
cleaves Mek1 and Mek2 proteins, kinases that are part of the
MAP-kinase signaling pathway. LF's proteolytic activity inactivates
the MAP-kinase signaling cascade through cleavage of mitogen
activated protein kinase kinases 1 or 2 (MEK1 or MEK2). (Leppla, S.
A. In The Comprehensive Sourcebook of Bacterial Protein Toxins. J.
E. Alouf and J. H. Freer, Eds. 2.sup.nd edition, San Diego,
Academic Press, 1999; pp 243-263.).
[0270] PA is capable of binding to the surface of many types of
cells. After PA binds to a specific receptor (Leppla, supra, 1991)
on the surface of susceptible cells, it is cleaved at a single site
by a cell surface protease, probably furin, to produce an
amino-terminal 19-kDa fragment that is released from the
receptor/PA complex (Singh et al., J. Biol. Chem. 264:19103-19107,
1989). Removal of this fragment from PA exposes a high-affinity
binding site for LF and EF on the receptor-bound 63-kDa
carboxyl-terminal fragment (PA63). The complex of PA63 and LF or EF
enters cells and probably passes through acidified endosomes to
reach the cytosol.
[0271] PA, the non-toxic, cell-binding component of the toxin, is
the essential component of the currently available human vaccine.
The vaccine is usually produced from batch cultures of the Sterne
strain of B. anthracis, which although avirulent, is still required
to be handled as a Class III pathogen. In addition to PA, the
vaccine contains small amounts of the anthrax toxin moieties, edema
factor and lethal factor, and a range of culture derived proteins.
All these factors contribute to the recorded reactogenicity of the
vaccine in some individuals. The vaccine is expensive and requires
a six month course of four vaccinations. Futhermore, present
evidence suggests that this vaccine may not be effective against
inhalation challenge with certain strains (M. G. Broster et al.,
Proceedings of the International Workshop on Anthrax, Apr. 11-13,
1989, Winchester UK. Salisbury med Bull Suppl No 68, (1990)
91-92).
[0272] Bradley et al. (Nature, 2001, 414: 225-229) disclose cloning
of the human anthrax receptor that binds to PA. The receptor, ATR
(anthrax toxin receptor) is a type I membrane protein consisting of
368 amino acids. The protein has a predicted signal peptide of 27
amino acids, an extracellular domain of 293 amino acids containing
three putative N-linked glycosylation sites, a putative
transmembrane region of 23 amino acids and a short cytoplasmic tail
of 25 amino acids. A notable feature of ATR is that the
extracellular domain consists of a von willebrand factor type A
(VWA) domain which is known to be important in protein-protein
interactions. This VWA domain is located at amino acids 44 to 216.
A soluble version of ATR comprising amino acids 41-227 was shown to
bind the anthrax toxin. Accordingly, the VWA domain of ATR binds
directly to PA.
[0273] The present invention provides an anthrax antitoxin
comprising the extracellular domain of ATR fused to transferrin or
modified transferrin molecule. The present invention also
contemplates fusion proteins comprising fragments thereof of the
extracellular domain of ATR that binds PA fused to transferrin or
modified transferrin molecule. Moreover, the present invention
contemplates fusion proteins comprising small molecule mimetics of
the extracellular domain of ATR that binds PA fused to transferrin
or modified transferrin molecule. Preferably, the present invention
provides amino acids 41-227 of ATR fused to transferrin or modified
transferrin molecule.
[0274] Clostridium Botulinum Toxin
[0275] The clostridial neurotoxins are the most poisonous
substance. Humans are exposed to the neurotoxin produced by
Clostridium tetani (tetanus toxin) as a result of wounds. Although
the tetanus toxin remains a serious public health problem in
developing countries around the world, nearly everyone in the
western world is protected from tetanus toxin as a consequence of
childhood immunizations. Humans usually come into contact with the
neurotoxin produced by Clostridium botulinum (botulinum toxin)
through food poisoning. However, there are rare incidents of wound
botulism and colonizing infection of neonates known as infant
botulism. Since botulinum poisoning is rare, immunization of the
general population is not warranted on the basis of cost and the
expected rates of adverse reaction to the vaccine. Therefore,
humans are not protected from botulinum toxins. Additionally, these
toxins are relatively to produce. Consequently, botulinum toxins
are likely biological warfare agents.
[0276] As discussed, the anaerobic, gram positive bacterium
Clostridium botulinum produces the most poisonous biological
neurotoxin known with a lethal human dose in the nanogram range.
The effect of the toxin ranges from diarrheal diseases that can
cause destruction of the colon, to paralytic effects that can cause
death. The spores of Clostridium botulinum are found in soil and
can grow in improperly sterilized and sealed food containers of
home based canneries which are the cause of many of the cases of
botulism. The symptoms of botulism typically appear 18 to 36 hours
after eating the foodstuffs infected with a Clostridium botulinum
culture or spores. The botulinum toxin can apparently pass
unattenuated through the lining of the gut and attack peripheral
motor neurons. Symptoms of botulinum toxin intoxication can
progress from difficulty walking, swallowing, and speaking to
paralysis of the respiratory muscles and death.
[0277] Botulism disease may be grouped into four types, based on
the method of introduction of toxin into the bloodstream.
Food-borne botulism results from ingesting improperly preserved and
inadequately heated food that contains botulinal toxin (i.e., the
toxin is pre-formed prior to ingestion). Wound-induced botulism
results from C. botulinum penetrating traumatized tissue and
producing toxin that is absorbed into the bloodstream. Since 1950,
thirty cases of wound botulism have been reported (Swartz,
"Anaerobic Spore-Forming Bacilli: The Clostridia," pp. 633-646, in
Davis et al., (eds.), Microbiology, 4th edition, J. B. Lippincott
Co. (1990)). Inhalation botulism results when the toxin is inhaled.
Inhalation botulism has been reported as the result of accidental
exposure in the laboratory (Holzer, Med. Klin., 41:1735 [1962]) and
is a potential danger if the toxin is used as an agent of
biological warfare (Franz et al., in Botulinum and Tetanus
Neurotoxins, DasGupta (ed.), Plenum Press, New York [1993], pp.
473-476). Infectious infant botulism results from C. botulinum
colonization of the infant intestine with production of toxin and
its absorption into the bloodstream.
[0278] Different strains of Clostridium botulinum each produce
antigenically distinct toxin designated by the letters A-G.
Serotype A toxin has been implicated in 26% of the cases of food
botulism; types B, E, and F have also been implicated in a smaller
percentage of the food botulism cases (Sugiyama, Microbiol. Rev.,
44:419 (1980)). Wound botulism has been reportedly caused by only
types A or B toxins (Sugiyama, supra). Nearly all cases of infant
botulism have been caused by bacteria producing either type A or
type B toxin (exceptionally, one New Mexico case was caused by
Clostridium botulinum producing type F toxin and another by
Clostridium botulinum producing a type B-type F hybrid) (Arnon,
Epidemiol. Rev., 3:45 (1981)). Type C toxin affects waterfowl,
cattle, horses and mink. Type D toxin affects cattle, and type E
toxin affects both humans and birds.
[0279] Clostridium botulinum neurotoxin acts on nerve endings to
block acetylcholine release. Binding of the neurotoxin to a
membrane receptor through its heavy chain is the first essential
step in its mode of toxin action. Li et al. (J Nat Toxins 1998,
7(3):215-26) purified Type E botulinum neurotoxin (BoNT/E) or type
A botulinum neurotoxin (BoNT/A) from rat brain synaptosomes
employing a neurotoxin affinity column chromatography. The protein
fraction eluted from the affinity column with 0.5 M NaCl contained
a 57 kDa protein as a major eluant. Immunoblotting the eluant with
anti-synaptotagmin antibodies revealed that the 57 kDa protein was
synaptotagmin I. Rat synaptotagmin I has been suggested as the
receptor for BoNT/B (Nishiki et al., J. Biol. Chem. 269,
10498-10503, 1994) in rat brain. Li et al. investigated the binding
of BoNT/A and BoNT/E to synaptotagmin I by a microtiter plate-based
method. Binding of synaptotagmin I to BoNT/A coated on the plate
was competitively reduced upon preincubation of the proteins with
BoNT/E, suggesting a competitive binding of BoNT/A and BoNT/E to
the receptor. Taken together, these results suggest that the same
receptor protein binds to all three BoNT serotypes tested.
[0280] Synaptotagmin I is a broad acting receptor of Clostridium
botulinum neurotoxin serotypes A, B, and E and possibly C, D, F,
and G. It is located on the motor neuronal cell. The N-terminal
fragment of synaptotagmin I, amino acids 1-53 (SEQ ID NO: 4), is
responsible for binding to the various neurotoxin serotypes. The
binding mediates translocation of the neurotoxin into the cell and
blocks neurotransmitter release which results in paralysis and in
extreme cases fatality. The N-terminal fragment may be produced by
recombinant means and used to bind neurotoxins in circulation. The
binding of the fragment to the neurotoxin prevents the neurotoxin
from binding its target receptor which results in neutralization of
the toxin.
[0281] The goal of the present invention is to provide an
anti-toxin of Clostridium botulinum with significantly increased
half-life as compared to the recombinantly produced 53 amino acid
fragment, so that the anti-toxin has sufficient time to find and
bind the neurotoxins as they enter the circulation. The present
invention provides a fusion protein comprising the N-terminal 53
amino acid fragment of synaptotagmin I fused to transferrin or
modified transferrin, thereby increasing the half-life of the
fragment without altering the binding properties of the fragment.
Alternatively, the fragment could be chemically pegylated to
prolong circulating life. The longer half-life of the anti-toxin
will make a given dose more effective. Unlike antibodies, the
present anti-toxin fusion protein is broad acting and bind to
several neurotoxin serotypes, specifically neurotoxin A, B, and
E.
[0282] In a preferred embodiment, the fusion protein is produced in
a highly efficient microbial production system which can provide
large quantities of the anti-toxin at a reasonable cost to treat
the population following mass exposure in acts of bioterrorism.
This fusion protein can also be used in a prophylactic mode prior
to exposure
[0283] The present invention also contemplates anti-toxins
comprising peptide fragments of amino acids 1-53 of synaptotagmin I
or small molecule mimetics of amino acids 1-53 of snynaptotagmin I
fused to transferrin or modified transferrin molecule.
[0284] The fusion protein can also be used to block botulism spread
through food or air contamination among the civilian
population.
[0285] In one aspect of the invention, the anti-toxin fusion
protein is used to treat wound botulism resulting from drug use and
accidental overdose of botulinum neurotoxin following the treatment
of various diseases such as migraine dystonia, and
hyperhidrosis.
[0286] In another aspect of the invention, the anti-toxin fusion
protein is used to treat botulism from food poisoning.
[0287] Diptheria Toxin Receptor
[0288] Diphtheria is caused by a bacterium, Corynebacterium
diphtheriae, which typically infects mucous membranes: the nose and
throat are favorite places for the infection to take hold, but
mucous membranes of the eyes or genitalia can be infected also. The
bacteria produce a toxin which causes damage to tissue both at the
site of the original infection and in other parts of the body once
the toxin is spread via the bloodstream. The most serious effects
of diphtheria toxin are on the heart (muscle damage leading to loss
of pumping ability), kidneys, and the nervous system.
[0289] Diphtheria can be treated by giving penicillin or other
antibiotics to kill the bacteria, and antitoxin to clear free toxin
in the body. However the antitoxin will not clear toxin that has
already bound to cells and started to damage them. The better
approach is to give toxoid to stimulate immunity to the toxin, thus
enabling the body to clear toxin as soon as it appears. Immunity to
a bacterial toxin such as diphtheria toxin (DT) may be acquired
naturally during the course of an infection, or artificially by
injection of a detoxified form of the toxin (i.e., a toxoid)
(Germanier, ed., Bacterial Vaccines, Academic Press, Orlando, Fla.,
1984). Toxoids have traditionally been prepared by chemical
modification of native toxins (e.g., with formalin or formaldehyde
(Lingood et al., Brit. J. Exp. Path. 44:177, 1963)), rendering them
nontoxic while retaining an antigenicity that protects the
vaccinated animal against subsequent challenges by the natural
toxin: an example of a chemically-inactivated DT is that described
by Michel and Dirkx (Biochem. Biophys. Acta 491:286-295, 1977), in
which Trp-153 of Fragment A is the modified residue. The toxoid is
given initially at ages 2, 4, and 6 months, again at ages 18 months
and 5 years, and regularly every 10 years after that.
[0290] Several years it appeared that diptheria was no longer a
major public health threat. However, recently, there has been a
resurgence of diphtheria in the New Independent States of the
former Soviet Union, Ecuador, Thailand, Algeria and other
countries. Although diphtheria patients have been treated with
equine antitoxin, which neutralizes unbound toxin, surviving
patients have often developed serum sickness, an immune
complex-type disease. Thus, there is a need to develop a better
treatment for diphtheria patients.
[0291] The DT molecule is produced as a single polypeptide of 535
amino acids that is readily spliced to form two subunits linked by
a disulfide bond, Fragment A (N-terminal of about 21 Kda) and
Fragment B (C-terminal of about 37 Kda), as a result of cleavage at
residue 190, 192, or 193 (Moskaug, et al., Biol Chem
264:15709-15713, 1989; Collier et al., Biol Chem, 246:1496-1503,
1971). Fragment A is the catalytically active portion of DT. It is
an NAD-dependent ADP-ribosyltransferase which specifically targets
a protein synthesis factor termed elongation factor 2 (EF-2),
thereby inactivating EF2 and shutting down protein synthesis in the
cell. Fragment A consists of the diphtheria toxin C domain.
Fragment A is linked to the diphtheria toxin Fragment B by a
polypeptide loop. Fragment B of DT possesses a receptor-binding
domain (the R domain) which recognizes and binds the toxin molecule
to a particular receptor structure found on the surfaces of many
types of mammalian cells. Once DT is bound to the cell via this
receptor structure, the receptor/DT complex is taken up by the cell
via receptor-mediated endocytosis. A second functional region on
Fragment B (the T domain) acts to translocate DT across the
membrane of the endocytic vesicle, releasing catalytically active
Fragment A into the cytosol of the cell. A single molecule of
Fragment A is sufficient to inactivate the protein synthesis
machinery in a given cell.
[0292] Naglich et al. (Cell, 1992, 69: 1051-1061) describe
expression cloning of diphtheria toxin receptor from highly
toxin-sensitive monkey Vero cells. The amino acid sequence of the
receptor was found to be identical to that of the cell
surface-expressed heparin-binding epidermal growth factor-like
growth factor (HB-EGF) precursor (proHB-EGF). Although proHB-EGF is
cleaved and released as soluble mature HB-EGF (Goishi et al., Mol.
Biol. Cell, 1995, 6:967-980), a significant amount of proHB-EGF
remains on the cell surface and functions as a juxtacrine growth
factor (Hagashiyama et al., Science, 251: 929-938) and as a DT
receptor (Iwamoto et al, EMBO J., 1994, 13:2322-2330; Naglich et
al., Cell, 1992, 69: 1051-1061).
[0293] Hooper et al. (Biochem. Biophys. Res. Commun., 1995, 206:
710-717) show that recombinant mature human HB-EGF consisting of
residues 63-148 (the extracellular domain or the mature growth
factor) strongly inhibits the binding of radiolabeled DT to toxin
receptor-bearing cells. This result suggests that it would be
possible to treat diphtheria patients with mature HB-EGF, a natural
growth factor which will not cause serum sickness. However, mature
HB-EGF might produce side effects due to its growth factor
activity.
[0294] Cha et al. (Infection and Immunity, 2002, 70(5): 2344-2350)
developed a treatment based on human DT receptor/HB-EGF precursor.
They teach a recombinant truncated HB-EGF, consisting of residues
106-149 and lacking most of the heparin binding domain, capable of
inhibiting binding of radioiodinated DT to cells. Moreover, they
showed that it was a more effective inhibitor of DT binding than
the recombinant mature HB-EGF. Further the investigators mutated
some residues in the EGF like domain of the recombinant truncated
HB-EGF to destroy some of its mitogenic effect. It was demonstrated
that the receptor analog (I117A/L148A) displayed a low mitogenic
effect. The truncated (I117A/L148A) HB-EGF protein retained high DT
binding affinity. The work of Cha et al. suggest that truncated
(I117A/L148A) HB-EGF protein could be a safe anti-toxin for EGF
receptor.
[0295] The present invention provides anti-toxin fusion protein
comprising truncated (I117A/L148A) HB-EGF protein fused to
transferrin or modified transferrin. The present invention also
provides transferrin/anti-toxin fusion proteins comprising
fragments thereof of truncated (I117A/L148A) HB-EGF protein that
bind DT and has minimal mitogenic activity. Additionally, the
invention provides transferrin/anti-toxin fusion proteins
comprising analogs of truncated HB-EGF protein that bind DT and has
minimal mitogenic activity.
[0296] Other Toxin Receptors
[0297] Bacterium Bacillus thuringiensis (BT) produces bacteriocidal
proteins that are toxic to a limited range of insects, mostly in
the orders Lepidoptera, Coleoptera and Diptera. Bt toxins have been
used to control pests, by applying Bacillus thuringiensis to plants
or transforming plants themselves so that they generate the toxins
by virtue of their transgenic character. The toxins themselves are
glycoprotein products of the cry gene as described by Hofte, H. et
al. Microbiol Rev (1989) 53:242. U.S. Pat. No. 5,693,491 discloses
the cDNA encoding a glycoprotein receptor from the tobacco hornworm
that binds a Bacillus thuringiensis toxin. The availability of this
cDNA permits the retrieval of DNAs encoding homologous receptors in
other insects and organisms as well as the design of assays for the
cytotoxicity and binding affinity of potential pesticides and the
development of methods to manipulate natural and/or introduced
homologous receptors and, thus, to destroy target cells, tissues
and/or organisms.
[0298] Most Vibrio cholerae vaccine candidates constructed by
deleting the ctxA gene encoding cholera toxin (CT) are able to
elicit high antibody responses, but more than one-half of the
vaccines still develop mild diarrhea (Levine et al., Infect.
Immun., 56(1):161-167 (1988)). Given the magnitude of the diarrhea
induced in the absence of CT, it was hypothesized that V. cholerae
produce other enterotoxigenic factors, which are still present in
strains deleted of the ctxA sequence (Levine et al., supra). As a
result, a second toxin, zonula occludens toxin (hereinafter "ZOT")
elaborated by V. cholerae, and which contribute to the residual
diarrhea, was discovered (Fasano et al., Proc. Nat. Acad. Sci.,
USA, 8:5242-5246 (1991)). The zot gene is located immediately
adjacent to the ctx genes. The high percent concurrence of the zot
gene with the ctx genes among V. cholerae strains (Johnson et al.,
J. Clin. Microb., 31/3:732-733 (1993); and Karasawa et al, FEBS
Microbiology Letters, 106:143-146 (1993)) suggests a possible
synergistic role of ZOT in the causation of acute dehydrating
diarrhea typical of cholera. The zot gene has also been identified
in other enteric pathogens (Tschape, 2nd Asian-Pacific Symposium on
Typhoid fever and other Salomellosis, 47(Abstr.) (1994)). U.S. Pat.
No. 5,864,014 discloses the purified receptor for zonula occludens
toxin.
[0299] Diarrhea can be caused by small, heat stable peptide toxins
(ST) produced by various pathogenic bacteria (Thompson, M. R.,
1987, Pathol. Immunopathol. Res. 6, 103-116). In developing
countries, such toxins may be responsible for 50% to 80% of the
reported cases of diarrhea (Giannella, R. A., 1981, Ann. Rev. Med.
32, 341-357). ST are also a major cause of diarrhea in laboratory
and domestic animals (Burgess et al., 1978, Infect. Immun. 21,
526-531). It has been shown that heat stable enterotoxins bind to a
cell surface receptor in the intestine which subsequently leads to
an activation of guanylyl cyclase (Field et al., 1978, Proc. Natl.
Acad. Sci. USA 75, 2800-2804; Guerrant et al.,1980, J. Infectios
Diseases 142, 220-228). The rise in cyclic GMP then stimulates
fluid secretion thereby causing diarrhea. It has been reported that
the ST receptor is a distinctly different protein than quanylyl
cyclase based on partial chromatographic separation of a
detergent-solubilized ST-binding protein from guanylyl cyclase
activity (Kuno et al., 1986, J. Biol. Chem. 261, 1470-1476;
Waldman, et al., 1986, Infect. Immun. 51, 320-326). U.S. Pat. No.
5,237,051 discloses cloning of the nucleic acid encoding the
intestinal receptor which recognizes heat stable enterotoxins and
has guanylyl cylase activity. Data shows that the receptor binds
enterotoxin and signals normally through the cyclic GMP second
messenger system.
[0300] Sepsis is most commonly caused by infection or trauma
induced by a toxin. The initial symptoms of sepsis typically
include chills, profuse sweat, irregularly remittent fever,
prostration and the like, followed by persistent fever, hypotension
leading to shock, neutropenia, leukopenia, disseminated
intravascular coagulation, adult respiratory distress syndrome and
multiple organ failure. Sepsis-inducing toxins have been found
associated with pathogenic bacteria, viruses, plants and venoms.
Among the well described bacterial toxins are the endotoxins or
lipopolysaccharides(LPS) of the gram-negative bacteria. These
molecules are glycolipids that are ubiquitous in the outer membrane
of all gram-negative bacteria. It has been reported that report
that membrane-fixed CD14 acts as a receptor for the protein-bound
endotoxin (LPS) complex and mediates the cellular effects of
endotoxin (Wright et al., 1990, Science, 249:1431). Soluble CD14
truncated at amino acid 71(N71) contains the lipolysaccharide
binding sequence. N71 has been shown to neutralize circulating LPS,
i.e., acting as an endotoxin antagonist (Higuchi et al.,
Pathobiology, 2002, 70: 103).
[0301] Methods of Delivering Antitoxin Fusion Protein
[0302] In one embodiment, the anti-toxin fusion proteins of the
present invention will be packaged in a single piston syringe with
two contiguous chambers. The first chamber will contain diluent and
the second will contain the lyophilized anti-toxin fusion protein.
As the plunger is pushed down the diluent will be driven into the
next chamber to dissolve the anti-toxin fusion protein which will
be expelled through a needle for direct intramuscular delivery. The
diluent can contain an anti-freeze such glycerol to act in freezing
conditions. The lyophilized product will remain stable in tropical
conditions.
[0303] The present invention contemplates deliverying the antitoxin
fusion proteins of the present invention in this manner to soldiers
entering into a combat situation where the risk of exposure to
toxins is high. The anti-toxin fusion protein can be used for
immediate treatment on the battlefied and as a prophylactic before
going on the battlefield.
[0304] Nucleic Acids
[0305] The present invention also provides nucleic acid molecules
encoding transferrin fusion proteins comprising a transferrin
protein or a portion of a transferrin protein covalently linked or
joined to a therapeutic protein, preferably a therapeutic protein.
As discussed in more detail below, any therapeutic protein may be
used. The fusion protein may further comprise a linker region, for
instance a linker less than about 50, 40, 30, 20, or 10 amino acid
residues. The linker can be covalently linked to and between the
transferrin protein or portion thereof and the therapeutic protein,
preferably the therapeutic protein. Nucleic acid molecules of the
invention may be purified or not.
[0306] Host cells and vectors for replicating the nucleic acid
molecules and for expressing the encoded fusion proteins are also
provided. Any vectors or host cells may be used, whether
prokaryotic or eukaryotic, but eukaryotic expression systems, in
particular yeast expression systems, may be preferred. Many vectors
and host cells are known in the art for such purposes. It is well
within the skill of the art to select an appropriate set for the
desired application.
[0307] DNA sequences encoding transferrin, portions of transferrin
and therapeutic proteins of interest may be cloned from a variety
of genomic or cDNA libraries known in the art. The techniques for
isolating such DNA sequences using probe-based methods are
conventional techniques and are well known to those skilled in the
art. Probes for isolating such DNA sequences may be based on
published DNA or protein sequences (see, for example, Baldwin, G.
S. (1993) Comparison of Transferrin Sequences from Different
Species. Comp. Biochem. Physiol. 106B/1:203-218 and all references
cited therein, which are hereby incorporated by reference in their
entirety). Alternatively, the polymerase chain reaction (PCR)
method disclosed by Mullis et al. (U.S. Pat. No. 4,683,195) and
Mullis (U.S. Pat. No. 4,683,202), incorporated herein by reference
may be used. The choice of library and selection of probes for the
isolation of such DNA sequences is within the level of ordinary
skill in the art.
[0308] As known in the art "similarity" between two polynucleotides
or polypeptides is determined by comparing the nucleotide or amino
acid sequence and its conserved nucleotide or amino acid
substitutes of one polynucleotide or polypeptide to the sequence of
a second polynucleotide or polypeptide. Also known in the art is
"identity" which means the degree of sequence relatedness between
two polypeptide or two polynucleotide sequences as determined by
the identity of the match between two strings of such sequences.
Both identity and similarity can be readily calculated
(Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part I, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991).
[0309] While there exist a number of methods to measure identity
and similarity between two polynucleotide or polypeptide sequences,
the terms "identity" and "similarity" are well known to skilled
artisans (Sequence Analysis in Molecular Biology, von Heinje, G.,
Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and
Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo,
H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods
commonly employed to determine identity or similarity between two
sequences include, but are not limited to those disclosed in Guide
to Huge Computers, Martin J. Bishop, ed., Academic Press, San
Diego, 1994, and Carillo, H., and Lipman, D., SIAM J. Applied Math.
48:1073 (1988).
[0310] Preferred methods to determine identity are designed to give
the largest match between the two sequences tested. Methods to
determine identity and similarity are codified in computer
programs. Preferred computer program methods to determine identity
and similarity between two sequences include, but are not limited
to, GCG program package (Devereux, et al., Nucl. Acid Res.
12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, et al., J. Mol.
Biol. 215:403 (1990)). The degree of similarity or identity
referred to above is determined as the degree of identity between
the two sequences, often indicating a derivation of the first
sequence from the second. The degree of identity between two
nucleic acid sequences may be determined by means of computer
programs known in the art such as GAP provided in the GCG program
package (Needleman and Wunsch J. Mol. Biol. 48:443-453 (1970)). For
purposes of determining the degree of identity between two nucleic
acid sequences for the present invention, GAP is used with the
following settings: GAP creation penalty of 5.0 and GAP extension
penalty of 0.3.
[0311] Codon Optimization
[0312] The degeneracy of the genetic code permits variations of the
nucleotide sequence of a transferrin protein and/or therapeutic
protein of interest, while still producing a polypeptide having the
identical amino acid sequence as the polypeptide encoded by the
native DNA sequence. The procedure, known as "codon optimization"
(described in U.S. Pat. No. 5,547,871 which is incorporated herein
by reference in its entirety) provides one with a means of
designing such an altered DNA sequence. The design of codon
optimized genes should take into account a variety of factors,
including the frequency of codon usage in an organism, nearest
neighbor frequencies, RNA stability, the potential for secondary
structure formation, the route of synthesis and the intended future
DNA manipulations of that gene. In particular, available methods
may be used to alter the codons encoding a given fusion protein
with those most readily recognized by yeast when yeast expression
systems are used.
[0313] The degeneracy of the genetic code permits the same amino
acid sequence to be encoded and translated in many different ways.
For example, leucine, serine and arginine are each encoded by six
different codons, while valine, proline, threonine, alanine and
glycine are each encoded by four different codons. However, the
frequency of use of such synonymous codons varies from genome to
genome among eukaryotes and prokaryotes. For example, synonymous
codon-choice patterns among mammals are very similar, while
evolutionarily distant organisms such as yeast (such as S.
cerevisiae), bacteria (such as E. coli) and insects (such as D.
melanogaster) reveal a clearly different pattern of genomic codon
use frequencies (Grantham, R., et al., Nucl. Acid Res., 8, 49-62
(1980); Grantham, R., et al., Nucl. Acid Res., 9, 43-74 (1981);
Maroyama, T., et al., Nucl. Acid Res., 14, 151-197 (1986); Aota,
S., et al., Nucl. Acid Res., 16, 315-402 (1988); Wada, K., et al.,
Nucl. Acid Res., 19 Supp., 1981-1985 (1991); Kurland, C. G., FEBS
Lett., 285, 165-169 (1991)). These differences in codon-choice
patterns appear to contribute to the overall expression levels of
individual genes by modulating peptide elongation rates. (Kurland,
C. G., FEBS Lett., 285, 165-169 (1991); Pedersen, S., EMBO J., 3,
2895-2898 (1984); Sorensen, M. A., J. Mol. Biol., 207, 365-377
(1989); Randall, L. L., et al., Eur. J. Biochem., 107, 375-379
(1980); Curran, J. F., and Yarus, M., J. Mol. Biol., 209, 65-77
(1989); Varenne, S., et al., J. Mol. Biol., 180, 549-576 (1984),
Varenne, S., et al., J. Mol, Biol., 180, 549-576 (1984); Garel,
J.-P., J. Theor. Biol., 43, 211-225 (1974); Ikemura, T., J. Mol.
Biol., 146, 1-21 (1981); Ikemura, T., J. Mol. Biol., 151, 389-409
(1981)).
[0314] The preferred codon usage frequencies for a synthetic gene
should reflect the codon usages of nuclear genes derived from the
exact (or as closely related as possible) genome of the
cell/organism that is intended to be used for recombinant protein
expression, particularly that of yeast species. As discussed above,
in one preferred embodiment the human Tf sequence is codon
optimized, before or after modification as herein described for
yeast expression as may be the therapeutic protein nucleotide
sequence(s).
[0315] Vectors
[0316] Expression units for use in the present invention will
generally comprise the following elements, operably linked in a 5'
to 3' orientation: a transcriptional promoter, a secretory signal
sequence, a DNA sequence encoding a modified Tf fusion protein
comprising transferrin protein or a portion of a transferrin
protein joined to a DNA sequence encoding a therapeutic protein or
peptide of interest and a transcriptional terminator. As discussed
above, any arrangement of the therapeutic protein or peptide fused
to or within the Tf portion may be used in the vectors of the
invention. The selection of suitable promoters, signal sequences
and terminators will be determined by the selected host cell and
will be evident to one skilled in the art and are discussed more
specifically below.
[0317] Suitable yeast vectors for use in the present invention are
described in U.S. Pat. No. 6,291,212 and include YRp7 (Struhl et
al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEp13 (Broach
et al. Gene 8: 121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature
275:104-108, 1978), pPPC0005, pSeCHSA, pScNHSA, pC4 and derivatives
thereof. Useful yeast plasmid vectors also include pRS403-406,
pRS413-416 and the Pichia vectors available from Stratagene Cloning
Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404,
pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and
incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
PlasmidspRS413.about.41.6 are Yeast Centromere plasmids (YCps).
[0318] Such vectors will generally include a selectable marker,
which may be one of any number of genes that exhibit a dominant
phenotype for which a phenotypic assay exists to enable
transformants to be selected. Preferred selectable markers are
those that complement host cell auxotrophy, provide antibiotic
resistance or enable a cell to utilize specific carbon sources, and
include LEU2 (Broach et al. ibid.), URA3 (Botstein et al., Gene 8:
17, 1979), HIS3 (Struhl et al., ibid.) or POT1 (Kawasaki and Bell,
EP 171,142). Other suitable selectable markers include the CAT
gene, which confers chloramphenicol resistance on yeast cells.
Preferred promoters for use in yeast include promoters from yeast
glycolytic genes (Hitzeman et al., J Biol. Chem. 225: 12073-12080,
1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982;
Kawasaki, U.S. Pat. No. 4,599,311) or alcohol dehydrogenase genes
(Young et al., in Genetic Engineering of Microorganisms for
Chemicals, Hollaender et al., (eds.), p. 355, Plenum, N.Y., 1982;
Ammerer, Meth. Enzymol. 101: 192-201, 1983). In this regard,
particularly preferred promoters are the TPI1 promoter (Kawasaki,
U.S. Pat. No. 4,599,311) and the ADH2-4.sup.C (see U.S. Pat. No.
6,291,212 promoter (Russell et al., Nature 304: 652-654, 1983). The
expression units may also include a transcriptional terminator. A
preferred transcriptional terminator is the TPI1 terminator (Alber
and Kawasaki, ibid.). Other preferred vectors and preferred
components such as promoters and terminators of a yeast expression
system are disclosed in European Patents EP 0258067, EP 0286424,
EP0317254, EP 0387319, EP 0386222, EP 0424117, EP 0431880, and EP
1002095; European Patent Publications EP 0828759, EP 0764209, EP
0749478, and EP 0889949; PCT Publication WO 00/44772 and WO
94/04687; and U.S. Pat. Nos. 5,739,007; 5,637,504; 5,302,697;
5,260,202; 5,667,986; 5,728,553; 5,783,423; 5,965,386; 6150,133;
6,379,924; and 5,714,377; which are herein incorporated by
reference in their entirety.
[0319] In addition to yeast, modified fusion proteins of the
present invention can be expressed in filamentous fungi, for
example, strains of the fungi Aspergillus. Examples of useful
promoters include those derived from Aspergillus nidulans
glycolytic genes, such as the adh3 promoter (McKnight et al., EMBO
J. 4: 2093-2099, 1985) and the tpiA promoter. An example of a
suitable terminator is the adh3 terminator (McKnight et al.,
ibid.). The expression units utilizing such components may be
cloned into vectors that are capable of insertion into the
chromosomal DNA of Aspergillus, for example.
[0320] Mammalian expression vectors for use in carrying out the
present invention will include a promoter capable of directing the
transcription of the modified Tf fusion protein. Preferred
promoters include viral promoters and cellular promoters. Preferred
viral promoters include the major late promoter from adenovirus 2
(Kaufman and Sharp, Mol. Cell. Biol. 2: 1304-13199, 1982) and the
SV40 promoter (Subramani et al., Mol. Cell. Biol. 1: 854-864,
1981). Preferred cellular promoters include the mouse
metallothionein 1 promoter (Palmiter et al., Science 222: 809-814,
1983) and a mouse V.kappa. (see U.S. Pat. No. 6,291,212) promoter
(Grant et al., Nuc. Acids Res. 15: 5496, 1987). A particularly
preferred promoter is a mouse V.sub.H (see U.S. Pat. No. 6,291,212)
promoter (Loh et al., ibid.). Such expression vectors may also
contain a set of RNA splice sites located downstream from the
promoter and upstream from the DNA sequence encoding the
transferrin fusion protein. Preferred RNA splice sites may be
obtained from adenovirus and/or immunoglobulin genes.
[0321] Also contained in the expression vectors is a
polyadenylation signal located downstream of the coding sequence of
interest. Polyadenylation signals include the early or late
polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the
polyadenylation signal from the adenovirus 5 E1B region and the
human growth hormone gene terminator (DeNoto et al., Nucl. Acid
Res. 9: 3719-3730, 1981). A particularly preferred polyadenylation
signal is the V.sub.H (see U.S. Pat. No. 6,291,212) gene terminator
(Loh et al., ibid.). The expression vectors may include a noncoding
viral leader sequence, such as the adenovirus 2 tripartite leader,
located between the promoter and the RNA splice sites. Preferred
vectors may also include enhancer sequences, such as the SV40
enhancer and the mouse .mu. (see U.S. Pat. No. 6,291,212) enhancer
(Gillies, Cell 33: 717-728, 1983). Expression vectors may also
include sequences encoding the adenovirus VA RNAs.
[0322] Transformation
[0323] Techniques for transforming fungi are well known in the
literature, and have been described, for instance, by Beggs
(ibid.), Hinnen et al. (Proc. Natl. Acad. Sci. USA 75: 1929-1933,
1978), Yelton et al., (Proc. Natl. Acad. Sci. USA 81: 1740-1747,
1984), and Russell (Nature 301: 167-169, 1983). Other techniques
for introducing cloned DNA sequences into fungal cells, such as
electroporation (Becker and Guarente, Methods in Enzymol. 194:
182-187, 1991) may be used. The genotype of the host cell will
generally contain a genetic defect that is complemented by the
selectable marker present on the expression vector. Choice of a
particular host and selectable marker is well within the level of
ordinary skill in the art.
[0324] Cloned DNA sequences comprising modified Tf fusion proteins
of the invention may be introduced into cultured mammalian cells
by, for example, calcium phosphate-mediated transfection (Wigler et
al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics
7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973.) Other
techniques for introducing cloned DNA sequences into mammalian
cells, such as electroporation (Neumann et al., EMBO J. 1: 841-845,
1982), or lipofection may also be used. In order to identify cells
that have integrated the cloned DNA, a selectable marker is
generally introduced into the cells along with the gene or cDNA of
interest. Preferred selectable markers for use in cultured
mammalian cells include genes that confer resistance to drugs, such
as neomycin, hygromycin, and methotrexate. The selectable marker
may be an amplifiable selectable marker. A preferred amplifiable
selectable marker is the DHFR gene. A particularly preferred
amplifiable marker is the DHFR.sup.r (see U.S. Pat. No. 6,291,212)
cDNA (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80:
2495-2499,1983). Selectable markers are reviewed by Thilly
(Mammalian Cell Technology, Butterworth Publishers, Stoneham,
Mass.) and the choice of selectable markers is well within the
level of ordinary skill in the art.
[0325] Host Cells
[0326] The present invention also includes a cell, preferably a
yeast cell transformed to express a modified transferrin fusion
protein of the invention. In addition to the transformed host cells
themselves, the present invention also includes 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.
[0327] Host cells for use in practicing the present invention
include eukaryotic cells, and in some cases prokaryotic cells,
capable of being transformed or transfected with exogenous DNA and
grown in culture, such as cultured mammalian, insect, fungal, plant
and bacterial cells.
[0328] Fungal cells, including species of yeast (e.g.,
Saccharomyces spp., Schizosaccharomyces spp., Pichia spp.) may be
used as host cells within the present invention. Examples of fungi
including yeasts contemplated to be useful in the practice, of the
present invention as hosts for expressing the, transferrin fusion
protein of the inventions are Pichia (some species of which were
formerly classified as Hansenula), Saccharomyces, Kluyveromyces,
Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces,
Citeromyces, Pachysolen, Zygosaccharomyces, Debaromyces,
Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia,
Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,
Sporidiobolus, Endomycopsis, and the like. Examples of
Saccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii.
Examples of Kluyveromyces spp. are K. fragilis, K. lactis and K.
marxianus. A suitable Torulaspora species is T. delbrueckii.
Examples of Pichia spp. are P. angusta (formerly H. polymorpha), P.
anomala (formerly H. anomala) and P. pastoris.
[0329] Particularly useful host cells to produce the Tf fusion
proteins of the invention are the methylotrophic Pichia pastoris
(Steinlein et al. (1995) Protein Express. Purif. 6:619-624). Pichia
pastoris has been developed to be an outstanding host for the
production of foreign proteins since its alcohol oxidase promoter
was isolated and cloned; its transformation was first reported in
1985. P. pastoris can utilize methanol as a carbon source in the
absence of glucose. The P. pastoris expression system can use the
methanol-induced alcohol oxidase (AOX1) promoter, which controls
the gene that codes for the expression of alcohol oxidase, the
enzyme which catalyzes the first step in the metabolism of
methanol. This promoter has been characterized and incorporated
into a series of P. pastoris expression vectors. Since the proteins
produced in P. pastoris are typically folded correctly and secreted
into the medium, the fermentation of genetically engineered P.
pastoris provides an excellent alternative to E. coli expression
systems. A number of proteins have been produced using this system,
including tetanus toxin fragment, Bordatella pertussis pertactin,
human serum albumin and lysozyme.
[0330] Strains of the yeast Saccharomyces cerevisiae are another
preferred host. In a preferred embodiment, a yeast cell, or more
specifically, a Saccharomyces cerevisiae host cell that contains a
genetic deficiency in a gene required for asparagine-linked
glycosylation of glycoproteins is used. S. cerevisiae host cells
having such defects may be prepared using standard techniques of
mutation and selection, although many available yeast strains have
been modified to prevent or reduce glycosylation or
hypermannosylation. Ballou et al. (J. Biol. Chem. 255: 5986-5991,
1980) have described the isolation of mannoprotein biosynthesis
mutants that are defective in genes which affect asparagine-linked
glycosylation. Gentzsch and Tanner (Glycobiology 7:481-486, 1997)
have described a family of at least six genes (PMT1-6) encoding
enzymes responsible for the first step in O-glycosylation of
proteins in yeast. Mutants defective in one or more of these genes
show reduced O-linked glycosylation and/or altered specificity of
O-glycosylation.
[0331] To optimize production of the heterologous proteins, it is
also preferred that the host strain carries a mutation, such as the
S. cerevisiae pep4 mutation (Jones, Genetics 85: 23-33, 1977),
which results in reduced proteolytic activity. Host strains
containing mutations in other protease encoding regions are
particularly useful to produce large quantities of the Tf fusion
proteins of the invention.
[0332] Host cells containing DNA constructs of the present
invention are grown in an appropriate growth medium. As used
herein, the term "appropriate growth medium" means a medium
containing nutrients required for the growth of cells. Nutrients
required for cell growth may include a carbon source, a nitrogen
source, essential amino acids, vitamins, minerals and growth
factors. The growth medium will generally select for cells
containing the DNA construct by, for example, drug selection or
deficiency in an essential nutrient which is complemented by the
selectable marker on the DNA construct or co-transfected with the
DNA construct. Yeast cells, for example, are preferably grown in a
chemically defined medium, comprising a carbon source, e.g.
sucrose, a non-amino acid nitrogen source, inorganic salts,
vitamins and essential amino acid supplements. The pH of the medium
is preferably maintained at a pH greater than 2 and less than 8,
preferably at pH 5.5-6.5. Methods for maintaining a stable pH
include buffering and constant pH control. Preferred buffering
agents include succinic acid and Bis-Tris (Sigma Chemical Co., St.
Louis, Mo.). Yeast cells having a defect in a gene required for
asparagine-linked glycosylation are preferably grown in a medium
containing an osmotic stabilizer. A preferred osmotic stabilizer is
sorbitol supplemented into the medium at a concentration between
0.1 M and 1.5 M., preferably at 0.5 M or 1.0 M.
[0333] Cultured mammalian cells are generally grown in commercially
available serum-containing or serum-free media. Selection of a
medium appropriate for the particular cell line used is within the
level of ordinary skill in the art. Transfected mammalian cells are
allowed to grow for a period of time, typically 1-2 days, to begin
expressing the DNA sequence(s) of interest. Drug selection is then
applied to select for growth of cells that are expressing the
selectable marker in a stable fashion. For cells that have been
transfected with an amplifiable selectable marker the drug
concentration may be increased in a stepwise manner to select for
increased copy number of the cloned sequences, thereby increasing
expression levels.
[0334] Baculovirus/insect cell expression systems may also be used
to produce the modified Tf fusion proteins of the invention. The
BacPAK.TM. Baculovirus Expression System (BD Biosciences
(Clontech)) expresses recombinant proteins at high levels in insect
host cells. The target gene is inserted into a transfer vector,
which is cotransfected into insect host cells with the linearized
BacPAK6 viral DNA. The BacPAK6 DNA is missing an essential portion
of the baculovirus genome. When the DNA recombines with the vector,
the essential element is restored and the target gene is
transferred to the baculovirus genome. Following recombination, a
few viral plaques are picked and purified, and the recombinant
phenotype is verified. The newly isolated recombinant virus can
then be amplified and used to infect insect cell cultures to
produce large amounts of the desired protein.
[0335] Tf fusion proteins of the present invention may also be
produced using transgenic plants and animals. For example, sheep
and goats can make the therapeutic protein in their milk. Or
tobacco plants can include the protein in their leaves. Both
transgenic plant and animal production of proteins comprises adding
a new gene coding the fusion protein into the genome of the
organism. Not only can the transgenic organism produce a new
protein, but it can also pass this ability onto its offspring.
[0336] Secretory Signal Sequences
[0337] The terms "secretory signal sequence" or "signal sequence"
or "secretion leader sequence" are used interchangeably and are
described, for example in U.S. Pat. No. 6,291,212 and U.S. Pat. No.
5,547,871, both of which are herein incorporated by reference in
their entirety. Secretory signal sequences or signal sequences or
secretion leader sequences encode secretory peptides. A secretory
peptide is an amino acid sequence that acts to direct the secretion
of a mature polypeptide or protein from a cell. Secretory peptides
are generally characterized by a core of hydrophobic amino acids
and are typically (but not exclusively) found at the amino termini
of newly synthesized proteins. Very often the secretory peptide is
cleaved from the mature protein during secretion. Secretory
peptides may contain processing sites that allow cleavage of the
signal peptide from the mature protein as it passes through the
secretory pathway. Processing sites may be encoded within the
signal peptide or may be added to the signal peptide by, for
example, in vitro mutagenesis.
[0338] Secretory peptides may be used to direct the secretion of
modified Tf fusion proteins of the invention. One such secretory
peptide that may be used in combination with other secretory
peptides is the alpha mating factor leader sequence. Secretory
signal sequences or signal sequences or secretion leader sequences
are required for a complex series of post-translational processing
steps which result in secretion of a protein. If an intact signal
sequence is present, the protein being expressed enters the lumen
of the rough endoplasmic reticulum and is then transported through
the Golgi apparatus to secretory vesicles and is finally
transported out of the cell. Generally, the signal sequence
immediately follows the initiation codon and encodes a signal
peptide at the amino-terminal end of the protein to be secreted. In
most cases, the signal sequence is cleaved off by a specific
protease, called a signal peptidase. Preferred signal sequences
improve the processing and export efficiency of recombinant protein
expression using viral, mammalian or yeast expression vectors. In
some cases, the native Tf signal sequence may be used to express
and secrete fusion proteins of the invention.
[0339] Linkers
[0340] The Tf moiety and the therapeutic protein of the modified
transferrin fusion proteins of the invention can be fused directly
or using a linker peptide of various lengths to provide greater
physical separation and allow more spatial mobility between the
fused proteins and thus maximize the accessibility of the
therapeutic protein, for instance, for binding to its cognate
receptor. The linker peptide may consist of amino acids that are
flexible or more rigid. For example, a linker such as but not
limited to a poly-glycine stretch. The linker can be less than
about 50, 40, 30, 20, or 10 amino acid residues. The linker can be
covalently linked to and between the transferrin protein or portion
thereof and the therapeutic protein.
[0341] Detection of Tf Fusion Proteins
[0342] Assays for detection of biologically active modified
transferrin-fusion protein may include Western transfer, protein
blot or colony filter as well as activity based assays that detect
the fusion protein comprising transferrin and therapeutic protein.
A Western transfer filter may be prepared using the method
described by Towbin et al. (Proc. Natl. Acad. Sci. USA 76:
4350-4354, 1979). Briefly, samples are electrophoresed in a sodium
dodecylsulfate polyacrylamide gel. The proteins in the gel are
electrophoretically transferred to nitrocellulose paper. Protein
blot filters may be prepared by filtering supernatant samples or
concentrates through nitrocellulose filters using, for example, a
Minifold (Schleicher & Schuell, Keene, N. H.). Colony filters
may be prepared by growing colonies on a nitrocellulose filter that
has been laid across an appropriate growth medium. In this method,
a solid medium is preferred. The cells are allowed to grow on the
filters for at least 12 hours. The cells are removed from the
filters by washing with an appropriate buffer that does not remove
the proteins bound to the filters. A preferred buffer comprises 25
mM Tris-base, 19 mM glycine, pH 8.3, 20% methanol.
[0343] Transferrin fusion proteins of the present invention may be
labeled with a radioisotope or other imaging agent and used for in
vivo diagnostic purposes. Preferred radioisotope imaging agents
include iodine-125 and technetium-99, with technetium-99 being
particularly preferred. Methods for producing protein-isotope
conjugates are well known in the art, and are described by, for
example, Eckelman et al. (U.S. Pat. No. 4,652,440), Parker et al.
(WO 87/05030) and Wilber et al. (EP 203,764). Alternatively, the
transferrin fusion proteins may be bound to spin label enhancers
and used for magnetic resonance (MR) imaging. Suitable spin label
enhancers include stable, sterically hindered, free radical
compounds such as nitroxides. Methods for labeling ligands for MR
imaging are disclosed by, for example, Coffman et al. (U.S. Pat.
No. 4,656,026).
[0344] Detection of a transferrin fusion protein of the present
invention can be facilitated by coupling (i.e., physically linking)
the therapeutic protein to a detectable substance. Examples of
detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, and radioactive materials. 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 .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0345] In one embodiment where one is assaying for the ability of a
transferrin fusion protein of the invention to bind or compete with
an antigen for binding to an 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), complement fixation
assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, the binding
of the transferrin fusion protein is detected by detecting a label
on the transferrin fusion protein. In another embodiment, the
transferrin fusion protein is detected by detecting binding of a
secondary antibody or reagent that interacts with the transferrin
fusion protein. In a further embodiment, the secondary antibody or
reagent is labeled. Many means are known in the art for detecting
binding in an immunoassay and are within the scope of the present
invention.
[0346] Fusion proteins of the invention may also be detected by
assaying for the activity of the therapeutic protein moiety.
Specifically, transferrin fusion proteins of the invention may be
assayed for functional activity (e.g., biological activity or
therapeutic activity) using assays known to one of ordinary skill
in the art. Additionally, one of skill in the art may routinely
assay fragments of a therapeutic protein corresponding to a
therapeutic protein portion of a fusion protein of the invention,
for activity using well-known assays. Further, one of skill in the
art may routinely assay fragments of a modified transferrin protein
for activity using assays known in the art.
[0347] For example, in one embodiment where one is assaying for the
ability of a transferrin fusion protein of the invention to bind or
compete with a therapeutic protein for binding to an
anti-therapeutic polypeptide antibody and/or anti-transferrin
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), 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 art for detecting binding in
an immunoassay and are within the scope of the present
invention.
[0348] In a further embodiment, where a binding partner (e.g., a
receptor or a ligand) of a therapeutic protein is identified,
binding to that binding partner by a transferrin fusion protein
containing 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. Other methods will be known to the skilled artisan and
are within the scope of the invention.
[0349] Production of Fusion Proteins
[0350] The present invention further provides methods for producing
a modified fusion protein of the invention using nucleic acid
molecules herein described. In general terms, the production of a
recombinant form of a protein typically involves the following
steps.
[0351] A nucleic acid molecule is first obtained that encodes a
transferrin fusion protein of the invention. The nucleic acid
molecule is then preferably placed in operable linkage with
suitable control sequences, as described above, to form an
expression unit containing the protein open reading frame. The
expression unit is used to transform a suitable host and the
transformed host is cultured under conditions that allow the
production of the recombinant protein. Optionally the recombinant
protein is isolated from the medium or from the cells; recovery and
purification of the protein may not be necessary in some instances
where some impurities may be tolerated.
[0352] Each of the foregoing steps can be accomplished in a variety
of ways. For example, the construction of expression vectors that
are operable in a variety of hosts is accomplished using
appropriate replicons and control sequences, as set forth above.
The control sequences, expression vectors, and transformation
methods are dependent on the type of host cell used to express the
gene and were discussed in detail earlier and are otherwise known
to persons skilled in the art. Suitable restriction sites can, if
not normally available, be added to the ends of the coding sequence
so as to provide an excisable gene to insert into these vectors. A
skilled artisan can readily adapt any host/expression system known
in the art for use with the nucleic acid molecules of the invention
to produce a desired recombinant protein.
[0353] As discussed above, any expression system may be used,
including yeast, bacterial, animal, plant, eukaryotic and
prokaryotic systems. In some embodiments, yeast, mammalian cell
culture and transgenic animal or plant production systems are
preferred. In other embodiments, yeast systems that have been
modified to reduce native yeast glycosylation, hyper-glycosylation
or proteolytic activity may be used.
[0354] Isolation/Purification of Modified Transferrin Fusion
Proteins
[0355] Secreted, biologically active, modified transferrin fusion
proteins may be isolated from the medium of host cells grown under
conditions that allow the secretion of the biologically active
fusion proteins. The cell material is removed from the culture
medium, and the biologically active fusion proteins are isolated
using isolation techniques known in the art. Suitable isolation
techniques include precipitation and fractionation by a variety of
chromatographic methods, including gel filtration, ion exchange
chromatography and affinity chromatography.
[0356] A particularly preferred purification method is affinity
chromatography on an iron binding or metal chelating column or an
immunoaffinity chromatography using an antigen directed against the
transferrin or therapeutic protein of the polypeptide fusion. The
antigen is preferably immobilized or attached to a solid support or
substrate. A particularly preferred substrate is CNBr-activated
Sepharose (Pharmacia LKB Technologies, Inc., Piscataway, N. J.). By
this method, the medium is combined with the antigen/substrate
under conditions that will allow binding to occur. The complex may
be washed to remove unbound material, and the transferrin fusion
protein is released or eluted through the use of conditions
unfavorable to complex formation. Particularly useful methods of
elution include changes in pH, wherein the immobilized antigen has
a high affinity for the transferrin fusion protein at a first pH
and a reduced affinity at a second (higher or lower) pH; changes in
concentration of certain chaotropic agents; or through the use of
detergents.
[0357] Delivery of a Drug or Therapeutic Protein to the Inside of a
Cell and/or Across the Blood Brain Barrier (BBB)
[0358] Within the scope of the invention, the modified transferrin
fusion proteins may be used as a carrier to deliver a molecule or
small molecule therapeutic complexed to the ferric ion of
transferrin to the inside of a cell or across the blood brain
barrier or other barriers including across the cell membrane of any
cell type that naturally or engineered to express a Tf receptor. In
these embodiments, the Tf fusion protein will typically be
engineered or modified to inhibit, prevent or remove glycosylation
to extend the serum half-life of the fusion protein and/or
therapeutic protein portion. The addition of a targeting peptide is
specifically contemplated to further target the Tf fusion protein
to a particular cell type, e.g., a cancer cell.
[0359] In one embodiment, the iron-containing, anti-anemic drug,
ferric-sorbitol-citrate complex is loaded onto a modified Tf fusion
protein of the invention. Ferric-sorbitol-citrate (FSC) has been
shown to inhibit proliferation of various murine cancer cells in
vitro and cause tumor regression in vivo, while not having any
effect on proliferation of non-malignant cells (Poljak-Blazi et al.
(June 2000) Cancer Biotherapy and Radiopharmaceuticals (United
States), 15/3:285-293).
[0360] In another embodiment, the antineoplastic drug
Adriamycin.RTM. (doxorubicin) and/or the chemotherapeutic drug
bleomycin, both of which are known to form complexes with ferric
ion, is loaded onto a Tf fusion protein of the invention. In other
embodiments, a salt of a drug, for instance, a citrate or carbonate
salt, may be prepared and complexed with the ferric iron that is
then bound to Tf. As tumor cells often display a higher turnover
rate for iron; transferrin modified to carry at least one
anti-tumor agent, may provide a means of increasing agent exposure
or load to the tumor cells. (Demant, E. J, (1983) Eur. J. Biochem.
137/(1-2):113-118; Padbury et al. (1985) J. Biol. Chem.
260/13:7820-7823).
[0361] Pharmaceutical Formulations and Treatment Methods
[0362] The modified fusion proteins comprising a modified
transferrin of the invention may be administered to a patient in
need thereof using standard administration protocols. For instance,
the modified Tf fusion proteins of the present invention can be
provided alone, or in combination, or in sequential combination
with other agents that modulate a particular pathological process.
As used herein, two agents are said to be administered in
combination when the two agents are administered simultaneously or
are administered independently in a fashion such that the agents
will act at the same or near the same time.
[0363] The fusion proteins of the present invention can be
administered via parenteral, subcutaneous, intravenous,
intramuscular, intraperitoneal, transdermal and buccal routes. For
example, an agent may be administered locally to a site of injury
via microinfusion. Alternatively, or concurrently, administration
may be noninvasive by either the oral, inhalation, nasal, or
pulmonary route. The dosage administered will be dependent upon the
age, health, and weight of the recipient, kind of concurrent
treatment, if any, frequency of treatment, and the nature of the
effect desired.
[0364] While any method of administration may be used to deliver
the mTF fusion proteins of the invention, administration or
delivery orally may be a preferred embodiment for certain classes
of fusion proteins or to treat certain conditions.
[0365] The present invention further provides compositions
containing one or more fusion proteins of the invention. While
individual needs vary, determination of optimal ranges of effective
amounts of each component is within the skill of the art. Typical
dosages comprise about 1 pg/kg to about 100 mg/kg body weight. The
preferred dosages for systemic administration comprise about 100
ng/kg to about 100 mg/kg body weight. The preferred dosages for
direct administration to a site via microinfusion comprise about 1
ng/kg to about 1 mg/kg body weight. When administered via direct
injection or microinfusion, modified fusion proteins of the
invention may be engineered to exhibit reduced or no binding of
iron to prevent, in part, localized iron toxicity.
[0366] In addition to the pharmacologically active fusion protein,
the compositions of the present invention may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries that facilitate processing of the active compounds into
preparations which can be used pharmaceutically for delivery to the
site of action. Suitable formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form, for example, water-soluble salts. In addition, suspensions of
the active compounds as appropriate oily injection suspensions may
be administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension and include, for example, sodium
carboxymethyl cellulose, sorbitol and dextran. Optionally, the
suspension may also contain stabilizers. Liposomes can also be used
to encapsulate the agent for delivery into the cell.
[0367] The pharmaceutical formulation for systemic administration
according to the invention may be formulated for enteral,
parenteral or topical administration. Indeed, all three types of
formulations may be used simultaneously to achieve systemic
administration of the active ingredient. Suitable formulations for
oral administration include hard or soft gelatin capsules, pills,
tablets, including coated tablets, elixirs, suspensions, syrups or
inhalations and controlled release forms thereof.
[0368] The pharmaceutical composition of the present invention can
be in unit dosage form, e.g. as tablets or capsules. In such form,
the composition is sub-divided in unit dose containing appropriate
quantities of the active ingredient; the unit dosage forms can be
packaged compositions, for example, packeted powders, vials,
ampoules, prefilled syringes or sachets containing liquids. The
unit dosage form can be, for example, a capsule or tablet itself,
or it can be the appropriate number of any such compositions in
package form. The dosage to be used in the treatment must be
subjectively determined by the physician.
[0369] In practicing the methods of this invention, the fusion
proteins of this invention may be used alone or in combination, or
in combination with other therapeutic or diagnostic agents. In
certain preferred embodiments, the compounds of this invention may
be co-administered along with other compounds typically prescribed
for these conditions according to generally accepted medical
practice. The compounds of this invention can be utilized in vivo,
ordinarily in mammals, such as humans, sheep, horses, cattle, pigs,
dogs, cats, rats and mice, or in vitro.
[0370] Oral Pharmaceutical Compositions and Delivery Methods
[0371] In the present invention, Tf fusion proteins, including but
not limited to modified Tf fusion proteins, may be formulated for
oral delivery. In particular, certain fusion proteins of the
invention that are used to treat certain classes of diseases or
medical conditions may be particularly amenable for oral
formulation and delivery. Such classes of diseases or conditions
include, but are not limited to, acute, chronic and recurrent
diseases. Chronic or recurrent diseases include, but are not
limited to, viral disease or infections, cancer, a metabolic
diseases, obesity, autoimmune diseases, inflammatory diseases,
allergy, graft-vs.-host disease, systemic microbial infection,
anemia, cardiovascular disease, psychosis, genetic diseases,
neurodegenerative diseases, disorders of hematopoietic cells,
diseases of the endocrine system or reproductive systems,
gastrointestinal diseases. Examples of these classes of disease
include diabetes, multiple sclerosis, asthma, HCV or HIV
infections, hypertension, hypercholesterolemia, arterial scherosis,
arthritis, and Alzheimer's disease. In many chronic diseases, oral
formulations of Tf fusion proteins of the invention and methods of
administration are particularly useful because they allow long-term
patient care and therapy via home oral administration without
reliance on injectable treatment or drug protocols.
[0372] Oral formulations and delivery methods comprising Tf fusion
proteins of the invention take advantage of, in part, transferrin
receptor mediated transcytosis across the gastrointestinal (GI)
epithelium. The Tf receptor is found at a very high density in the
human GI epithelium, transferrin is highly resistant to tryptic and
chymotryptic digestion and Tf chemical conjugates have been used to
successfully deliver proteins and peptides across the GI epithelium
(Xia et al., (2000) J. Pharmacol. Experiment. Therap., 295:594-600;
Xia et al. (2001) Pharmaceutical Res., 18(2):191-195; and Shah et
al. (1996) J. Pharmaceutical Sci., 85(12):1306-1311, all of which
are herein incorporated by reference in their entirety). Once
transported across the GI epithelium, Tf fusion proteins of the
invention exhibit extended half-life in serum, that is, the
therapeutic protein or peptide(s) attached or inserted into Tf
exhibit an extended serum half-life compared to the protein or
peptide in its non-fused state.
[0373] Oral formulations of Tf fusion proteins of the invention may
be prepared so that they are suitable for transport to the GI
epithelium and protection of the Tf fusion protein component and
other active components in the stomach. Such formulations may
include carrier and dispersant components and may be in any
suitable form, including aerosols (for oral or pulmonary delivery),
syrups, elixirs, tablets, including chewable tablets, hard or soft
capsules, troches, lozenges, aqueous or oily suspensions,
emulsions, cachets or pellets granulates, and dispersible powders.
Preferably, Tf fusion protein formulations are employed in solid
dosage forms suitable for simple, and preferably oral,
administration of precise dosages. Solid dosage forms for oral
administration are preferably tablets, capsules, or the like.
[0374] For oral administration in the form of a tablet or capsule,
care should be taken to ensure that the composition enables
sufficient active ingredient to be absorbed by the host to produce
an effective response. Thus, for example, the amount of Tf fusion
protein may be increased over that theoretically required or other
known measures such as coating or encapsulation may be taken to
protect the polypeptides from enzymatic action in the stomach.
[0375] Traditionally, peptide and protein drugs have been
administered by injection because of the poor bioavailability when
administered non-parenterally, and in particular orally. These
drugs are prone to chemical and conformational instability and are
often degraded by the acidic conditions in the stomach, as well as
by enzymes in the stomach and gastrointestinal tract. In response
to these delivery problems, certain technologies for oral delivery
have been developed, such as encapsulation in nanoparticles
composed of polymers with a hydrophobic backbone and hydrophilic
branches as drug carriers, encapsulation in microparticles,
insertion into liposomes in emulsions, and conjugation to other
molecules. All of which may be used with the Tf fusion molecules of
the present invention.
[0376] Examples of nanoparticles include mucoadhesive nanoparticles
coated with chitosan and Carbopol (Takeuchi et al., Adv. Drug
Deliv. Rev. 47(1):39-54, 2001) and nanoparticles containing charged
combination polyesters, poly(2-sulfobutyl-vinyl alcohol) and
poly(D,L-lactic-co-glycolic acid) (Jung et al., Eur. J. Pharm.
Biopharm. 50(1):147-160, 2000). Nanoparticles containing surface
polymers with poly-N-isopropylacrylamide regions and cationic
poly-vinylamine groups showed improved absorption of salmon
calcitonin when administered orally to rats.
[0377] Drug delivery particles composed of alginate and pectin,
strengthened with polylysine, are relatively acid and base
resistant and can be used as a carrier for drugs. These particles
combine the advantages of bioadhesion, enhanced absorption and
sustained release (Liu et al., J. Pharm. Pharmacol. 51(2):141-149,
1999).
[0378] Additionally, lipoamino acid groups and liposaccharide
groups conjugated to the N- and C-termini of peptides such as
synthetic somatostatin, creating an amphipathic surfactant, were
shown to produce a composition that retained biological activity
(Toth et al., J. Med. Chem. 42(19):4010-4013, 1999).
[0379] Examples of other peptide delivery technologies include
carbopol-coated mucoadhesive emulsions containing the peptide of
interest and either nitroso-N-acetyl-D,L-penicillamine and
carbolpol or taurocholate and carbopol. These were shown to be
effective when orally administered to rats to reduce serum calcium
concentrations (Ogiso et al., Biol. Pharm. Bull. 24(6):656-661,
2001). Phosphatidylethanol, derived from phosphatidylcholine, was
used to prepare liposomes containing phosphatidylethanol as a
carrier of insulin. These liposomes, when administered orally to
rats, were shown to be active (Kisel et al., Int. J. Pharm.
216(1-2):105-114, 2001).
[0380] Insulin has also been formulated in poly(vinyl alcohol)-gel
spheres containing insulin and a protease inhibitor, such as
aprotinin or bacitracin. The glucose-lowering properties of these
gel spheres have been demonstrated in rats, where insulin is
released largely in the lower intestine (Kimura et al., Biol.
Pharm. Bull. 19(6):897-900, 1996.
[0381] Oral delivery of insulin has also been studied using
nanoparticles made of poly(alkyl cyanoacrylate).that were dispersed
with a surfactant in an oily phase (Damge et al., J. Pharm. Sci.
86(12):1403-1409, 1997) and using calcium alginate beads coated
with chitosan (Onal et al., Artif. Cells Blood Substit. Immobil.
Biotechnol. 30(3):229-237, 2002).
[0382] In other methods, the N- and C-termini of a peptide are
linked to polyethylene glycol and then to allyl chains to form
conjugates with improved resistance to enzymatic degradation and
improved diffusion through the GI wall (www.nobexcorp.com).
[0383] BioPORTER.RTM. is a cationic lipid mixture, which interacts
non-covalently with peptides to create a protective coating or
layer. The peptide-lipid complex can fuse to the plasma membrane of
cells, and the peptides are internalized into the cells
(www.genetherapysystems.com).
[0384] In a process using liposomes as a starting material,
cochleate-shaped particles have been developed as a pharmaceutical
vehicle. A peptide is added to a suspension of liposomes containing
mainly negatively charged lipids. The addition of calcium causes
the collapse and fusion of the liposomes into large sheets composed
of lipid bilayers, which then spontaneously roll up or stack into
cochleates (U.S. Pat. No. 5,840,707;
http://www.biodeliverysciences.com).
[0385] Compositions comprising Tf fusion protein intended for oral
use may be prepared according to any method known to the art for
the manufacture of pharmaceutical compositions and such
compositions may contain one or more agents selected from the group
consisting of sweetening agents in order to provide a
pharmaceutically elegant and palatable preparation. For example, to
prepare orally deliverable tablets, Tf fusion protein is mixed with
at least one pharmaceutical excipient, and the solid formulation is
compressed to form a tablet according to known methods, for
delivery to the gastrointestinal tract. The tablet composition is
typically formulated with additives, e.g. a saccharide or cellulose
carrier, a binder such as starch paste or methyl cellulose, a
filler, a disintegrator, or other additives typically usually used
in the manufacture of medical preparations. To prepare orally
deliverable capsules, DHEA is mixed with at least one
pharmaceutical excipient, and the solid formulation is placed in a
capsular container suitable for delivery to the gastrointestinal
tract. Compositions comprising Tf fusion protein may be prepared as
described generally in Remington's Pharmaceutical Sciences, 18th
Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89,
which is herein incorporated by reference.
[0386] As described above, many of the oral formulations of the
invention may contain inert ingredients which allow for protection
against the stomach environment, and release of the biologically
active material in the intestine. Such formulations, or enteric
coatings, are well known in the art. For example, tablets
containing Tf fusion protein in admixture with non-toxic
pharmaceutically acceptable excipients which are suitable for
manufacture of tablets may be used. These excipients may be inert
diluents, such as calcium carbonate, sodium carbonate, lactose,
calcium phosphate or sodium phosphate; granulating and
disintegrating agents, for example, maize starch, gelatin or
acacia, and lubricating agents, for example, magnesium stearate,
stearic acid, or talc.
[0387] The tablets may be uncoated or they may be coated with known
techniques to delay disintegration and absorption in the
gastrointestinal track and thereby provide a sustained action over
a longer period of time. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate alone or with a wax
may be employed.
[0388] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate, or kaolin or as soft gelatin capsules wherein the active
ingredient is mixed with an aqueous or an oil medium, for example,
arachis oil, peanut oil, liquid paraffin or olive oil.
[0389] Aqueous suspensions may contain Tf fusion protein in the
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example,
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally occurring phosphatide, for
example, lecithin, or condensation products of an alkylene oxide
with fatty acids, for example, polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example, heptadecylethyloxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example
polyoxyethylene sorbitan monooleate. The aqueous suspensions may
also contain one or more preservatives for example, ethyl or
n-propyl p-hydroxybenzoate, one or more coloring agents, one or
more flavoring agents and one or more sweetening agents such as
sucrose or saccharin.
[0390] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example, arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oil suspensions may contain a thickening agent, for
example, beeswax, hard paraffin or cetyl alcohol. Sweetening
agents, such as those set forth above, and flavoring agents may be
added to provide a palatable oral preparation. These compositions
may be preserved by the addition of an antioxidant such as ascorbic
acid.
[0391] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient and admixture with dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example,
sweetening, flavoring and coloring agents, may also be present.
[0392] The pharmaceutical compositions containing Tf fusion protein
may also be in the form of oil-in-water emulsions. The oil phase
may be a vegetable oil, for example, olive oil or arachis oil, or a
mineral oil for example, gum acacia or gum tragacanth,
naturally-occurring phosphotides, for example soybean lecithin, and
esters or partial esters derived from fatty acids and hexitol
anhydrides, for example, sorbitan monooleate, and condensation
products of the same partial esters with ethylene oxide, for
example, polyoxyethylene sorbitan monooleate. The emulsions may
also contain sweetening and flavoring agents.
[0393] Syrups and elixirs containing Tf fusion protein may be
formulated with sweetening agents, for example, glycerol, sorbitol
or sucrose. Such formulations may also contain a demulcent, a
preservative and flavoring and coloring agents. The pharmaceutical
compositions may be in the form of a sterile injectable
preparation, for example, as a sterile injectable aqueous or
oleaginous suspension. This suspension may be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents which have been mentioned above. The sterile
injectable preparations may also be a sterile injectable solution
or suspension in a non-toxic parenterally-acceptable diluent or
solvate, for example as a solution in 1,3-butanediol. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this period any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid find use in the
preparation of injectables.
[0394] Pharmaceutical compositions may also be formulated for oral
delivery using polyester microspheres, zein microspheres,
proteinoid microspheres, polycyanoacrylate microspheres, and
lipid-based systems (see, for example, DiBase and Morrel, Oral
Delivery of Microencapsulated Proteins, in Protein Delivery:
Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum
Press 1997)).
[0395] The proportion of pharmaceutically active Tf fusion protein
to carrier and/or other substances may vary from about 0.5 to about
100 wt. % (weight percent). For oral use, the pharmaceutical
formulation will generally contain from about 5 to about 100% by
weight of the active material. For other uses, the formulation will
generally have from about 0.5 to about 50 wt. % of the active
material.
[0396] Tf fusion protein formulations employed in the invention
provide an effective amount of Tf fusion protein upon
administration to an individual. As used in this context, an
"effective amount" of Tf fusion is an amount that is effective to
ameliorate a symptom of a disease.
[0397] The Tf fusion protein composition of the present invention
may be, though not necessarily, administered daily, in an effective
amount to ameliorate a symptom. Generally, the total daily dosage
will be at least about 50 mg, preferably at least about 100 mg, and
more preferably at least about 200 mg, and preferably not more than
500 mg per day, administered orally, e.g., in 4 capsules or
tablets, each containing 50 mg Tf fusion protein. Capsules or
tablets for oral delivery can conveniently contain up to a full
daily oral dose, e.g., 200 mg or more.
[0398] In a particularly preferred embodiment, oral pharmaceutical
compositions comprising Tf fusion protein are formulated in
buffered liquid form which is then encapsulated into soft or
hard-coated gelatin capsules which are then coated with an
appropriate enteric coating. For the oral pharmaceutical
compositions of the invention, the location of release may be
anywhere in the GI system, including the small intestine (the
duodenum, the jejunum, or the ileum), or the large intestine.
[0399] In other embodiments, oral compositions of the invention are
formulated to slowly release the active ingredients, including the
Tf fusion proteins of the invention, in the GI system using known
delayed release formulations.
[0400] Tf fusion proteins of the invention for oral delivery are
capable of binding the Tf receptor found in the GI epithelium. To
facilitate this binding and receptor mediated transport, Tf fusion
proteins of the invention are typically produced with iron and in
some instances carbonate, bound to the Tf moiety. Processes and
methods to load the Tf moiety of the fusion protein compositions of
the invention with iron and carbonate are known in the art
[0401] In some pharmaceutical formulations of the invention, the Tf
moiety of the Tf fusion protein may be modified to increase the
affinity or avidity of the Tf moiety to iron. Such methods are
known in the art. For instance, mutagenesis can be used to produce
mutant transferrin moieties that bind iron more avidly than natural
transferrin. In human serum transferrin, the amino acids which are
ligands for metal ion chelation include, but are not limited to N
lobe amino acids Asp63, Tyr 95, Tyr188, Lys206, His207 and His249;
and C lobe amino acids Asp392, Tyr426, Tyr517 and His585 of SEQ ID
NO: 3 (the number beside the amino acid indicates the position of
the amino acid residue in the primary amino acid sequence where the
valine of the mature protein is designated position 1). See U.S.
Pat. No. 5,986,067, which is herein incorporated be reference. In
one embodiment, the Lys206 and His207 residues within the N lobe
are replaced with Gln and Glu, respectively.
[0402] In some pharmaceutical formulations of the invention, the Tf
fusion protein is engineered to contain a cleavage site between the
therapeutic protein or peptide and the Tf moiety. Such cleavable
sites or linkers are known in the art.
[0403] Pharmaceutical compositions of the invention and methods of
the invention may include the addition of a transcytosis enhancer
to facilitate transfer of the fusion protein across the GI
epithelium. Such enhancers are known in the art. See Xia et al.,
(2000) J. Pharmacol. Experiment. Therap., 295:594-600; and Xia et
al. (2001) Pharmaceutical Res., 18(2):191-195.
[0404] In preferred embodiments of the invention, oral
pharmaceutical formulations include Tf fusion proteins comprising a
modified Tf moiety exhibiting reduced or no glycosylation fused at
the N terminal end to an insulin or GLP-1 protein or peptide as
described above. Such pharmaceutical compositions may be used to
treat glucose imbalance disorders such as diabetes by oral
administration of the pharmaceutical composition comprising an
effective dose of fusion protein.
[0405] The effective dose of fusion protein may be measured in a
numbers of ways, including dosages calculated to alleviate symptoms
associated with a specific disease state in a patient, such as the
symptoms of diabetes. In other formulations, dosages are calculated
to comprise an effective amount of fusion protein to induce a
detectable change in blood glucose levels in the patient. Such
detectable changes in blood glucose may include a decrease in blood
glucose levels of between about 1% and 90%, or between about 5% and
about 80%. These decreases in blood glucose levels will be
dependent on the disease condition being treated and pharmaceutical
compositions or methods of administration may be modified to
achieve the desired result for each patient. In other instances,
the pharmaceutical compositions are formulated and methods of
administration modified to detect an increase in the activity level
of the therapeutic protein or peptide in the patient, for instance,
detectable increases in the activities of insulin or GLP-1. Such
formulations and methods may deliver between about 1 pg to about
100 mg /kg body weight of fusion protein, about 100 ng to about 100
.mu.g/kg body weight of fusion protein, about 100 .mu.g/to about
100 mg/kg body weight of fusion protein, about 1 .mu.g to about 1 g
of fusion protein, about 10 .mu.g to about 100 mg of fusion protein
or about 10 mg to about 50 mg of fusion protein. Formulations may
also be calculated using a unit measurement of therapeutic protein
activity, such as about 5 to about 500 units of human insulin or
about 10 to about 100 units of human insulin. The measurements by
weight or activity can be calculated using known standards for each
therapeutic protein or peptide fused to Tf.
[0406] The invention also includes methods of orally administering
the pharmaceutical compositions of the invention. Such methods may
include, but are not limited to, steps of orally administering the
compositions by the patient or a caregiver. Such administration
steps may include administration on intervals such as once or twice
per day depending on the Tf fusion protein, disease or patient
condition or individual patient. Such methods also include the
administration of various dosages of the individual Tf fusion
protein. For instance, the initial dosage of a pharmaceutical
composition may be at a higher level to induce a desired effect,
such as reduction in blood glucose levels. Subsequent dosages may
then be decreased once a desired effect is achieved. These changes
or modifications to administration protocols may be done by the
attending physician or health care worker. In some instances, the
changes in the administration protocol may be done by the
individual patient, such as when a patient is monitoring blood
glucose levels and administering a mTf-insulin or mTf-GLP-1 oral
composition of the invention.
[0407] The invention also includes methods of producing oral
compositions or medicant compositions of the invention comprising
formulating a Tf fusion protein of the invention into an orally
administerable form. In other instances, the invention includes
methods of producing compositions or medicant compositions of the
invention comprising formulating a Tf fusion protein of the
invention into a form suitable for oral administration.
[0408] Moreover, the present invention includes pulmonary delivery
of the Tf fusion protein formulations. Pulmonary delivery is
particularly promising for the delivery of macromolecules which are
difficult to deliver by other routes of administration. Such
pulmonary delivery can be effective both for systemic delivery and
for localized delivery to treat diseases of the lungs, since drugs
delivered to the lung are readily absorbed through the alveolar
region directly into the blood circulation.
[0409] The present invention provides compositions suitable for
forming a drug dispersion for oral inhalation (pulmonary delivery)
to treat various conditions or diseases. The Tf fusion protein
formulation could be delivered by different approaches such as
liquid nebulizers, aerosol-based metered dose inhalers (MDI's), and
dry powder dispersion devices. In formulating compositions for
pulmonary delivery, pharmaceutically acceptable carriers including
surface active agents or surfactants and bulk carriers are commonly
added to provide stability, dispersibility, consistency, and/or
bulking characteristics to enhance uniform pulmonary delivery of
the composition to the subject.
[0410] Surface active agents or surfactants promotes absorption of
polypeptide through mucosal membrane or lining. Useful surface
active agents or surfactants include fatty acids and salts thereof,
bile salts, phospholipid, or an alkyl saccharide. Examples of fatty
acids and salts thereof include sodium, potassium and lysine salts
of caprylate (C.sub.8), caprate (C.sub.10), laurate (C.sub.12) and
myristate (C.sub.14). Examples of bile salts include cholic acid,
chenodeoxycholic acid, glycocholic acid, taurocholic acid,
glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic
acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic
acid, and ursodeoxycholic acid.
[0411] Examples of phospholipids include single-chain
phospholipids, such as lysophosphatidylcholine,
lysophosphatidylglycerol, lysophosphatidylethanolamine,
lysophosphatidylinositol and lysophosphatidylserine; or
double-chain phospholipids, such as diacylphosphatidylcholines,
diacylphosphatidylglycerols, diacylphosphatidylethanolamines,
diacylphosphatidylinositols and diacylphosphatidylserines. Examples
of alkyl saccharides include alkyl glucosides or alkyl maltosides,
such as decyl glucoside and dodecyl maltoside.
[0412] Pharmaceutical excipients that are useful as carriers
include stabilizers such as human serum albumin (HSA); bulking
agents such as carbohydrates, amino acids and polypeptides; pH
adjusters or buffers; salts such as sodium chloride; and the like.
These carriers may be in a crystalline or amorphous form or may be
a mixture of the two.
[0413] Examples of carbohydrates for use as bulking agents include
monosaccharides such as galactose, D-mannose, sorbose, and the
like; disaccharides, such as lactose, trehalose, and the like;
cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and
polysaccharides, such as raffinose, maltodextrins, dextrans, and
the like; alditols, such as mannitol, xylitol, and the like.
Examples of polypeptides for use as bulking agents include
aspartame. Amino acids include alanine and glycine, with glycine
being preferred.
[0414] Additives, which are minor components of the composition,
may be included for conformational stability during spray drying
and for improving dispersibility of the powder. These additives
include hydrophobic amino acids such as tryptophan, tyrosine,
leucine, phenylalanine, and the like.
[0415] Suitable pH adjusters or buffers include organic salts
prepared from organic acids and bases, such as sodium citrate,
sodium ascorbate, and the like; sodium citrate is preferred.
[0416] The Tf fusion compositions for pulmonary delivery may be
packaged as unit doses where a therapeutically effective amount of
the composition is present in a unit dose receptacle, such as a
blister pack, gelatin capsule, or the like. The manufacture of
blister packs or gelatin capsules is typically carried out by
methods that are generally well known in the packaging art.
[0417] U.S. Pat. No. 6,524,557 discloses a pharmaceutical aerosol
formulation comprising (a) a HFA propellant; (b) a pharmaceutically
active polypeptide dispersible in the propellant; and (c) a
surfactant which is a C.sub.8-C.sub.16 fatty acid or salt thereof,
a bile salt, a phospholipid, or an alkyl saccharide, which
surfactant enhances the systemic absorption of the polypeptide in
the lower respiratory tract. The invention also provides methods of
manufacturing such formulations and the use of such formulations in
treating patients.
[0418] One approach for the pulmonary delivery of dry powder drugs
utilizes a hand-held device with a hand pump for providing a source
of pressurized gas. The pressurized gas is abruptly released
through a powder dispersion device, such as a venturi nozzle, and
the dispersed powder made available for patient inhalation.
[0419] Dry powder dispersion devices are described in several
patents. U.S. Pat. No. 3,921,637 describes a manual pump with
needles for piercing through a single capsule of powdered medicine.
The use of multiple receptacle disks or strips of medication is
described in European Patent Application No. EP 0 467 172;
International Patent Publication Nos. WO 91/02558; and WO 93/09832;
U.S. Pat. Nos. 4,627,432; 4,811,731; 5,035,237; 5,048,514;
4,446,862; 5,048,514, and 4,446,862.
[0420] The aerosolization of protein therapeutic agents is
disclosed in European Patent Application No. EP 0 289 336.
Therapeutic aerosol formulations are disclosed in International
Patent Publication No. WO 90/09781.
[0421] The present invention provides formulating Tf fusion protein
for oral inhalation. The formulation comprises Tf fusion protein
and suitable pharmaceutical excipients for pulmonary delivery. The
present invention also provides administering the Tf fusion protein
composition via oral inhalation to subjects in need thereof.
[0422] Transgenic Animals
[0423] The production of transgenic non-human animals that contain
a modified transferrin fusion construct with increased serum
half-life increased serum stability or increased bioavailability of
the instant invention is contemplated in one embodiment of the
present invention. In some embodiments, lactoferrin may be used as
the Tf portion of the fusion protein so that the fusion protein is
produced and secreted in milk.
[0424] The successful production of transgenic, non-human animals
has been described in a number of patents and publications, such
as, for example U.S. Pat. No. 6,291,740 (issued Sep. 18, 2001);
U.S. Pat. No. 6,281,408 (issued Aug. 28, 2001); and U.S. Pat. No.
6,271,436 (issued Aug. 7, 2001) the contents of which are hereby
incorporated by reference in their entireties.
[0425] The ability to alter the genetic make-up of animals, such as
domesticated mammals including cows, pigs, goats, horses, cattle,
and sheep, allows a number of commercial applications. These
applications include the production of animals which express large
quantities of exogenous proteins in an easily harvested form (e.g.,
expression into the milk or blood), the production of animals with
increased weight gain, feed efficiency, carcass composition, milk
production or content, disease resistance and resistance to
infection by specific microorganisms and the production of animals
having enhanced growth rates or reproductive performance. Animals
which contain exogenous DNA sequences in their genome are referred
to as transgenic animals.
[0426] The most widely used method for the production of transgenic
animals is the microinjection of DNA into the pronuclei of
fertilized embryos (Wall et al., J. Cell. Biochem. 49:113 [1992]).
Other methods for the production of transgenic animals include the
infection of embryos with retroviruses or with retroviral vectors.
Infection of both pre- and post-implantation mouse embryos with
either wild-type or recombinant retroviruses has been reported
(Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]; Janenich et
al., Cell 24:519 [1981]; Stuhlmann et al., Proc. Natl. Acad. Sci.
USA 81:7151 [1984]; Jahner et al., Proc. Natl. Acad Sci. USA
82:6927 [1985]; Van der Putten et al., Proc. Natl. Acad Sci. USA
82:6148-6152 [1985]; Stewart et al., EMBO J. 6:383-388 [1987]).
[0427] An alternative means for infecting embryos with retroviruses
is the injection of virus or virus-producing cells into the
blastocoele of mouse embryos (Jahner, D. et al., Nature 298:623
[1982]). The introduction of transgenes into the germline of mice
has been reported using intrauterine retroviral infection of the
midgestation mouse embryo (Jahner et al., supra [1982]). Infection
of bovine and ovine embryos with retroviruses or retroviral vectors
to create transgenic animals has been reported. These protocols
involve the micro-injection of retroviral particles or growth
arrested (i.e., mitomycin C-treated) cells which shed retroviral
particles into the perivitelline space of fertilized eggs or early
embryos (PCT International Application WO 90/08832 [1990]; and
Haskell and Bowen, Mol. Reprod. Dev., 40:386 [1995]. PCT
International Application WO 90/08832 describes the injection of
wild-type feline leukemia virus B into the perivitelline space of
sheep embryos at the 2 to 8 cell stage. Fetuses derived from
injected embryos were shown to contain multiple sites of
integration.
[0428] U.S. Pat. No. 6,291,740 (issued Sep. 18, 2001) describes the
production of transgenic animals by the introduction of exogenous
DNA into pre-maturation oocytes and mature, unfertilized oocytes
(i.e., pre-fertilization oocytes) using retroviral vectors which
transduce dividing cells (e.g., vectors derived from murine
leukemia virus [MLV]). This patent also describes methods and
compositions for cytomegalovirus promoter-driven, as well as mouse
mammary tumor LTR expression of various recombinant proteins.
[0429] U.S. Pat. No. 6,281,408 (issued Aug. 28, 2001) describes
methods for producing transgenic animals using embryonic stem
cells. Briefly, the embryonic stem cells are used in a mixed cell
co-culture with a morula to generate transgenic animals. Foreign
genetic material is introduced into the embryonic stem cells prior
to co-culturing by, for example, electroporation, microinjection or
retroviral delivery. ES cells transfected in this manner are
selected for integrations of the gene via a selection marker such
as neomycin.
[0430] U.S. Pat. No. 6,271,436 (issued Aug. 7, 2001) describes the
production of transgenic animals using methods including isolation
of primordial germ cells, culturing these cells to produce
primordial germ cell-derived cell lines, transforming both the
primordial germ cells and the cultured cell lines, and using these
transformed cells and cell lines to generate transgenic animals.
The efficiency at which transgenic animals are generated is greatly
increased, thereby allowing the use of homologous recombination in
producing transgenic non-rodent animal species.
[0431] Gene Therapy
[0432] The use of modified transferrin fusion constructs for gene
therapy wherein a modified transferrin protein or transferrin
domain is joined to a therapeutic protein or peptide is
contemplated in one embodiment of this invention. The modified
transferrin fusion constructs with increased serum half-life or
serum stability of the instant invention are ideally suited to gene
therapy treatments.
[0433] The successful use of gene therapy to express a soluble
fusion protein has been described. Briefly, gene therapy via
injection of an adenovirus vector containing a gene encoding a
soluble fusion protein consisting of cytotoxic lymphocyte antigen 4
(CTLA4) and the Fc portion of human immunoglubulin G1 was recently
shown in Ijima et al. (Jun. 10, 2001) Human Gene Therapy (United
States) 12/9:1063-77. In this application of gene therapy, a murine
model of type II collagen-induced arthritis was successfully
treated via intraarticular injection of the vector.
[0434] Gene therapy is also described in a number of U.S. patents
including U.S. Pat. No. 6,225,290 (issued May 1, 2001); U.S. Pat.
No. 6,187,305 (issued Feb. 13, 2001); and U.S. Pat. No. 6,140,111
(issued Oct. 31, 2000).
[0435] U.S. Pat. No. 6,225,290 provides methods and constructs
whereby intestinal epithelial cells of a mammalian subject are
genetically altered to operatively incorporate a gene which
expresses a protein which has a desired therapeutic effect.
Intestinal cell transformation is accomplished by administration of
a formulation composed primarily of naked DNA, and the DNA may be
administered orally. Oral or other intragastrointestinal routes of
administration provide a simple method of administration, while the
use of naked nucleic acid avoids the complications associated with
use of viral vectors to accomplish gene therapy. The expressed
protein is secreted directly into the gastrointestinal tract and/or
blood stream to obtain therapeutic blood levels of the protein
thereby treating the patient in need of the protein. The
transformed intestinal epithelial cells provide short or long term
therapeutic cures for diseases associated with a deficiency in a
particular protein or which are amenable to treatment by
overexpression of a protein.
[0436] U.S. Pat. No. 6,187,305 provides methods of gene or DNA
targeting in cells of vertebrate, particularly mammalian, origin.
Briefly, DNA is introduced into primary or secondary cells of
vertebrate origin through homologous recombination or targeting of
the DNA, which is introduced into genomic DNA of the primary or
secondary cells at a preselected site.
[0437] U.S. Pat. No. 6,140,111 (issued Oct. 31, 2000) describes
retroviral gene therapy vectors. The disclosed retroviral vectors
include an insertion site for genes of interest and are capable of
expressing high levels of the protein derived from the genes of
interest in a wide variety of transfected cell types. Also
disclosed are retroviral vectors lacking a selectable marker, thus
rendering them suitable for human gene therapy in the treatment of
a variety of disease states without the co-expression of a marker
product, such as an antibiotic. These retroviral vectors are
especially suited for use in certain packaging cell lines. The
ability of retroviral vectors to insert into the genome of
mammalian cells has made them particularly promising candidates for
use in the genetic therapy of genetic diseases in humans and
animals. Genetic therapy typically involves (1) adding new genetic
material to patient cells in vivo, or (2) removing patient cells
from the body, adding new genetic material to the cells and
reintroducing them into the body, i.e., in vitro gene therapy.
Discussions of how to perform gene therapy in a variety of cells
using retroviral vectors can be found, for example, in U.S. Pat.
No. 4,868,116, issued Sep. 19, 1989, and U.S. Pat. No. 4,980,286,
issued Dec. 25, 1990 (epithelial cells), WO 89/07136 published Aug.
10, 1989 (hepatocyte cells), EP 378,576 published Jul. 25, 1990
(fibroblast cells), and WO 89/05345 published Jun. 15, 1989 and
WO/90/06997, published Jun. 28, 1990 (endothelial cells), the
disclosures of which are incorporated herein by reference.
[0438] Kits Containing Transferrin Fusion Proteins
[0439] In a further embodiment, the present invention provides kits
containing transferrin fusion proteins, which can be used, for
instance, for the therapeutic or non-therapeutic applications. The
kit comprises a container with a label. Suitable containers
include, for example, bottles, vials, and test tubes. The
containers may be formed from a variety of materials such as glass
or plastic. The container holds a composition which includes a
transferrin fusion protein that is effective for therapeutic or
non-therapeutic applications, such as described above. The active
agent in the composition is the therapeutic protein. The label on
the container indicates that the composition is used for a specific
therapy or non-therapeutic application, and may also indicate
directions for either in vivo or in vitro use, such as those
described above.
[0440] The kit of the invention will typically comprise the
container described above and one or more other containers
comprising materials desirable from a commercial and user
standpoint, including buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0441] Without further description, it is believed that a person of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the present
invention and practice the claimed methods. For example, a skilled
artisan would readily be able to determine the biological activity,
both in vitro and in vivo, for the fusion protein constructs of the
present invention as compared with the comparable activity of the
therapeutic moiety in its unfused state. Similarly, a person
skilled in the art could readily determine the serum half life and
serum stability of constructs according to the present invention.
The following working examples therefore, specifically point out
the 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
T-20/Transferrin Fusion Protein
[0442] T-20 is a HIV fusogenic inhibitor peptide with the following
amino acid sequence: YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID
NO: 17). The present invention provides fusion proteins comprising
T-20 peptide and modified transferrin (mTf) with increased
half-life and pharmaceutical compositions of such fusion proteins
for the treatment of diseases associated with the transmission of a
virus. The example described below may also be used to generate
transferrin fusion proteins with analogs of the T-20 peptides.
[0443] The transferrin fusion protein with anti-HIV activity of the
present invention was produced by fusing T-20 to modified
transferrin (mTf) using Saccharomyces cerevisiae. Accordingly, in
the first step, SEQ ID NO: 17 was back translated into DNA codon
optimized for Saccharomyces cerevisiae and used to produce fusion
constructs of T-20 at the N- or C-terminus of mTf.
5' Fusion
[0444] As an example, T-20 is fused to the 5' end of mTf using
overlapping primers with restriction site overhangs at each end,
such as XbaI and KpnI sites for ligation into an appropriate
vector. Alternatively, overlapping primers with other restriction
site overhangs could be generated or the primers could be annealed
to adapters with appropriate restriction site overhangs for
ligation into a specifically designed vector.
[0445] A vector is specifically designed with restriction sites
such as XbaI and KpnI sites to allow fusion of therapeutic
molecules into the N-terminus of mTf. The primers are annealed and
cloned into this vector using the XbaI and KpnI sites or other
appropriate restriction sites at the 5' end of the modified
transferrin (mTf) vector. The cassette encoding the T-20/mTf fusion
protein is then removed from the vector and cloned into a yeast
vector for protein expression
[0446] Specifically, the following overlapping primers with
restriction overhangs are designed for producing the 5' T-20
fusion: ##STR1##
[0447] The overlapping primers have the following sequences:
TABLE-US-00004 P0038: CTAGAGAAAAGGTACACTAGCTTAATACAC (SEQ ID NO:
18) P0039: TGCGATTCTTCAATTAAGGAGTGTATTAAGCTAGTGTACCTTTTCT (SEQ ID
NO: 19) P0040: TCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATG (SEQ ID NO:
20) P0041: TAATTCCAATAATTCTTGTTCATTCTTTTCTTGCTGGTTT (SEQ ID NO: 21)
P0042: AACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTG (SEQ ID
NO: 22) TAC P0043: AAACCAATTCCACAAACTTGCCCATTTATC (SEQ ID NO:
23)
[0448] The primers are annealed at 65.degree. C. and ligated into
the specifically designed vector pREX0004, cut with XbaI and KpnI
to create pREX0016. Following identification of a correct clone by
sequencing, the correct clone is bulked up and digested with
another pair of appropriate restriction enzymes such as PsiI and
AgeI to remove the cassette encoding the T-20/mTf fusion protein
out of pREX0016. The cassette is ligated into a yeast vector, such
as pSAC3, cut with PsiI and AgeI. pSAC3 containing the T-20/mTf
N-terminus fusion protein is then electroporated into yeast for
protein production.
3' Fusion
[0449] In the same manner, T-20 fusion is fused to the 3' end of
T-20 using overlapping primers with restriction site overhangs,
such as SalI and HindIII or other restriction enzymes, at each end.
Once annealed, this fragment is cloned into a specifically designed
vector, pREX0001, using restriction sites such as SalI and HindIII
sites. Following identification of the correct clone through
sequencing, the plasmid is cut with SalI and HindIII and the
fragment is sub-cloned into pREX0004 which is specifically designed
to with unique SalI and HindIII sites to allow fusion of
therapeutic protein to the C-terminus of mTf. The resulting plasmid
is pREX0017. pREX0017 is then cut with appropriate enzymes such as
PsiI and AgeI to remove the T-20/mTf cassette out of the vector.
The T-20/m-Tf cassette is sub-cloned into a yeast vector, such as
pSAC3, for expression in yeast.
[0450] Specifically, the following overlapping primers with
restriction overhangs are designed for producing the T-20 fusion to
the C-terminus of mTf: ##STR2##
[0451] The overlapping primers have the following sequences:
TABLE-US-00005 P0044: TCGACCTTACAGTAGCTTAATACAC (SEQ ID NO: 24)
P0045: TGCGATTCTTCAATTAAGGAGTGTATTAAGCTAGTGTAAGG (SEQ ID NO: 25)
P0040: TCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATG (SEQ ID NO: 26)
P0041: TAATTCCAATAATTCTTGTTCATTCTTTTCTTGCTGGTTT (SEQ ID NO: 27)
P0046: AACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTT (SEQ ID
NO: 28) AATA P0047: AGCTTATTAAAACCAATTCCACAAACTTGCCCATTTATC (SEQ ID
NO: 29)
[0452] The primers are annealed at 65.degree. C. and ligated into
the specifically designed vector cut with SalI and HindIII.
Following identification of a correct clone by sequencing, the
correct clone is bulked up and digested with another pair of
appropriate restriction enzymes such as PsiI and AgeI to remove the
cassette encoding the T-20/mTf C-terminus fusion protein out of the
vector. This cassette is ligated into a yeast vector, such as
pSAC3, cut with PsiI and AgeI. pSAC3 containing the T-20/mTf fusion
protein is then electroporated into yeast for protein
production.
C-Terminus Modifications
[0453] Modifications are made at the 3' end of mTf to determine if
they would effect an increase in fusion expression levels, T-20
fusion activity, and/or improve mobility of the peptide at the C
terminus of mTf.
RRP Deletion
[0454] In the first step, proline at the 3' end of mTf is removed.
In addition to the proline, the adjoining two arginine residues are
removed since they may present a potential Kex2p protease cleavage
site. The deleted sequence is shown in the alignment below of
unmodified 3' T-20 mTf fusion insert (pREX0017, SEQ ID NO: 31) and
3' T-20 mTf fusion insert with RRP deletion (pREX0032, SEQ ID NO:
30): TABLE-US-00006 ##STR3##
[0455] Specifically, the RRP sequence is deleted using primers
homologous to adjoining regions of the sequence. The primers used
for generating the deletion are: TABLE-US-00007 P0060: (SEQ ID NO:
32) TCATCACTCCTGGAAGCCTGCACTTTCTACACTAGCTTAATACACTCCTT P0061: (SEQ
ID NO: 33) AAGGAGTGTATTAAGCTAGTGTAGAAAGTGCAGGCTTCCAGGAGTGATGA
[0456] A mutagenic PCR reaction is performed using the external
primers and pREX0017 as the template to generate a product missing
the 9 bases coding for RRP. This product is then cut with
SphI/HindIII and cloned into SphI/HindIII cut pREX0017 to make
pREX0032. pREX0032 was then cut with AgeI/PsiI, sub-cloned into a
yeast vector such as pSAC3 and transformed into yeast for protein
expression.
C402-C674 Disulfide Removal
[0457] In the next step, the disulfide bond that stretches between
Cys402 and Cys674 in the mTF molecule, is removed. This is
accomplished by mutating both cysteine residues to glycine
residues. First, using mutagenic primers Cys402 is mutated to Gly.
The mutagenic primers are: TABLE-US-00008 P0064: (SEQ ID NO: 34)
TTGTCTACATAGCGGGCAAGGGTGGTCTGGTGCCTGTCTTG P0065: (SEQ ID NO: 35)
CAAGACAGGCACCAGACCACCCTTGCCCGCTATGTAGACAA
[0458] A PCR reaction is performed using the external primers and
pREX0017 as the template to generate the product with the desired
mutation. This product is then cut with HpaI/PstI and cloned back
into pREX0017 making a Gly402 intermediate, pREX0039. Next, Cys674
is mutated into Gly674 following the same procedure and using
pREX0004 as a template and the following primers: TABLE-US-00009
P0068: (SEQ ID NO: 36)
TCCACCTCATCACTCCTGGAAGCCGGTACTTTCCGTCGACCTTAA P0069: (SEQ ID NO:
37) CTTATTAAGGTCGACGGAAAGTACCGGCTTCCAGGAGTGATGAGGTGG
[0459] The resulting product is cut with SphI/HindIII and
sub-cloned into the new Gly402 intermediate, pREX0039, making
pREX0038. This plasmid does not contain the T-20 peptide making it
useful in the future for other mTf fusions. To insert the T-20
peptide into pREX0038, the SalI/AgeI fragment of pREX0017 is cloned
to form pREX0034. pREX0034 was then cut with AgeI/Psi I. The
cassette is sub-cloned into a yeast vector, such as pSAC3, and
transformed into yeast for protein expression. The mutations are
shown in the alignment below of unmodified 3' T-20 mTf fusion
plasmid insert (pREX0017) and 3' T-20 mTf fusion insert with
C402-C674 disulfide deletion: TABLE-US-00010 ##STR4##
Disulfide and RRP Removal Combination
[0460] In the third step, RRP deletion and the C402-C674 disulfide
deletion are combined. To begin, an intermediate plasmid, pREX0041,
is made in the manner as pREX0032 with the exception that the
Cys674 mutation was present in the primers. The following primers
are used: TABLE-US-00011 P0066: (SEQ ID NO: 42)
TCCACCTCATCACTCCTGGAAGCCGGCACTTTCTACACTAGCTTAATA P0067: (SEQ ID NO:
43) GTGTATTAAGCTAGTGTAGAAAGTACCGGCTTCCAGGAGTGATGAGGT
[0461] pREX0041 is then cut with SphI/HindIII and sub-cloned into
pREX0039 to create pREX0033. pREX0033 is then cut with AgeI/PsiI.
The cassette is sub-cloned into a yeast vector, such as pSAC3, and
then transformed into yeast for protein expression. The following
alignment of the mTf 3' T-20 fusion of pREX0033 with pREX0017 shows
the mutations of the resulting product. TABLE-US-00012 ##STR5##
Pharmacokinetics of mTf-T20 in Rabbits
[0462] T20 was fused to the C-terminus of mTf by the method
described above to generate the mTf-T-20 fusion protein. 50 ug of
the mTf-T20 fusion protein was injected intravenously in New
Zealand white rabbits and at indicated times (see FIG. 4) 3 ml of
blood was removed and plasma was separated and frozen. Plasma
samples were later analyzed in an ELISA assay which is specific for
human transferrin and does not cross react with endogenous rabbit
transferrin. The concentration of mTf-T20 measured at 2 hours after
injection is normally the highest and was considered as 100%. All
other concentrations where compared to that of the two-hour sample.
The elimination half-life was calculated to be 44.+-.2 hrs. FIG. 4
shows the pharmacokinetics of mTf-T20 in two rabbits.
[0463] mTf has half-life of about 14 days in man and 60 hrs in
rabbits. Therefore, a half-life of over 7 days for mTf-T20 in man
is anticipated which is significantly longer than the reported 2
hrs for T20.
Example 2
EMP1/Transferrin Fusion Protein
[0464] EMP1 (SEQ ID NO: 11) has been shown to mimic EPO activity by
causing dimerization of the EPO receptor. The peptide, which is
cyclic, has no homology to EPO. To become active, the peptide has
to act in concert with another peptide, i.e. as a dimer, such that
two copies of the receptor are brought in close enough proximity to
form an active complex. As with many peptides, the peptide dimer
suffers from short half life and would benefit from the longevity
that fusion to transferrin would give. The present invention
provides fusion protein with EPO mimetic activity. As an example,
the fusion protein of the present invention comprises EMP1 peptide
(SEQ ID NO: 11) and modified transferrin (mTf) with increased
half-life. The present invention also provides pharmaceutical
compositions of such fusion proteins for the treatment of diseases
associated with low or defective red blood cell production.
EMP1-mTF Fusions and Insertions
[0465] The initial fusions to mTf comprises fusions to the N-, C-,
and N- and C-termini of mTf. The individual fusions will bind the
receptor but not cause activation of the receptor. The dual fusion,
one of which must be a different codon composition than the other
to prevent recombination, will enable binding to the receptor and
cause activation.
[0466] Examination of the N-domain of human Tf (PDB identifier
1A8E) and the full Tf model AAAaoTfwo, generated using the ExPasy
Swiss Model Server with the rabbit model 1JNF as template, reveals
a number of potential sites for insertion of a peptide, either
directly or by replacement of a number of residues. These sites are
duplicated by their equivalent sites in the C domain.
TABLE-US-00013 N.sub.1 N.sub.2 Asp33 Ser105 Asn55 Glu141 Asn75
Asp166 Asp90 Gln184 Gly257 Asp197 Lys280 Lys217 His289 Thr231
Ser298 Cys241
[0467] Two of these loops are the preferred sites into which the
EMP1 peptide may be inserted: N.sub.1 His289 (286-292) and N.sub.2
Asp166 (162-170). These positions give the correct orientation
required for binding to the two halves of the EPO receptor. As the
insertion sites are on the N.sub.1 and N.sub.2 domains of the N
domain, they have the flexibility of the hinge between these two
sub domains, which allows them to work their way into the
receptor.
[0468] Due to the structural similarity between the N and C domain
the equivalent insertion sites on the C domain (C.sub.1 489-495,
C.sub.2 623-628) may also be used to make the molecule multivalent.
This is done using a variety of the potential insert sites
indicated above either on just the N or C domain or by a
combination of sites on both domains. TABLE-US-00014 ##STR6##
Steps for Producing the EMP1/mTf Fusion Protein
[0469] In this Example, two EMP1 peptides (SEQ ID NO: 11) are
engineered into the transferrin scaffold using the encoding nucleic
acids of the peptides and mTf. TABLE-US-00015 1 ggtggtactt
actcttgtca ttttggtcca ttgacttggg g g t y s c h f g p l t w
tttgtaagcc acaaggtggt v c k p q g g nucleic acid sequence: SEQ ID
NO: 45 amino acid sequence: SEQ ID NO: 11
[0470] A EMP1 peptide is engineered into mTf between His289 and
Gly290. The duplication inherent to the transferrin molecule, with
the two domains mirroring each other, makes it possible to engineer
a second EMP1 peptide into the duplicate region of the C domain,
between Glu625 and Thr626. TABLE-US-00016 ##STR7##
[0471] As an example, for each insertion two overlapping mutagenic
primers are synthesized (see below). Using a vector containing the
nucleic acid encoding mTf, such as pREX0010, as a template,
reactions are performed with each mutagenic primer and an external
primer from the 5' or 3' of the Tf cDNA. The products from these
two reactions were then mixed and a further reaction performed with
the external primers to join the two products together. The
His289-Gly290 insert PCR product is digested with XbaI and HpaI for
ligation into XbaI/HpaI digested pREX0010. The resulting vector is
then digested with HpaI and SalI for ligation of HpaI/SalI digested
Glu625-Thr626 insert PCR product. TABLE-US-00017 His289-Gly290
insert (SEQ ID NO: 46) ------------- 2031 agacaaatca[aaagaatttc
aactattcag ctctcctcat ggtggtactt actcttgtca ttttggtcca tctgtttagt
tttcttaaag ttgataagtc gagaggagta ccaccatgaa[tgagaacagt aaaaccaggt
>>.............EMOm..............>
>....................................Tf...............................-
.....> >.................................N
domain.................................> 2101 ttgacttggg
tttgtaagcc]acaaggtggt gggaaggacc tgctgtttaa ggactctgcc cacgggtttt
aactgaaccc aaacattcgg tgttccacca cccttcctgg acgacaaatt
cctgagacgg]gtgcccaaaa -----------.fwdarw.
>............EMOm.............>>
>....................................Tf...............................-
.....> >.................................N
domain.................................> Glu625-Thr626 insert
(SEQ ID NO: 47) ------------- 3081 cctatttgga agcaacgtaa
ctgactgctc[gggcaacttt tgtttgttcc ggtcggaagg tggtacttac ggataaacct
tcgttgcatt gactgacgag cccgttgaaa acaaacaagg ccagccttcc accatgaat[g
>>...EPOm...> >.................................C
domain.................................>
>....................................Tf...............................-
.....> KpnI ------- 3151 tcttgtcatt ttggtccatt gacttgggtt
tgtaagccac]aaggtggtac caaggacctt ctgttcagag agaacagtaa aaccaggtaa
ctgaacccaa acattcggtg ttccaccatg gttcctggaa gacaagtctc
-----------.fwdarw.
>......................EPOm.......................>>
>.................................C
domain.................................>
>....................................Tf...............................-
.....> 3221 atgacacagt atgtttggcc aaacttcatg acagaaacac
atatgaaaaa tacttaggag aagaatatgt tactgtgtca]tacaaaccgg tttgaagtac
tgtctttgtg tatacttttt atgaatcctc ttcttataca
>.................................C
domain.................................>
>....................................Tf...............................-
.....>
[0472] The cassette containing the EMP1/mTF fusion protein is cut
out of the vector with appropriate restriction enzymes and ligated
into a yeast vector, such as pSAC35. pSAC35 is transformed into
yeast for protein expression.
[0473] Alternative points for insertion of the EPO mimetic
peptide(s), or any other peptide(s) are the two glycosylation sites
on the C domain of Transferrin at N413 and N611. The advantage of
these sites is that once insertion is achieved, glycosylation is
prevented by through disruption of the N--X--S/T sequence.
Example 3
GLP-1/Transferrin Fusion Protein
[0474] GLP-1 is a peptide that regulates insulin secretion. It
possesses anti-diabetic activity in human subjects suffering
diabetes, especially type II diabetes. Like other peptides, GLP-1
has a short plasma half-life in humans. The present invention
provides fusion proteins with GLP-1 fused to mTf with increased
half-life and pharmaceutical compositions of such fusion proteins
for the treatment of diseases associated with abnormal glucose
levels that can be administered orally.
[0475] The present invention also provides fusion proteins
comprising an GLP-1 analog and mTF. In one embodiment of the
invention, the GLP-1 analog comprises an additional His residue at
the N-terminus. The His residue could be added to the N-terminus of
GLP-1 or inserted after the His residue at the N-terminus of GLP-1.
In another embodiment, the GLP-1 analog comprises an amino acid
substitution at position 2. For example, the Ala in GLP-1(7-36) or
GLP-1(7-37) peptide (SEQ ID NO: 6) is substituted with another
amino acid.
[0476] In this example, the steps for producing a GLP-1/mTf fusion
protein are described. The same steps may be used to generate
transferrin fusion proteins with analogs of the GLP-1 peptides.
[0477] To produce the GLP-1/mTf fusion protein, the amino acid
sequence of GLP-1(7-36) and GLP-1(7-37) may be used. TABLE-US-00018
haegtftsdvssylegqaakefiawlvkgr (SEQ ID NO: 48)
haegtftsdvssylegqaakefiawlvkgrg (SEQ ID NO: 64)
[0478] For example, the peptide sequence of GLP-1(7-36) may be back
translated into DNA and codon optimized for yeast: TABLE-US-00019
catgctgaaggtacttttacttctgatgtttcttcttatttggaaggtcaagctgctaaagaa h a
e g t f t s d v s s y l e g q a a k e tttattgcttggttggttaaaggtaga
(SEQ ID NO: 49) f i a w l v k g r (SEQ ID NO: 48)
[0479] The primers were specifically designed to form 5' XbaI and
3' KpnI sticky ends after annealing and to enable direct ligation
into XbaI/KpnI cut pREX0052, just 5' of the end of the leader
sequence and at the N-terminus of mTf. Alternatively, other sticky
ends may be engineered for ligations into other vectors.
TABLE-US-00020 XbaI -+----- 1 aggtctctag agaaaaggca tgctgaaggt
acttttactt ctgatgtttc ttcttatttg tccagagatc tcttttccgt acgacttcca
tgaaaatgaa gactacaaag aagaataaac >>......FL.......>> r
s l e k r (SEQ ID NO: 52)
>>..................GLP-1....................> (SEQ ID NO:
48) h a e g t f t s d v s s y l KpnI ------+ 61 gaaggtcaag
ctgctaaaga atttattgct tggttggtta aaggtagggt acctgata cttccagttc
gacgatttct taaataacga accaaccaat ttccatccca tggactat
>......................GLP-1......................>> e g q
a a k e f i a w l v k g r >>..mTf..>> v p d Top strand:
SEQ ID NO: 50 Bottom strand: SEQ ID NO: 51 Top strand primer: P0056
(nucleotides 7-112 of SEQ ID NO: 50) Bottom strand primer: P0057
(nucleotides 9-108 of SEQ ID NO: 51)
[0480] After annealing and ligation, the clones were sequenced to
confirm correct insertion. This vector was designated pREX0094. The
cassette was cut out of pREX0094 with NotI and sub-cloned into NotI
cut yeast vector, pSAC35, to make pREX0100.
[0481] This plasmid was then electroporated into the host
Saccharomyces yeast strains and transformants selected for leucine
prototrohy on minimal media plates. Expression was determined by
growth in liquid minimal media and analysis of supernatant by
SDS-PAGE, western blot, and ELISA.
Example 4
.beta.-IFN/Transferrin Protein
[0482] .beta.-IFN is effective in the treatment of various
diseases, such as, but not limited to, multiple sclerosis, brain
tumor, skin cancer, and hepatitis B and C. Like most cytokines,
.beta.-IFN has a short circulation half-life. The present invention
provides fusion proteins comprising .beta.-IFN fused to mTf with
increased half-life and efficacy in patients. This example
describes the steps in generating a .beta.-IFN/mTf fusion protein
that may be administered orally.
[0483] In this example, IFN.beta.-1 is fused to modified
transferrin at either the N- or C-termini. The IFN.beta.-1 clone
was obtained from the ATCC (No. 39517). Specifically designed
primers were used to confirm the DNA sequence of the IFN.beta.-1
clone. These primers were external to the IFN.beta.-1 DNA sequence
and designed to read in from the vector such that the full-length
sequence of the clone was obtained. The primers used were:
TABLE-US-00021 P0070 GCTATGACCAACAAGTGTCTC (SEQ ID NO: 53) P0071
CGCACCTGTGGCGCCGGTGATG (SEQ ID NO: 54)
N-Terminal Fusion
[0484] Once the DNA sequence was confirmed, primers were designed
for fusion of IFN.beta.-1 to mTf. The N-terminal fusion was a two
step process. A straight fusion using primers with XbaI and KpnI
sites would have destroyed the KpnI site and clipped the beginning
of mTf. A linker, primers P0082 (nucleotides 18-48 of SEQ ID NO:
55) and P0083 (nucleotides 17-39 SEQ ID NO: 56), was designed to
create an internal KpnI site at the 3' end of IFN.beta.-1, by a
single silent mutation of bp 486 from T to G (bold), and with a 5'
XbaI overhang and 3' GTAC which would anneal with a KpnI site. The
overhang destroyed the existing KpnI site in pREX0052. The linkers
were annealed and ligated into pREX0054 cut with XbaI/KpnI,
creating an intermediate vector with mTf untouched and a KpnI site
that could be used to fuse the IFN.beta.-1 gene at the N-terminus
of mTf. TABLE-US-00022 XbaI KpnI -+----- -----+
>>............P0082.............>> ctgcttactc
taggtctcta gagaaaacag ggtacctccg aaacgtacct gataaaactg gacgaatgag
atacagagat ctcttttgtc ccatggaggc tttgcatgga ctattttgac
>>........P0083........>>
>>.........MFa-1..........>> a y s r s l e k (SEQ ID
NO: 57) >>....IFN-B-1.....>> t g y l r n (SEQ ID NO:
58) >>.....mTf......> v p d k t (SEQ ID NO: 59) Top
Strand: SEQ ID NO: 55 Bottom Strand: SEQ ID NO: 56
[0485] A second set of primers, P0084 (SEQ ID NO: 60) and P0085
(SEQ ID NO: 61), were designed to tailor the ends of the
IFN.beta.-1 gene by mutagenic PCR for subsequent insertion into the
intermediate vector via the XbaI and KpnI sites. A XbaI/KpnI digest
of this tailored gene removed the last 5 amino acids of
IFN.beta.-1; however, these were already engineered into the
intermediate vector. The resulting construct, pREX0048, was created
by ligating the IFN.beta.-1 gene cut with XbaI/KpnI into the
XbaI/KpnI cut intermediate vector. TABLE-US-00023 ##STR8##
[0486] After the pREX0048 construct was created, it was sequenced
to confirm correct insertion. The expression cassette, as a NotI
fragment, was then sub-cloned into NotI cut yeast vector, pSAC35,
to make the pREX0050.
C-Terminal Fusion
[0487] Specifically designed primers, P0086 (SEQ ID NO: 62) and
P0087 (SEQ ID NO: 63), were used to PCR amplify the original clone
and, in addition, to tailor the ends of IFN.beta.-1 to have SalI
and HindIII sites at the 5' and 3' ends, respectively. The newly
tailored product was ligated into SalI/HindIII cut pREX0052 to
create pREX0049. TABLE-US-00024 ##STR9##
[0488] After the Prex0049 construct was created, it was sequenced
to confirm correct insertion. The expression cassette, as a
NotIfragment, is then sub-cloned into NotIcut yeast vector, such as
pSAC35, to make pREX0051.
[0489] In one embodiment of the invention, .beta.-IFN-1 (GenBank
Acc. No. NM.sub.--002176, SEQ ID NO: 65) is made more stable and
soluble by mutating Cys17 (in the mature protein) to Ser. The
mutation of Cys17 to Ser can be performed by routine mutagenic
reactions such as a mutagenic PCR reaction using specifically
designed primers and the nucleic acid encoding .beta.-IFN-1 as the
template.
[0490] Further, the .beta.-IFN-1 is modified to prevent
glycosylation by modifying the N-linked glycosylation site, NES
(residues 80 to 82 of SEQ ID NO: 65)/T. As an example, N could be
mutagenized to Q and S/T could be mutagenized to Ala or other amino
acid acids. Such mutagenesis could be performed with mutagenic PCR
reaction using specifically designed primers and the nucleic acid
encoding .beta.-IFN-1 as the template.
Example 5
Insulin-Transferrin Fusion Proteins
[0491] Insulin is a peptide hormone that is secreted by the islets
of Langerhans in the pancreas and that regulates the metabolism of
carbohydrates and fats, in particular the conversion of glucose to
glycogen. It is given to humans suffering from type I and type II
diabetes, as well as to diabetic animals. Currently, insulin must
be administered by subcutaneous injection and has a short plasma
half-life in humans. The present invention provides fusion proteins
of insulin fused to mTf that have increased half-life and
pharmaceutical compositions of such fusion proteins for the
treatment of diseases associated with abnormal glucose levels that
can be administered orally.
[0492] In this example, the steps for producing an insulin/mTf
fusion protein are described. Similar steps may be followed to
generate transferrin fusion proteins with analogs of insulin
peptides.
[0493] For expression in Saccharomyces constructs were initially
made in the base vector pREX0052, comprising an E. coli cloning
vector with a cassette for the expression of mTf in yeast, as
either inserts between the 5' XbaI/KpnI sites for the N-terminal
fusion, or 3' SalI/HindIII sites for the C-terminal fusion.
TABLE-US-00025 XbaI KpnI -+----- ------+ aggtctctag agaagagggt
acctgata (SEQ ID NO: 67) tccagagatc tcttctccca tggactat
>>......FL.......>> r s l e k r (SEQ ID NO: 69)
>>..mTf..>> v p d SalI HindIII -+----- --+----
actttccgtc gaccttaata agcttaattc (SEQ ID NO: 68) tgaaaggcag
ctggaattat tcgaattaag >>........mTf........>> t f r r p
- - (SEQ ID NO: 70) mADHlt >>.....>>
[0494] These constructs form an expression cassette with (5' to 3')
the yeast PRB1 promoter, leader sequence directing secretion into
the growth media, (N-terminal fusion), mTf sequence, (C-terminal
fusion), stop codons and the ADH1 terminator sequence. Once
constructed, the expression cassettes were recovered as NotI
fragments and inserted into NotI digested pSAC35, an E. coli/yeast
shuttle vector.
[0495] The insulin sequence used corresponds to that of Genbank
Accession No. NM.sub.--000207, SEQ ID NOS: 71 (DNA) and 72
(protein), as shown below. TABLE-US-00026 1 gctgcatcag aagaggccat
caagcacatc actgtccttc tgccatggcc ctgtggatgc cgacgtagtc ttctccggta
gttcgtgtag tgacaggaag acggtaccgg gacacctacg
>>.....INS......> >>...leader.....> m a l w m 61
gcctcctgcc cctgctggcg ctgctggccc tctggggacc tgacccagcc gcagcctttg
cggaggacgg ggacgaccgc gacgaccggg agacccctgg actgggtcgg cgtcggaaac
>..............................INS..............................>
>..........................leader..........................>>
r l l p l l a l l a l w g p d p a a a >>.> f 121
tgaaccaaca cctgtgcggc tcacacctgg tggaagctct ctacctagtg tgcggggaac
acttggttgt ggacacgccg agtgtggacc accttcgaga gatggatcac acgccccttg
>..............................INS..............................>
>...............................B...............................>
v n q h l c g s h l v e a l y l v c g e 181 gaggcttctt ctacacaccc
aagacccgcc gggaggcaga ggacctgcag gtggggcagg ctccgaagaa gatgtgtggg
ttctgggcgg ccctccgtct cctggacgtc caccccgtcc
>..............................INS..............................>
r r e a e d l q v g q >............B............>> r g f f
y t p k t 241 tggagctggg cgggggccct ggtgcaggca gcctgcagcc
cttggccctg gaggggtccc acctcgaccc gcccccggga ccacgtccgt cggacgtcgg
gaaccgggac ctccccaggg
>..............................INS..............................>
v e l g g g p g a g s l q p l a l e g s 301 tgcagaagcg tggcattgtg
gaacaatgct gtaccagcat ctgctccctc taccagctgg acgtcttcgc accgtaacac
cttgttacga catggtcgta gacgagggag atggtcgacc
>..............................INS..............................>
l q k r
>>........................A.........................> g i
v e q c c t s i c s l y q l 361 agaactactg caactagacg cagcccgcag
gcagcccccc acccgccgcc tcctgcaccg tcttgatgac gttgatctgc gtcgggcgtc
cgtcgggggg tgggcggcgg aggacgtggc >......INS......>>
>......A.....>> e n y c n 421 agagagatgg aataaagccc
ttgaaccagc tctctctacc ttatttcggg aacttggtcg
[0496] The cDNA for the above sequence can be generated in a number
of ways, e.g., by RT-PCR, from a cDNA pool, or by overlapping
synthetic oligonucleotides. To generate a clone from a cDNA pool,
two primers were synthesized and used as PCR primers.
TABLE-US-00027 (SEQ ID NO: 73) 5' primer
5'-tttgtgaaccaacacctgtgcggc-3' (SEQ ID NO: 74) 3' primer
3'-gacgagggagatggtcgacctcttgatgacgttg-5'
[0497] To make the N-terminal insert, a 5' mutagenic primer was
used to create a second PCR product using the first PCR product as
template. This primer inserted the last 5 amino acids of the leader
sequence and the XbaI site. The KpnI site could not be inserted by
this method, as an amino acid change would have resulted from the
creation of the KpnI site. Instead, the PCR product was digested
with XbaI/PvuII. A linker was then made of two overlapping oligos
with a PvuII 5' end and 3' overhang which would ligate to the KpnI
overhang on KpnI digested pREX0052. By annealing and ligating this
linker to the digested PCR fragment and ligating the resulting
product into XbaI/KpnI digested pREX0052 the plasmid pREX0052
N-insulin (SEQ ID NOS: 75 and 76, DNA and protein, respectively)
was generated. TABLE-US-00028 XbaI -+---- 1 gcttactcta ggtctctaga
taagaggttt gtgaaccaac acctgtgcgg ctcacacctg cgaatgagat ccagagatct
attctccaaa cacttggttg tggacacgcc gagtgtggac
>>...........FL............>> a y s r s l d k r
>>..............Ins................>
>>...............B.................> f v n q h l c g s h l
61 gtggaagctc tctacctagt gtgcggggaa cgaggcttct tctacacacc
caagacccgc caccttcgag agatggatca cacgcccctt gctccgaaga agatgtgtgg
gttctgggcg
>..............................Ins..............................>
r
>.............................B.............................>>
v e a l y l v c g e r g f f y t p k t 121 cgggaggcag aggacctgca
ggtggggcag gtggagctgg gcgggggccc tggtgcaggc gccctccgtc tcctggacgt
ccaccccgtc cacctcgacc cgcccccggg accacgtccg
>..............................Ins..............................>
r e a e d l q v g q v e l g g g p g a g 181 agcctgcagc ccttggccct
ggaggggtcc ctgcagaagc gtggcattgt ggaacaatgc tcggacgtcg ggaaccggga
cctccccagg gacgtcttcg caccgtaaca ccttgttacg
>..............................Ins..............................>
s l q p l a l e g s l q k r >>.......A........> g i v e q
c PvuII --+--- 241 tgtaccagca tctgctccct ctaccagctg gagaactact
gcaacgtac acatggtcgt agacgaggga gatggtcgac ctcttgatga cgttg
>......................Ins.....................>>
>.......................A......................>> c t s i
c s l y q l e n y c n >>mTf v
5' primer: 5'-gcttactctaggtctctagataagaggtttgtgaaccaacacctgtgcg-3'
(SEQ ID NO: 77) Linkers: 5'-ctggagaactactgcaacgtac-3' (SEQ ID NO:
78) 3'-gacctcttgatgacgttg-5' (SEQ ID NO: 79)
[0498] To make the C terminal insert, 5' and 3' mutagenic primers
were used to create a second PCR product using the first PCR
product as template. This product was then digested with
SalI/HindIII and ligated into SalI/HindIII digested pREX0052. This
resulted in the plasmid pREX0052 C-insulin (SEQ ID NOS: 80 and 81).
TABLE-US-00029 SalI -+---- 1 tgcactttcc gtcgaccttt tgtgaaccaa
cacctgtgcg gctcacacct ggtggaagct acgtgaaagg cagctggaaa acacttggtt
gtggacacgc cgagtgtgga ccaccttcga >>......mTf......>> c
t f r r p >>...................Ins.....................>
>>....................B......................> f v n q h l
c g s h l v e a 61 ctctacctag tgtgcgggga acgaggcttc ttctacacac
ccaagacccg ccgggaggca gagatggatc acacgcccct tgctccgaag aagatgtgtg
ggttctgggc ggccctccgt
>..............................Ins..............................>
r r e a
>........................B........................>> l y l
v c g e r g f f y t p k t 121 gaggacctgc aggtggggca ggtggagctg
ggcgggggcc ctggtgcagg cagcctgcag ctcctggacg tccaccccgt ccacctcgac
ccgcccccgg gaccacgtcc gtcggacgtc
>..............................Ins..............................>
e d l q v g q v e l g g g p g a g s l q 181 cccttggccc tggaggggtc
cctgcagaag cgtggcattg tggaacaatg ctgtaccagc gggaaccggg acctccccag
ggacgtcttc gcaccgtaac accttgttac gacatggtcg
>..............................Ins..............................>
p l a l e g s l q k r >>............A.............> g i v
e q c c t s HindIII -+---- 241 atctgctccc tctaccagct ggagaactac
tgcaactaat aagcttaatt tagacgaggg agatggtcga cctcttgatg acgttgatta
ttcgaattaa >.................Ins................>>
>..................A.................>> i c s l y q l e n
y c n mADHl >>....>> a - 5'
5'-tgcactttccgtcgaccttttgtgaaccaacacctgtgcg-3' (SEQ ID NO: 82)
primer: 3' 3'-gacctcttgatgacgttgattattcgaattaa-5' (SEQ ID NO: 83)
primer:
[0499] Once the DNA sequence for both the N- and C-terminal inserts
had been checked and confimed, the plasmids pREX0052 N-insulin and
pREX0052 C-insulin were digested with NotI and the expression
cassettes recovered. These were then ligated into NotI digested
pSAC35 to give pSAC35 N-insulin and pSAC35 C-insulin. These
plasmids were then electroporated into the host Saccharomyces yeast
strains and transformants selected for leucine prototrohy on
minimal media plates. Expression was determined by growth in liquid
minimal media and analysis of superanatant by SDS-PAGE, western
blot, ELISA and BIAcore.
[0500] These fusion constructs result in the production of
proinsulin attached to transferrin. Proteases in yeast may convert
the proinsulin to insulin as it is being made and secreted,
although the final expression product may contain only proinsulin.
In that case, the proinsulin can be converted to insulin
post-expression using an appropriate purified protease.
Oral Administration of Insulin/Modified Transferrin Fusion Protein
to Rats
[0501] To test the insulin activity of the insulin/mTf fusion
protein, diabetic rats are first prepared. Female Sprague-Dawley
rats are fasted for 24 hours and their blood glucose level
determined to establish a baseline. The rats are then injected
intraperitoneally with a solution of streptozotocin (STZ), 60
mg/ml, at a dosage of 60 mg/kg. I.p. injections of STZ are
continued for four more days, and rats with a fasting blood glucose
level above 300 mg/dl are selected as diabetic rats.
[0502] Solutions of the fusion protein and of insulin alone are
prepared in PBS or sodium bicarbonate to provide dosages of 7 to 80
units of insulin/kg when administered to rats. As a control, rats
are also treated with PBS alone. The solutions or PBS are
administered by oral gavage to rats following a 12 hour fast, and
blood samples are collected from the tail after 0, 30 and 60
minutes, and then at 2-hour intervals. Blood glucose levels at 0,
0.5, 1, 3, 5, 7, 9 and 11 hours after dosing are measured with a
blood glucose monitoring device designed for diabetics, and the
rats are fed again at 11 hours post-dose.
[0503] The activity of the insulin is determined by measuring the
decrease in blood glucose level over time, correcting the decrease
by any increases or decreases in the PBS-only samples. The insulin
activities of the fusion protein versus unfused insulin are
compared.
[0504] To examine the uptake of the fusion protein by transferrin
receptors in the intestinal mucosa, fusion protein and unfused
insulin as a control are administered to diabetes induced rats as
described above and transport measured using standard sandwich
ELISAs and serum samples. Alternatively, .sup.125I-labeled fusion
protein or .sup.125I-labeled unfused insulin is administered to
diabetes-induced rats at dosage of 80 U insulin/kg by oral gavage,
as described above. Blood samples are collected from the tail after
0, 30 and 60 minutes, then at 2-hour intervals, also as described
above, and serum samples are analyzed by HPLC, using, for example a
Sephacryl column and eluting samples with PBS. Standards containing
.sup.125I-labeled transferrin, .sup.125I-labeled insulin and
.sup.125I-labeled fusion protein are also run on the Sephacryl
column to determine their peak elution times and fraction numbers.
The radioactivity of each fraction is measured with a gamma
counter, and the protein content of each fraction is measured by
the absorbance at 280 nm. Serum samples from rats treated with the
fusion protein may not show the appearance of the fusion protein
immediately, as there may be a delay of a few hours.
Example 6
Preparation of Therapeutic mTf Fusion Proteins with Increased Iron
Affinity
[0505] Therapeutic mTf fusion protein with increased iron affinity
may be prepared. As an example for preparing modified transferrin
fusion proteins with increased iron binding ability, the procedure
in Example 5 above may be carried out with the following
modification. These fusion proteins may be used to facilitate
uptake and transfer of the fusion protein across the
gastrointestinal epithelium.
[0506] A cloning vector such as pREX0052, described above, which
contains the mTf sequence is cut with a restriction enzyme, or a
pair of restriction enzymes, to remove a portion of the mTf gene.
Using techniques standard in the art, this fragment is then
subjected to site-directed mutagenesis using primers that introduce
a mutation at a position corresponding to nucleotide 723 of SEQ ID
NO: 1, converting the codon AAG (Lys) to CAG (Gln) or GAG (Glu).
Similarly, primers are used that introduce mutations at positions
corresponding to nucleotides 726 and 728 of SEQ ID NO: 1,
converting the codon CAC (His) to CAG (Gln) or GAG (Glu). Primers
may also be used that introduce mutations at all three nucleotide
positions, resulting in the substitution of two adjacent amino
acids. These nucleotide positions correspond to amino acids 225 and
226 of the protein encoded with the leader sequence and to amino
acids 206 and 207 of the mature protein. The mutated fragment is
then amplified by RT-PCR and religated into the cloning vector.
This vector containing the mutation or mutations is used in a
subsequent step for introduction of a DNA molecule coding for the
therapeutic protein. As described in Example 5, above, the mTf
fusion protein sequence may be introduced into yeast expression
vectors and transformed into Saccharomyces or other yeasts for
protein production.
[0507] As discussed previously, other amino acids may also be
mutated to obtain therapeutic mTf proteins with increased iron
affinity.
Example 7
Soluble Toxin Receptor/Transferrin Protein
[0508] Clostridial neurotoxins are poisonous substance.
Synaptotagmin I is a broad acting receptor of Clostridium botulinum
neurotoxin serotypes. Amino acids 1-53 (SEQ ID NO: 4) of
synaptotagmin I is responsible for binding to various neurotoxin
serotypes. Like other peptides, a soluble toxin receptor, such as
amino acids 1-53, has a short half-life. The present invention
provides fusion protein comprising amino acids 1-53 fused mTf with
increased half-life as compared to the soluble toxin receptor
having SEQ ID NO: 1-53.
[0509] The present invention provides fusion proteins with
anti-toxin activity and increased half-life. Specifically, in this
example, a fusion protein comprising modified Tf and a peptide
consisting of amino acids 1-53 (SEQ ID NO: 4) of synaptotagmin I,
is produced by fusing one or more copies of the nucleotide sequence
encoding the peptide to the nucleotide sequence of Tf to produce a
fusion protein with a peptide fused to the N- or C-terminus of
Tf.
[0510] To insert the sequence encoding SEQ ID NO: 4, the vector
pREX0010 with the modified transferrin cDNA, is digested with the
restriction enzymes Xba I/KpnI for insertion at the 5' end and Sal
I/Hind III for insertion at the 3' end.
[0511] For the 5' insertion, two overlapping oligos that form an
Xba I overhang at the 5' end and a Kpn I overhang at the 3' end of
the nucleic acid encoding SEQ ID NO: 4 are synthesized. These
oligos are then annealed together and ligated into the Xba I/Kpn I
digested pREX0010 vector.
[0512] Transformation, selection, and expression are then performed
in yeast.
[0513] Although the present invention has been described in detail
with reference to examples above, it is understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims. All cited patents, patent applications and
publications referred to in this application are herein
incorporated by reference in their entirety.
Sequence CWU 1
1
90 1 2318 DNA Homo sapiens CDS (51)..(2147) GenBank Acc. No.
NM_001063, transferrin gene and protein sig_peptide (51)..(107) 1
gcacagaagc gagtccgact gtgctcgctg ctcagcgccg cacccggaag atg agg 56
Met Arg 1 ctc gcc gtg gga gcc ctg ctg gtc tgc gcc gtc ctg ggg ctg
tgt ctg 104 Leu Ala Val Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu
Cys Leu 5 10 15 gct gtc cct gat aaa act gtg aga tgg tgt gca gtg tcg
gag cat gag 152 Ala Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser
Glu His Glu 20 25 30 gcc act aag tgc cag agt ttc cgc gac cat atg
aaa agc gtc att cca 200 Ala Thr Lys Cys Gln Ser Phe Arg Asp His Met
Lys Ser Val Ile Pro 35 40 45 50 tcc gat ggt ccc agt gtt gct tgt gtg
aag aaa gcc tcc tac ctt gat 248 Ser Asp Gly Pro Ser Val Ala Cys Val
Lys Lys Ala Ser Tyr Leu Asp 55 60 65 tgc atc agg gcc att gcg gca
aac gaa gcg gat gct gtg aca ctg gat 296 Cys Ile Arg Ala Ile Ala Ala
Asn Glu Ala Asp Ala Val Thr Leu Asp 70 75 80 gca ggt ttg gtg tat
gat gct tac ctg gct ccc aat aac ctg aag cct 344 Ala Gly Leu Val Tyr
Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro 85 90 95 gtg gtg gca
gag ttc tat ggg tca aaa gag gat cca cag act ttc tat 392 Val Val Ala
Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr 100 105 110 tat
gct gtt gct gtg gtg aag aag gat agt ggc ttc cag atg aac cag 440 Tyr
Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln 115 120
125 130 ctt cga ggc aag aag tcc tgc cac acg ggt cta ggc agg tcc gct
ggg 488 Leu Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala
Gly 135 140 145 tgg aac atc ccc ata ggc tta ctt tac tgt gac tta cct
gag cca cgt 536 Trp Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro
Glu Pro Arg 150 155 160 aaa cct ctt gag aaa gca gtg gcc aat ttc ttc
tcg ggc agc tgt gcc 584 Lys Pro Leu Glu Lys Ala Val Ala Asn Phe Phe
Ser Gly Ser Cys Ala 165 170 175 cct tgt gcg gat ggg acg gac ttc ccc
cag ctg tgt caa ctg tgt cca 632 Pro Cys Ala Asp Gly Thr Asp Phe Pro
Gln Leu Cys Gln Leu Cys Pro 180 185 190 ggg tgt ggc tgc tcc acc ctt
aac caa tac ttc ggc tac tcg gga gcc 680 Gly Cys Gly Cys Ser Thr Leu
Asn Gln Tyr Phe Gly Tyr Ser Gly Ala 195 200 205 210 ttc aag tgt ctg
aag gat ggt gct ggg gat gtg gcc ttt gtc aag cac 728 Phe Lys Cys Leu
Lys Asp Gly Ala Gly Asp Val Ala Phe Val Lys His 215 220 225 tcg act
ata ttt gag aac ttg gca aac aag gct gac agg gac cag tat 776 Ser Thr
Ile Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr 230 235 240
gag ctg ctt tgc ctg gac aac acc cgg aag ccg gta gat gaa tac aag 824
Glu Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys 245
250 255 gac tgc cac ttg gcc cag gtc cct tct cat acc gtc gtg gcc cga
agt 872 Asp Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala Arg
Ser 260 265 270 atg ggc ggc aag gag gac ttg atc tgg gag ctt ctc aac
cag gcc cag 920 Met Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn
Gln Ala Gln 275 280 285 290 gaa cat ttt ggc aaa gac aaa tca aaa gaa
ttc caa cta ttc agc tct 968 Glu His Phe Gly Lys Asp Lys Ser Lys Glu
Phe Gln Leu Phe Ser Ser 295 300 305 cct cat ggg aag gac ctg ctg ttt
aag gac tct gcc cac ggg ttt tta 1016 Pro His Gly Lys Asp Leu Leu
Phe Lys Asp Ser Ala His Gly Phe Leu 310 315 320 aaa gtc ccc ccc agg
atg gat gcc aag atg tac ctg ggc tat gag tat 1064 Lys Val Pro Pro
Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr Glu Tyr 325 330 335 gtc act
gcc atc cgg aat cta cgg gaa ggc aca tgc cca gaa gcc cca 1112 Val
Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro 340 345
350 aca gat gaa tgc aag cct gtg aag tgg tgt gcg ctg agc cac cac gag
1160 Thr Asp Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His His
Glu 355 360 365 370 agg ctc aag tgt gat gag tgg agt gtt aac agt gta
ggg aaa ata gag 1208 Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser
Val Gly Lys Ile Glu 375 380 385 tgt gta tca gca gag acc acc gaa gac
tgc atc gcc aag atc atg aat 1256 Cys Val Ser Ala Glu Thr Thr Glu
Asp Cys Ile Ala Lys Ile Met Asn 390 395 400 gga gaa gct gat gcc atg
agc ttg gat gga ggg ttt gtc tac ata gcg 1304 Gly Glu Ala Asp Ala
Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala 405 410 415 ggc aag tgt
ggt ctg gtg cct gtc ttg gca gaa aac tac aat aag agc 1352 Gly Lys
Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Asn Lys Ser 420 425 430
gat aat tgt gag gat aca cca gag gca ggg tat ttt gct gta gca gtg
1400 Asp Asn Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala
Val 435 440 445 450 gtg aag aaa tca gct tct gac ctc acc tgg gac aat
ctg aaa ggc aag 1448 Val Lys Lys Ser Ala Ser Asp Leu Thr Trp Asp
Asn Leu Lys Gly Lys 455 460 465 aag tcc tgc cat acg gca gtt ggc aga
acc gct ggc tgg aac atc ccc 1496 Lys Ser Cys His Thr Ala Val Gly
Arg Thr Ala Gly Trp Asn Ile Pro 470 475 480 atg ggc ctg ctc tac aat
aag atc aac cac tgc aga ttt gat gaa ttt 1544 Met Gly Leu Leu Tyr
Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe 485 490 495 ttc agt gaa
ggt tgt gcc cct ggg tct aag aaa gac tcc agt ctc tgt 1592 Phe Ser
Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys 500 505 510
aag ctg tgt atg ggc tca ggc cta aac ctg tgt gaa ccc aac aac aaa
1640 Lys Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn
Lys 515 520 525 530 gag gga tac tac ggc tac aca ggc gct ttc agg tgt
ctg gtt gag aag 1688 Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg
Cys Leu Val Glu Lys 535 540 545 gga gat gtg gcc ttt gtg aaa cac cag
act gtc cca cag aac act ggg 1736 Gly Asp Val Ala Phe Val Lys His
Gln Thr Val Pro Gln Asn Thr Gly 550 555 560 gga aaa aac cct gat cca
tgg gct aag aat ctg aat gaa aaa gac tat 1784 Gly Lys Asn Pro Asp
Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr 565 570 575 gag ttg ctg
tgc ctt gat ggt acc agg aaa cct gtg gag gag tat gcg 1832 Glu Leu
Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala 580 585 590
aac tgc cac ctg gcc aga gcc ccg aat cac gct gtg gtc aca cgg aaa
1880 Asn Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr Arg
Lys 595 600 605 610 gat aag gaa gct tgc gtc cac aag ata tta cgt caa
cag cag cac cta 1928 Asp Lys Glu Ala Cys Val His Lys Ile Leu Arg
Gln Gln Gln His Leu 615 620 625 ttt gga agc aac gta act gac tgc tcg
ggc aac ttt tgt ttg ttc cgg 1976 Phe Gly Ser Asn Val Thr Asp Cys
Ser Gly Asn Phe Cys Leu Phe Arg 630 635 640 tcg gaa acc aag gac ctt
ctg ttc aga gat gac aca gta tgt ttg gcc 2024 Ser Glu Thr Lys Asp
Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala 645 650 655 aaa ctt cat
gac aga aac aca tat gaa aaa tac tta gga gaa gaa tat 2072 Lys Leu
His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr 660 665 670
gtc aag gct gtt ggt aac ctg aga aaa tgc tcc acc tca tca ctc ctg
2120 Val Lys Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu
Leu 675 680 685 690 gaa gcc tgc act ttc cgt aga cct taa aatctcagag
gtagggctgc 2167 Glu Ala Cys Thr Phe Arg Arg Pro 695 caccaaggtg
aagatgggaa cgcagatgat ccatgagttt gccctggttt cactggccca 2227
agtggtttgt gctaaccacg tctgtcttca cagctctgtg ttgccatgtg tgctgaacaa
2287 aaaataaaaa ttattattga ttttatattt c 2318 2 698 PRT Homo sapiens
2 Met Arg Leu Ala Val Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu 1
5 10 15 Cys Leu Ala Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser
Glu 20 25 30 His Glu Ala Thr Lys Cys Gln Ser Phe Arg Asp His Met
Lys Ser Val 35 40 45 Ile Pro Ser Asp Gly Pro Ser Val Ala Cys Val
Lys Lys Ala Ser Tyr 50 55 60 Leu Asp Cys Ile Arg Ala Ile Ala Ala
Asn Glu Ala Asp Ala Val Thr 65 70 75 80 Leu Asp Ala Gly Leu Val Tyr
Asp Ala Tyr Leu Ala Pro Asn Asn Leu 85 90 95 Lys Pro Val Val Ala
Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr 100 105 110 Phe Tyr Tyr
Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met 115 120 125 Asn
Gln Leu Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser 130 135
140 Ala Gly Trp Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu
145 150 155 160 Pro Arg Lys Pro Leu Glu Lys Ala Val Ala Asn Phe Phe
Ser Gly Ser 165 170 175 Cys Ala Pro Cys Ala Asp Gly Thr Asp Phe Pro
Gln Leu Cys Gln Leu 180 185 190 Cys Pro Gly Cys Gly Cys Ser Thr Leu
Asn Gln Tyr Phe Gly Tyr Ser 195 200 205 Gly Ala Phe Lys Cys Leu Lys
Asp Gly Ala Gly Asp Val Ala Phe Val 210 215 220 Lys His Ser Thr Ile
Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp 225 230 235 240 Gln Tyr
Glu Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu 245 250 255
Tyr Lys Asp Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala 260
265 270 Arg Ser Met Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn
Gln 275 280 285 Ala Gln Glu His Phe Gly Lys Asp Lys Ser Lys Glu Phe
Gln Leu Phe 290 295 300 Ser Ser Pro His Gly Lys Asp Leu Leu Phe Lys
Asp Ser Ala His Gly 305 310 315 320 Phe Leu Lys Val Pro Pro Arg Met
Asp Ala Lys Met Tyr Leu Gly Tyr 325 330 335 Glu Tyr Val Thr Ala Ile
Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu 340 345 350 Ala Pro Thr Asp
Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His 355 360 365 His Glu
Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys 370 375 380
Ile Glu Cys Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile 385
390 395 400 Met Asn Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe
Val Tyr 405 410 415 Ile Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala
Glu Asn Tyr Asn 420 425 430 Lys Ser Asp Asn Cys Glu Asp Thr Pro Glu
Ala Gly Tyr Phe Ala Val 435 440 445 Ala Val Val Lys Lys Ser Ala Ser
Asp Leu Thr Trp Asp Asn Leu Lys 450 455 460 Gly Lys Lys Ser Cys His
Thr Ala Val Gly Arg Thr Ala Gly Trp Asn 465 470 475 480 Ile Pro Met
Gly Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp 485 490 495 Glu
Phe Phe Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser 500 505
510 Leu Cys Lys Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn
515 520 525 Asn Lys Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys
Leu Val 530 535 540 Glu Lys Gly Asp Val Ala Phe Val Lys His Gln Thr
Val Pro Gln Asn 545 550 555 560 Thr Gly Gly Lys Asn Pro Asp Pro Trp
Ala Lys Asn Leu Asn Glu Lys 565 570 575 Asp Tyr Glu Leu Leu Cys Leu
Asp Gly Thr Arg Lys Pro Val Glu Glu 580 585 590 Tyr Ala Asn Cys His
Leu Ala Arg Ala Pro Asn His Ala Val Val Thr 595 600 605 Arg Lys Asp
Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln 610 615 620 His
Leu Phe Gly Ser Asn Val Thr Asp Cys Ser Gly Asn Phe Cys Leu 625 630
635 640 Phe Arg Ser Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val
Cys 645 650 655 Leu Ala Lys Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr
Leu Gly Glu 660 665 670 Glu Tyr Val Lys Ala Val Gly Asn Leu Arg Lys
Cys Ser Thr Ser Ser 675 680 685 Leu Leu Glu Ala Cys Thr Phe Arg Arg
Pro 690 695 3 679 PRT Homo sapiens MISC_FEATURE Mature Transferrin
Protein 3 Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His
Glu Ala 1 5 10 15 Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser
Val Ile Pro Ser 20 25 30 Asp Gly Pro Ser Val Ala Cys Val Lys Lys
Ala Ser Tyr Leu Asp Cys 35 40 45 Ile Arg Ala Ile Ala Ala Asn Glu
Ala Asp Ala Val Thr Leu Asp Ala 50 55 60 Gly Leu Val Tyr Asp Ala
Tyr Leu Ala Pro Asn Asn Leu Lys Pro Val 65 70 75 80 Val Ala Glu Phe
Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr Tyr 85 90 95 Ala Val
Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln Leu 100 105 110
Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp 115
120 125 Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg
Lys 130 135 140 Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser
Cys Ala Pro 145 150 155 160 Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu
Cys Gln Leu Cys Pro Gly 165 170 175 Cys Gly Cys Ser Thr Leu Asn Gln
Tyr Phe Gly Tyr Ser Gly Ala Phe 180 185 190 Lys Cys Leu Lys Asp Gly
Ala Gly Asp Val Ala Phe Val Lys His Ser 195 200 205 Thr Ile Phe Glu
Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr Glu 210 215 220 Leu Leu
Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys Asp 225 230 235
240 Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala Arg Ser Met
245 250 255 Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln Ala
Gln Glu 260 265 270 His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu
Phe Ser Ser Pro 275 280 285 His Gly Lys Asp Leu Leu Phe Lys Asp Ser
Ala His Gly Phe Leu Lys 290 295 300 Val Pro Pro Arg Met Asp Ala Lys
Met Tyr Leu Gly Tyr Glu Tyr Val 305 310 315 320 Thr Ala Ile Arg Asn
Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro Thr 325 330 335 Asp Glu Cys
Lys Pro Val Lys Trp Cys Ala Leu Ser His His Glu Arg 340 345 350 Leu
Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys Ile Glu Cys 355 360
365 Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn Gly
370 375 380 Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile
Ala Gly 385 390 395 400 Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn
Tyr Asn Lys Ser Asp 405 410 415 Asn Cys Glu Asp Thr Pro Glu Ala Gly
Tyr Phe Ala Val Ala Val Val 420 425 430 Lys Lys Ser Ala Ser Asp Leu
Thr Trp Asp Asn Leu Lys Gly Lys Lys 435 440 445 Ser Cys His Thr Ala
Val Gly Arg Thr Ala Gly Trp Asn Ile Pro Met 450 455 460 Gly Leu Leu
Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe Phe 465 470 475 480
Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys Lys 485
490 495 Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn Lys
Glu 500 505 510 Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val
Glu Lys Gly 515 520 525 Asp Val Ala Phe Val Lys His Gln Thr Val Pro
Gln Asn Thr Gly Gly 530 535 540 Lys Asn
Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr Glu 545 550 555
560 Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala Asn
565 570 575 Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr Arg
Lys Asp 580 585 590 Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln
Gln His Leu Phe 595 600 605 Gly Ser Asn Val Thr Asp Cys Ser Gly Asn
Phe Cys Leu Phe Arg Ser 610 615 620 Glu Thr Lys Asp Leu Leu Phe Arg
Asp Asp Thr Val Cys Leu Ala Lys 625 630 635 640 Leu His Asp Arg Asn
Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr Val 645 650 655 Lys Ala Val
Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu Leu Glu 660 665 670 Ala
Cys Thr Phe Arg Arg Pro 675 4 53 PRT Homo sapiens misc_feature
Amino acids 1-53 of synaptotagmin I 4 Met Val Ser Glu Ser His His
Glu Ala Leu Ala Ala Pro Pro Val Thr 1 5 10 15 Thr Val Ala Thr Val
Leu Pro Ser Asn Ala Thr Glu Pro Ala Ser Pro 20 25 30 Gly Glu Gly
Lys Glu Asp Ala Phe Ser Lys Leu Lys Glu Lys Phe Met 35 40 45 Asn
Glu Leu His Lys 50 5 12 PRT Artificial sequence Neutrophil
lactoferrin splice variant 5 Glu Asp Cys Ile Ala Leu Lys Gly Glu
Ala Asp Ala 1 5 10 6 31 PRT Homo sapiens MISC_FEATURE Glucagon-Like
Peptide misc_feature (31)..(31) Xaa can be any naturally occurring
amino acid MISC_FEATURE (31)..(31) Xaa can be Gly in GLP-1(7-37) or
absent in GLP-1(7-36) 6 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Arg Xaa 20 25 30 7 28 PRT Artificial sequence GLP-1
molecule having insulinotropic activity 7 His Ala Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys 20 25 8 29 PRT Artificial sequence
GLP-1 molecule having insulinotropic activity 8 His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly 20 25 9 29 PRT Artificial
sequence GLP-1 analog MISC_FEATURE (1)..(1) Xaa can be Ala, Gly,
Val, Thr, Ile, or alpha-methyl-Ala MISC_FEATURE (14)..(14) Xaa can
be Glu, Gln, Ala, Thr, Ser, or Gly MISC_FEATURE (20)..(20) Xaa can
be Glu, Gln, Ala, Thr, Ser or Gly 9 Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Xaa Gly Gln 1 5 10 15 Ala Ala Lys Xaa Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 10 10 PRT Artificial sequence EPO
domain 10 Cys Arg Ile Gly Pro Ile Thr Trp Val Cys 1 5 10 11 20 PRT
Artificial sequence EMP1 peptide 11 Gly Gly Thr Tyr Ser Cys His Phe
Gly Pro Leu Thr Trp Val Cys Lys 1 5 10 15 Pro Gln Gly Gly 20 12 13
PRT Artificial sequence EMP20 peptide 12 Tyr Ser Cys His Phe Gly
Pro Leu Thr Trp Val Cys Lys 1 5 10 13 47 PRT Homo sapiens
misc_feature N1 subdomain of transferrin 13 Asp Lys Ser Lys Glu Phe
Gln Leu Phe Ser Ser Pro His Gly Lys Asp 1 5 10 15 Leu Leu Phe Lys
Asp Ser Ala His Gly Phe Leu Lys Val Pro Pro Arg 20 25 30 Met Asp
Ala Lys Met Tyr Leu Gly Tyr Glu Tyr Val Thr Ala Ile 35 40 45 14 45
PRT Homo sapiens misc_feature N2 subdomain of Transferrin 14 Pro
Glu Pro Arg Lys Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser 1 5 10
15 Gly Ser Cys Ala Pro Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys
20 25 30 Gln Leu Cys Pro Gly Cys Gly Cys Ser Thr Leu Asn Gln 35 40
45 15 42 PRT Homo sapiens misc_feature C1 subdomain of transferrin
15 Asn His Cys Arg Phe Asp Glu Phe Phe Ser Glu Gly Cys Ala Pro Gly
1 5 10 15 Ser Lys Lys Asp Ser Ser Leu Cys Lys Leu Cys Met Gly Ser
Gly Leu 20 25 30 Asn Leu Cys Glu Pro Asn Asn Lys Glu Gly 35 40 16
49 PRT Homo sapiens misc_feature C2 subdomain of transferrin 16 Asn
Val Thr Asp Cys Ser Gly Asn Phe Cys Leu Phe Arg Ser Glu Thr 1 5 10
15 Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala Lys Leu His
20 25 30 Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr Val
Lys Ala 35 40 45 Val 17 36 PRT Human immunodeficiency virus HIV
T-20 antifusogenic peptide 17 Tyr Thr Ser Leu Ile His Ser Leu Ile
Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu
Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 18
30 DNA Artificial sequence Primer P0038 18 ctagagaaaa ggtacactag
cttaatacac 30 19 46 DNA Artificial sequence Primer P0039 19
tgcgattctt caattaagga gtgtattaag ctagtgtacc ttttct 46 20 40 DNA
Artificial sequence Primer P0040 20 tccttaattg aagaatcgca
aaaccagcaa gaaaagaatg 40 21 40 DNA Artificial sequence Primer P0041
21 taattccaat aattcttgtt cattcttttc ttgctggttt 40 22 54 DNA
Artificial sequence Primer P0042 22 aacaagaatt attggaatta
gataaatggg caagtttgtg gaattggttt gtac 54 23 30 DNA Artificial
sequence Primer P0043 23 aaaccaattc cacaaacttg cccatttatc 30 24 25
DNA Artificial sequence Primer P0044 24 tcgaccttac actagcttaa tacac
25 25 41 DNA Artificial sequence Primer P0045 25 tgcgattctt
caattaagga gtgtattaag ctagtgtaag g 41 26 40 DNA Artificial sequence
Primer P0040 26 tccttaattg aagaatcgca aaaccagcaa gaaaagaatg 40 27
40 DNA Artificial sequence Primer P0041 27 taattccaat aattcttgtt
cattcttttc ttgctggttt 40 28 55 DNA Artificial sequence Primer P0046
28 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt taata 55
29 39 DNA Artificial sequence Primer P0047 29 agcttattaa aaccaattcc
acaaacttgc ccatttatc 39 30 175 DNA Artificial sequence pREX0032
insert 30 caaggctgtt ggtaacctga gaaaatgctc cacctcatca ctcctggaag
cctgcacttt 60 ctacactagc ttaatacact ccttaattga agaatcgcaa
aaccagcaag aaaagaatga 120 acaagaatta ttggaattag ataaatgggc
aagtttgtgg aattggtttt aataa 175 31 184 DNA Artificial sequence
pREX0017 insert 31 caaggctgtt ggtaacctga gaaaatgctc cacctcatca
ctcctggaag cctgcacttt 60 ccgtcgacct tacactagct taatacactc
cttaattgaa gaatcgcaaa accagcaaga 120 aaagaatgaa caagaattat
tggaattaga taaatgggca agtttgtgga attggtttta 180 ataa 184 32 50 DNA
Artificial sequence Primer P0060 32 tcatcactcc tggaagcctg
cactttctac actagcttaa tacactcctt 50 33 50 DNA Artificial sequence
Primer P0061 33 aaggagtgta ttaagctagt gtagaaagtg caggcttcca
ggagtgatga 50 34 41 DNA Artificial sequence Primer P0064 34
ttgtctacat agcgggcaag ggtggtctgg tgcctgtctt g 41 35 41 DNA
Artificial sequence Primer P0065 35 caagacaggc accagaccac
ccttgcccgc tatgtagaca a 41 36 45 DNA Artificial sequence Primer
P0068 36 tccacctcat cactcctgga agccggtact ttccgtcgac cttaa 45 37 48
DNA Artificial sequence Primer P0069 37 cttattaagg tcgacggaaa
gtaccggctt ccaggagtga tgaggtgg 48 38 50 DNA Artificial sequence
pREX0017 plasmid starting at 1501 38 tagcgggcaa gtgtggtctg
gtgcctgtct tggcagaaaa ctacaataag 50 39 50 DNA Artificial sequence
pREX0034 plasmid starting at 1501 39 tagcgggcaa gggtggtctg
gtgcctgtct tggcagaaaa ctacaataag 50 40 50 DNA Artificial sequence
pREX0017 plasmid starting at 2301 40 tgctccacct catcactcct
ggaagcctgc actttccgtc gaccttacac 50 41 50 DNA Artificial sequence
pREX0034 plasmid starting at 2301 41 tgctccacct catcactcct
ggaagccggt actttccgtc gaccttacac 50 42 48 DNA Artificial sequence
Primer P0066 42 tccacctcat cactcctgga agccggcact ttctacacta
gcttaata 48 43 48 DNA Artificial sequence Primer P0067 43
gtgtattaag ctagtgtaga aagtaccggc ttccaggagt gatgaggt 48 44 41 DNA
Artificial sequence pREX0033 plasmid starting at 2301 44 tgctccacct
catcactcct ggaagccggt actttctaca c 41 45 60 DNA Artificial sequence
synthetic oligonucleotide encoding peptide with EPO activity 45
ggtggtactt actcttgtca ttttggtcca ttgacttggg tttgtaagcc acaaggtggt
60 46 140 DNA Artificial sequence His289-Gly290 insert PCR product
46 agacaaatca aaagaatttc aactattcag ctctcctcat ggtggtactt
actcttgtca 60 ttttggtcca ttgacttggg tttgtaagcc acaaggtggt
gggaaggacc tgctgtttaa 120 ggactctgcc cacgggtttt 140 47 210 DNA
Artificial sequence Glu625-Thr626 insert PCR product 47 cctatttgga
agcaacgtaa ctgactgctc gggcaacttt tgtttgttcc ggtcggaagg 60
tggtacttac tcttgtcatt ttggtccatt gacttgggtt tgtaagccac aaggtggtac
120 caaggacctt ctgttcagag atgacacagt atgtttggcc aaacttcatg
acagaaacac 180 atatgaaaaa tacttaggag aagaatatgt 210 48 30 PRT
Artificial sequence glucagon-like peptide-1 48 His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 49 90 DNA
Artificial sequence sequence encoding glucagon-like peptide-1 49
catgctgaag gtacttttac ttctgatgtt tcttcttatt tggaaggtca agctgctaaa
60 gaatttattg cttggttggt taaaggtaga 90 50 118 DNA Artificial
sequence Top Strand Synthetic Oligonucleotide misc_feature
(7)..(112) Top Strand Primer P0056 50 aggtctctag agaaaaggca
tgctgaaggt acttttactt ctgatgtttc ttcttatttg 60 gaaggtcaag
ctgctaaaga atttattgct tggttggtta aaggtagggt acctgata 118 51 118 DNA
Artificial sequence Bottom Strand Synthetic Oligonucleotide
misc_feature (9)..(108) Bottom Strand Primer P0057 51 tatcaggtac
cctaccttta accaaccaag caataaattc tttagcagct tgaccttcca 60
aataagaaga aacatcagaa gtaaaagtac cttcagcatg ccttttctct agagacct 118
52 6 PRT Artificial sequence N-terminal flanking peptide encoded by
pREX00052 52 Arg Ser Leu Glu Lys Arg 1 5 53 21 DNA Artificial
sequence Primer P0070 53 gctatgacca acaagtgtct c 21 54 22 DNA
Artificial sequence Primer P0071 54 cgcacctgtg gcgccggtga tg 22 55
60 DNA Artificial sequence Sequence for fusion of IFN Beta-1 to mTf
misc_feature (18)..(48) Primer P0082 sequence 55 ctgcttactc
taggtctcta gagaaaacag ggtacctccg aaacgtacct gataaaactg 60 56 60 DNA
Artificial sequence Sequence for fusion of IFN Beta-1 to mTf
misc_feature (17)..(39) Primer P0083 sequence 56 cagttttatc
aggtacgttt cggaggtacc ctgttttctc tagagaccta gagtaagcag 60 57 8 PRT
Artificial sequence MFa-1 sequence 57 Ala Tyr Ser Arg Ser Leu Glu
Lys 1 5 58 6 PRT Artificial sequence IFN-B-1 sequence 58 Thr Gly
Tyr Leu Arg Asn 1 5 59 5 PRT Artificial sequence mTf sequence 59
Val Pro Asp Lys Thr 1 5 60 43 DNA Artificial sequence Primer P0084
60 ctctaggtct ctagagaaaa ggagctacaa cttgcttgga ttc 43 61 25 DNA
Artificial sequence Primer P0085 61 gtctgaatgt cccatggagg ctttg 25
62 36 DNA Artificial sequence Primer P0086 62 actttccgtc gacctagcta
caacttgctt ggattc 36 63 50 DNA Artificial sequence Primer P0087 63
tgaatgtcca atggaggctt tgattatttc gaattaagaa tactaaatac 50 64 31 PRT
Artificial sequence GLP-1(7-37) amino acid sequence 64 His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 65
757 DNA Homo sapiens CDS (1)..(564) GenBank No. NM_002176,
interferon-beta 1 65 atg acc aac aag tgt ctc ctc caa att gct ctc
ctg ttg tgc ttc tcc 48 Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu
Leu Leu Cys Phe Ser 1 5 10 15 act aca gct ctt tcc atg agc tac aac
ttg ctt gga ttc cta caa aga 96 Thr Thr Ala Leu Ser Met Ser Tyr Asn
Leu Leu Gly Phe Leu Gln Arg 20 25 30 agc agc aat ttt cag tgt cag
aag ctc ctg tgg caa ttg aat ggg agg 144 Ser Ser Asn Phe Gln Cys Gln
Lys Leu Leu Trp Gln Leu Asn Gly Arg 35 40 45 ctt gaa tat tgc ctc
aag gac agg atg aac ttt gac atc cct gag gag 192 Leu Glu Tyr Cys Leu
Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu 50 55 60 att aag cag
ctg cag cag ttc cag aag gag gac gcc gca ttg acc atc 240 Ile Lys Gln
Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile 65 70 75 80 tat
gag atg ctc cag aac atc ttt gct att ttc aga caa gat tca tct 288 Tyr
Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser 85 90
95 agc act ggc tgg aat gag act att gtt gag aac ctc ctg gct aat gtc
336 Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val
100 105 110 tat cat cag ata aac cat ctg aag aca gtc ctg gaa gaa aaa
ctg gag 384 Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys
Leu Glu 115 120 125 aaa gaa gat ttt acc agg gga aaa ctc atg agc agt
ctg cac ctg aaa 432 Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser
Leu His Leu Lys 130 135 140 aga tat tat ggg agg att ctg cat tac ctg
aag gcc aag gag tac agt 480 Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu
Lys Ala Lys Glu Tyr Ser 145 150 155 160 cac tgt gcc tgg acc ata gtc
aga gtg gaa atc cta agg aac ttt tac 528 His Cys Ala Trp Thr Ile Val
Arg Val Glu Ile Leu Arg Asn Phe Tyr 165 170 175 ttc att aac aga ctt
aca ggt tac ctc cga aac tga agatctccta 574 Phe Ile Asn Arg Leu Thr
Gly Tyr Leu Arg Asn 180 185 gcctgtccct ctgggactgg acaattgctt
caagcattct tcaaccagca gatgctgttt 634 aagtgactga tggctaatgt
actgcaaatg aaaggacact agaagatttt gaaattttta 694 ttaaattatg
agttattttt atttatttaa attttatttt ggaaaataaa ttatttttgg 754 tgc 757
66 187 PRT Homo sapiens 66 Met Thr Asn Lys Cys Leu Leu Gln Ile Ala
Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Met Ser Tyr
Asn Leu Leu Gly Phe Leu Gln Arg 20 25 30 Ser Ser Asn Phe Gln Cys
Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg 35 40 45 Leu Glu Tyr Cys
Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu 50 55 60 Ile Lys
Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile 65 70 75 80
Tyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser 85
90 95 Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn
Val 100 105 110 Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu
Lys Leu Glu 115 120 125 Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser
Ser Leu His Leu Lys 130 135 140 Arg Tyr Tyr Gly Arg Ile Leu His Tyr
Leu Lys Ala Lys Glu Tyr Ser 145 150 155 160 His Cys Ala Trp Thr Ile
Val Arg Val Glu Ile Leu Arg Asn Phe Tyr 165 170 175 Phe Ile Asn Arg
Leu Thr Gly Tyr Leu Arg Asn 180 185 67 28 DNA Artificial Sequence
Description of Artificial Sequence XbaI/KpnI region of pREX0052 67
aggtctctag agaagagggt acctgata 28 68 30 DNA Artificial Sequence
Description of Artificial Sequence SalI/HindIII region of pREX0052
68 actttccgtc gaccttaata agcttaattc 30 69 9 PRT Artificial Sequence
Description of Artificial Sequence Amino acid sequence encoded by
XbaI/KpnI region of pREX0052 69 Arg Ser Leu Glu Lys Arg Val Pro Asp
1 5 70 5 PRT Artificial Sequence Description of Artificial Sequence
Amino acid sequence encoded by SalI/HindIII region of pREX0052 70
Thr Phe Arg Arg Pro 1 5 71 450 DNA Homo sapiens CDS (45)..(377)
GenBank Accession No. NM_000207, human insulin 71 gctgcatcag
aagaggccat caagcacatc actgtccttc tgcc atg gcc ctg tgg 56 Met Ala
Leu Trp 1 atg cgc ctc ctg ccc ctg ctg gcg ctg ctg gcc ctc tgg gga
cct gac 104
Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu Trp Gly Pro Asp 5
10 15 20 cca gcc gca gcc ttt gtg aac caa cac ctg tgc ggc tca cac
ctg gtg 152 Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly Ser His
Leu Val 25 30 35 gaa gct ctc tac cta gtg tgc ggg gaa cga ggc ttc
ttc tac aca ccc 200 Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe
Phe Tyr Thr Pro 40 45 50 aag acc cgc cgg gag gca gag gac ctg cag
gtg ggg cag gtg gag ctg 248 Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln
Val Gly Gln Val Glu Leu 55 60 65 ggc ggg ggc cct ggt gca ggc agc
ctg cag ccc ttg gcc ctg gag ggg 296 Gly Gly Gly Pro Gly Ala Gly Ser
Leu Gln Pro Leu Ala Leu Glu Gly 70 75 80 tcc ctg cag aag cgt ggc
att gtg gaa caa tgc tgt acc agc atc tgc 344 Ser Leu Gln Lys Arg Gly
Ile Val Glu Gln Cys Cys Thr Ser Ile Cys 85 90 95 100 tcc ctc tac
cag ctg gag aac tac tgc aac tag acgcagcccg caggcagccc 397 Ser Leu
Tyr Gln Leu Glu Asn Tyr Cys Asn 105 110 cccacccgcc gcctcctgca
ccgagagaga tggaataaag cccttgaacc agc 450 72 110 PRT Homo sapiens 72
Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu 1 5
10 15 Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys
Gly 20 25 30 Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu
Arg Gly Phe 35 40 45 Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu
Asp Leu Gln Val Gly 50 55 60 Gln Val Glu Leu Gly Gly Gly Pro Gly
Ala Gly Ser Leu Gln Pro Leu 65 70 75 80 Ala Leu Glu Gly Ser Leu Gln
Lys Arg Gly Ile Val Glu Gln Cys Cys 85 90 95 Thr Ser Ile Cys Ser
Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 100 105 110 73 24 DNA
Artificial Sequence Description of Artificial Sequence 5' primer
for cloning insulin sequence 73 tttgtgaacc aacacctgtg cggc 24 74 34
DNA Artificial Sequence Description of Artificial Sequence 3'
primer for cloning insulin sequence 74 gttgcagtag ttctccagct
ggtagaggga gcag 34 75 289 DNA Artificial Sequence Description of
Artificial Sequence Plasmid pREX0052 N-insulin CDS (1)..(288) 75
gct tac tct agg tct cta gat aag agg ttt gtg aac caa cac ctg tgc 48
Ala Tyr Ser Arg Ser Leu Asp Lys Arg Phe Val Asn Gln His Leu Cys 1 5
10 15 ggc tca cac ctg gtg gaa gct ctc tac cta gtg tgc ggg gaa cga
ggc 96 Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg
Gly 20 25 30 ttc ttc tac aca ccc aag acc cgc cgg gag gca gag gac
ctg cag gtg 144 Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp
Leu Gln Val 35 40 45 ggg cag gtg gag ctg ggc ggg ggc cct ggt gca
ggc agc ctg cag ccc 192 Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala
Gly Ser Leu Gln Pro 50 55 60 ttg gcc ctg gag ggg tcc ctg cag aag
cgt ggc att gtg gaa caa tgc 240 Leu Ala Leu Glu Gly Ser Leu Gln Lys
Arg Gly Ile Val Glu Gln Cys 65 70 75 80 tgt acc agc atc tgc tcc ctc
tac cag ctg gag aac tac tgc aac gta c 289 Cys Thr Ser Ile Cys Ser
Leu Tyr Gln Leu Glu Asn Tyr Cys Asn Val 85 90 95 76 96 PRT
Artificial Sequence Description of Artificial Sequence Plasmid
pREX0052 N-insulin 76 Ala Tyr Ser Arg Ser Leu Asp Lys Arg Phe Val
Asn Gln His Leu Cys 1 5 10 15 Gly Ser His Leu Val Glu Ala Leu Tyr
Leu Val Cys Gly Glu Arg Gly 20 25 30 Phe Phe Tyr Thr Pro Lys Thr
Arg Arg Glu Ala Glu Asp Leu Gln Val 35 40 45 Gly Gln Val Glu Leu
Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro 50 55 60 Leu Ala Leu
Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys 65 70 75 80 Cys
Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn Val 85 90
95 77 49 DNA Artificial Sequence Description of Artificial Sequence
5' insulin cloning primer 77 gcttactcta ggtctctaga taagaggttt
gtgaaccaac acctgtgcg 49 78 22 DNA Artificial Sequence Description
of Artificial Sequence Linker for cloning insulin 78 ctggagaact
actgcaacgt ac 22 79 18 DNA Artificial Sequence Description of
Artificial Sequence Linker for cloning insulin 79 gttgcagtag
ttctccag 18 80 290 DNA Artificial Sequence Description of
Artificial Sequence Plasmid pREX0052 C-insulin CDS (1)..(276) 80
tgc act ttc cgt cga cct ttt gtg aac caa cac ctg tgc ggc tca cac 48
Cys Thr Phe Arg Arg Pro Phe Val Asn Gln His Leu Cys Gly Ser His 1 5
10 15 ctg gtg gaa gct ctc tac cta gtg tgc ggg gaa cga ggc ttc ttc
tac 96 Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe
Tyr 20 25 30 aca ccc aag acc cgc cgg gag gca gag gac ctg cag gtg
ggg cag gtg 144 Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val
Gly Gln Val 35 40 45 gag ctg ggc ggg ggc cct ggt gca ggc agc ctg
cag ccc ttg gcc ctg 192 Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu
Gln Pro Leu Ala Leu 50 55 60 gag ggg tcc ctg cag aag cgt ggc att
gtg gaa caa tgc tgt acc agc 240 Glu Gly Ser Leu Gln Lys Arg Gly Ile
Val Glu Gln Cys Cys Thr Ser 65 70 75 80 atc tgc tcc ctc tac cag ctg
gag aac tac tgc aac taataagctt aatt 290 Ile Cys Ser Leu Tyr Gln Leu
Glu Asn Tyr Cys Asn 85 90 81 92 PRT Artificial Sequence Description
of Artificial Sequence Plasmid pREX0052 C-insulin 81 Cys Thr Phe
Arg Arg Pro Phe Val Asn Gln His Leu Cys Gly Ser His 1 5 10 15 Leu
Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr 20 25
30 Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val
35 40 45 Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu
Ala Leu 50 55 60 Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln
Cys Cys Thr Ser 65 70 75 80 Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr
Cys Asn 85 90 82 40 DNA Artificial Sequence Description of
Artificial Sequence 5' insulin cloning primer 82 tgcactttcc
gtcgaccttt tgtgaaccaa cacctgtgcg 40 83 32 DNA Artificial Sequence
Description of Artificial Sequence 3' insulin cloning primer 83
aattaagctt attagttgca gtagttctcc ag 32 84 676 PRT Oryctolagus
cuniculus 84 Val Thr Glu Lys Thr Val Arg Trp Cys Ala Val Asn Asp
His Glu Ala 1 5 10 15 Ser Lys Cys Ala Asn Phe Arg Asp Ser Met Lys
Lys Val Leu Pro Glu 20 25 30 Asp Gly Pro Arg Ile Ile Cys Val Lys
Lys Ala Ser Tyr Leu Asp Cys 35 40 45 Ile Lys Ala Ile Ala Ala His
Glu Ala Asp Ala Val Thr Leu Asp Ala 50 55 60 Gly Leu Val His Glu
Ala Gly Leu Thr Pro Asn Asn Leu Lys Pro Val 65 70 75 80 Val Ala Glu
Phe Tyr Gly Ser Lys Glu Asn Pro Lys Thr Phe Tyr Tyr 85 90 95 Ala
Val Ala Leu Val Lys Lys Gly Ser Asn Phe Gln Leu Asn Glu Leu 100 105
110 Gln Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp
115 120 125 Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro
Arg Lys 130 135 140 Pro Leu Glu Lys Ala Val Ala Ser Phe Phe Ser Gly
Ser Cys Val Pro 145 150 155 160 Cys Ala Asp Gly Ala Asp Phe Pro Gln
Leu Cys Gln Leu Cys Pro Gly 165 170 175 Cys Gly Cys Ser Ser Val Gln
Pro Tyr Phe Gly Tyr Ser Gly Ala Phe 180 185 190 Lys Cys Leu Lys Asp
Gly Leu Gly Asp Val Ala Phe Val Lys Gln Glu 195 200 205 Thr Ile Phe
Glu Asn Leu Pro Ser Lys Asp Glu Arg Asp Gln Tyr Glu 210 215 220 Leu
Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Glu Gln 225 230
235 240 Cys His Leu Ala Arg Val Pro Ser His Ala Val Val Ala Arg Ser
Val 245 250 255 Asp Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln
Ala Gln Glu 260 265 270 His Phe Gly Lys Asp Lys Ser Gly Asp Phe Gln
Leu Phe Ser Ser Pro 275 280 285 His Gly Lys Asn Leu Leu Phe Lys Asp
Ser Ala Tyr Gly Phe Phe Lys 290 295 300 Val Pro Pro Arg Met Asp Ala
Asn Leu Tyr Leu Gly Tyr Glu Tyr Val 305 310 315 320 Thr Ala Val Arg
Asn Leu Arg Glu Gly Ile Cys Pro Asp Pro Leu Gln 325 330 335 Asp Glu
Cys Lys Ala Val Lys Trp Cys Ala Leu Ser His His Glu Arg 340 345 350
Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Cys 355
360 365 Glu Ser Ala Glu Thr Pro Glu Asp Cys Ile Ala Lys Ile Met Asn
Gly 370 375 380 Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Tyr Val Tyr
Ile Ala Gly 385 390 395 400 Gln Cys Gly Leu Val Pro Val Leu Ala Glu
Asn Tyr Glu Ser Thr Asp 405 410 415 Cys Lys Lys Ala Pro Glu Glu Gly
Tyr Leu Ser Val Ala Val Val Lys 420 425 430 Lys Ser Asn Pro Asp Ile
Asn Trp Asn Asn Leu Glu Gly Lys Lys Ser 435 440 445 Cys His Thr Ala
Val Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly 450 455 460 Leu Leu
Tyr Asn Arg Ile Asn His Cys Arg Phe Asp Glu Phe Phe Arg 465 470 475
480 Gln Gly Cys Ala Pro Gly Ser Gln Lys Asn Ser Ser Leu Cys Glu Leu
485 490 495 Cys Ile Gly Pro Ser Val Cys Ala Pro Asn Asn Arg Glu Gly
Tyr Tyr 500 505 510 Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys
Gly Asp Val Ala 515 520 525 Phe Val Lys Ser Gln Thr Val Leu Gln Asn
Thr Gly Gly Arg Asn Ser 530 535 540 Glu Pro Trp Ala Lys Asp Leu Lys
Glu Glu Asp Phe Glu Leu Leu Cys 545 550 555 560 Leu Asp Gly Thr Arg
Lys Pro Val Ser Glu Ala His Asn Cys His Leu 565 570 575 Ala Lys Ala
Pro Asn His Ala Val Val Ser Arg Lys Asp Lys Ala Ala 580 585 590 Cys
Val Lys Gln Lys Leu Leu Asp Leu Gln Val Glu Tyr Gly Asn Thr 595 600
605 Val Ala Asp Cys Ser Ser Lys Phe Cys Met Phe His Ser Lys Thr Lys
610 615 620 Asp Leu Leu Phe Arg Asp Asp Thr Lys Cys Leu Val Asp Leu
Arg Gly 625 630 635 640 Lys Asn Thr Tyr Glu Lys Tyr Leu Gly Ala Asp
Tyr Ile Lys Ala Val 645 650 655 Ser Asn Leu Arg Lys Cys Ser Thr Ser
Arg Leu Leu Glu Ala Cys Thr 660 665 670 Phe His Lys His 675 85 676
PRT Rattus norvegicus 85 Val Pro Asp Lys Thr Val Lys Trp Cys Ala
Val Ser Glu His Glu Asn 1 5 10 15 Thr Lys Cys Ile Ser Phe Arg Asp
His Met Lys Thr Val Leu Pro Ala 20 25 30 Asp Gly Pro Arg Leu Pro
Cys Val Lys Lys Thr Ser Tyr Gln Asp Cys 35 40 45 Ile Lys Ala Ile
Ser Gly Gly Glu Ala Asp Ala Ile Thr Leu Asp Gly 50 55 60 Gly Trp
Val Tyr Asp Ala Gly Leu Thr Pro Asn Asn Leu Lys Pro Val 65 70 75 80
Ala Ala Glu Phe Tyr Gly Ser Leu Glu His Arg Gln Thr His Tyr Leu 85
90 95 Ala Val Ala Val Val Lys Lys Gly Thr Asp Phe Gln Leu Asn Gln
Leu 100 105 110 Gln Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser
Ala Gly Trp 115 120 125 Ile Ile Pro Ile Gly Leu Leu Phe Cys Asn Leu
Pro Glu Pro Arg Lys 130 135 140 Pro Leu Glu Lys Ala Val Ala Ser Phe
Phe Ser Gly Ser Cys Val Pro 145 150 155 160 Cys Ala Asp Pro Val Ala
Phe Pro Gln Leu Cys Gln Leu Cys Pro Gly 165 170 175 Cys Gly Cys Ser
Pro Thr Gln Pro Phe Phe Gly Tyr Val Gly Ala Phe 180 185 190 Lys Cys
Leu Arg Asp Gly Gly Gly Asp Val Ala Phe Val Lys His Thr 195 200 205
Thr Ile Phe Glu Val Leu Pro Gln Lys Ala Asp Arg Asp Gln Tyr Glu 210
215 220 Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Gln Tyr Glu
Asp 225 230 235 240 Cys Tyr Leu Ala Arg Ile Pro Ser His Ala Val Val
Ala Arg Asn Gly 245 250 255 Asp Gly Lys Glu Asp Leu Ile Trp Glu Ile
Leu Lys Val Ala Gln Glu 260 265 270 His Phe Gly Lys Gly Lys Ser Lys
Asp Phe Gln Leu Phe Gly Ser Pro 275 280 285 Leu Gly Lys Asp Leu Leu
Phe Lys Asp Ser Arg Phe Gly Leu Leu Arg 290 295 300 Ala Pro Lys Asp
Gly Leu Gln Ala Val Pro Arg Pro Gln Leu Cys His 305 310 315 320 Cys
His Ser Lys Ser Ala Gly Ser Cys Pro Asp Ala Ile Asp Ser Ala 325 330
335 Pro Val Lys Trp Cys Ala Leu Ser His Gln Glu Arg Ala Lys Cys Asp
340 345 350 Glu Trp Ser Val Thr Gly Asn Gly Gln Ile Glu Cys Glu Ser
Ala Glu 355 360 365 Ser Thr Glu Asp Cys Ile Asp Lys Ile Val Asn Gly
Glu Ala Asp Ala 370 375 380 Met Ser Leu Asp Gly Gly His Ala Tyr Ile
Ala Gly Gln Cys Gly Leu 385 390 395 400 Val Pro Val Met Ala Glu Asn
Tyr Asp Ile Ser Ser Cys Thr Asn Pro 405 410 415 Gln Ser Asp Val Phe
Pro Lys Gly Tyr Tyr Ala Val Ala Val Val Lys 420 425 430 Ala Ser Asp
Ser Ser Ile Asn Trp Asn Asn Leu Lys Gly Lys Lys Ser 435 440 445 Cys
His Thr Gly Val Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly 450 455
460 Leu Leu Phe Ser Arg Ile Asn His Cys Lys Phe Asp Glu Phe Phe Ser
465 470 475 480 Gln Gly Cys Ala Pro Gly Tyr Lys Lys Asn Ser Thr Leu
Cys Asp Leu 485 490 495 Cys Ile Gly Pro Ala Lys Cys Ala Pro Asn Asn
Arg Glu Gly Tyr Asn 500 505 510 Gly Tyr Thr Gly Ala Phe Gln Cys Leu
Val Glu Lys Gly Asp Val Ala 515 520 525 Phe Val Lys His Gln Thr Val
Leu Glu Asn Thr Asn Gly Lys Asn Thr 530 535 540 Ala Ala Trp Ala Lys
Asp Leu Lys Gln Glu Asp Phe Gln Leu Leu Cys 545 550 555 560 Pro Asp
Gly Thr Lys Lys Pro Val Thr Glu Phe Ala Thr Cys His Leu 565 570 575
Ala Gln Ala Pro Asn His Val Val Val Ser Arg Lys Glu Lys Ala Ala 580
585 590 Arg Val Ser Thr Val Leu Thr Ala Gln Lys Asp Leu Phe Trp Lys
Gly 595 600 605 Asp Lys Asp Cys Thr Gly Asn Phe Cys Leu Phe Arg Ser
Ser Thr Lys 610 615 620 Asp Leu Leu Phe Arg Asp Asp Thr Lys Cys Leu
Thr Lys Leu Pro Glu 625 630 635 640 Gly Thr Thr Tyr Glu Glu Tyr Leu
Gly Ala Glu Tyr Leu Gln Ala Val 645 650 655 Gly Asn Ile Arg Lys Cys
Ser Thr Ser Arg Leu Leu Glu Ala Cys Thr 660 665 670 Phe His Lys Ser
675 86 677 PRT Mus musculus 86 Val Pro Asp Lys Thr Val Lys Trp Cys
Ala Val Ser Glu His Glu Asn 1 5 10 15 Thr Lys Cys Ile Ser Phe Arg
Asp His Met Lys Thr Val Leu Pro Pro 20 25 30 Asp Gly Pro Arg Leu
Ala Cys Val Lys Lys Thr Ser Tyr Pro Asp Cys 35 40 45 Ile Lys Ala
Ile Ser Ala Ser Glu Ala Asp Ala Met Thr Leu Asp Gly 50 55 60 Gly
Trp Val Tyr Asp Ala Gly Leu Thr Pro
Asn Asn Leu Lys Pro Val 65 70 75 80 Ala Ala Glu Phe Tyr Gly Ser Val
Glu His Pro Gln Thr Tyr Tyr Tyr 85 90 95 Ala Val Ala Val Val Lys
Lys Gly Thr Asp Phe Gln Leu Asn Gln Leu 100 105 110 Glu Gly Lys Lys
Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp 115 120 125 Val Ile
Pro Ile Gly Leu Leu Phe Cys Lys Leu Ser Glu Pro Arg Ser 130 135 140
Pro Leu Glu Lys Ala Val Ser Ser Phe Phe Ser Gly Ser Cys Val Pro 145
150 155 160 Cys Ala Asp Pro Val Ala Phe Pro Lys Leu Cys Gln Leu Cys
Pro Gly 165 170 175 Cys Gly Cys Ser Ser Thr Gln Pro Phe Phe Gly Tyr
Val Gly Ala Phe 180 185 190 Lys Cys Leu Lys Asp Gly Gly Gly Asp Val
Ala Phe Val Lys His Thr 195 200 205 Thr Ile Phe Glu Val Leu Pro Glu
Lys Ala Asp Arg Asp Gln Tyr Glu 210 215 220 Leu Leu Cys Leu Asp Asn
Thr Arg Lys Pro Val Asp Gln Tyr Glu Asp 225 230 235 240 Cys Tyr Leu
Ala Arg Ile Pro Ser His Ala Val Val Ala Arg Lys Asn 245 250 255 Asn
Gly Lys Glu Asp Leu Ile Trp Glu Ile Leu Lys Val Ala Gln Glu 260 265
270 His Phe Gly Lys Gly Lys Ser Lys Asp Phe Gln Leu Phe Ser Ser Pro
275 280 285 Leu Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala Phe Gly Leu
Leu Arg 290 295 300 Val Pro Pro Arg Met Asp Tyr Arg Leu Tyr Leu Gly
His Asn Tyr Val 305 310 315 320 Thr Ala Ile Arg Asn Gln Gln Glu Gly
Val Cys Pro Glu Gly Ser Ile 325 330 335 Asp Asn Ser Pro Val Lys Trp
Cys Ala Leu Ser His Leu Glu Arg Thr 340 345 350 Lys Cys Asp Glu Trp
Ser Ile Ile Ser Glu Gly Lys Ile Glu Cys Glu 355 360 365 Ser Ala Glu
Thr Thr Glu Asp Cys Ile Glu Lys Ile Val Asn Gly Glu 370 375 380 Ala
Asp Ala Met Thr Leu Asp Gly Gly His Ala Tyr Ile Ala Gly Gln 385 390
395 400 Cys Gly Leu Val Pro Val Met Ala Glu Tyr Tyr Glu Ser Ser Asn
Cys 405 410 415 Ala Ile Pro Ser Gln Gln Gly Ile Phe Pro Lys Gly Tyr
Tyr Ala Val 420 425 430 Ala Val Val Lys Ala Ser Asp Thr Ser Ile Thr
Trp Asn Asn Leu Lys 435 440 445 Gly Lys Lys Ser Cys His Thr Gly Val
Asp Arg Thr Ala Gly Trp Asn 450 455 460 Ile Pro Met Gly Met Leu Tyr
Asn Arg Ile Asn His Cys Lys Phe Asp 465 470 475 480 Glu Phe Phe Ser
Gln Gly Cys Ala Pro Gly Tyr Glu Lys Asn Ser Thr 485 490 495 Leu Cys
Asp Leu Cys Ile Gly Pro Leu Lys Cys Ala Pro Asn Asn Lys 500 505 510
Glu Glu Tyr Asn Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys 515
520 525 Gly Asp Val Ala Phe Val Lys His Gln Thr Val Leu Asp Asn Thr
Glu 530 535 540 Gly Lys Asn Pro Ala Glu Trp Ala Lys Asn Leu Lys Gln
Glu Asp Phe 545 550 555 560 Glu Leu Leu Cys Pro Asp Gly Thr Arg Lys
Pro Val Lys Asp Phe Ala 565 570 575 Ser Cys His Leu Ala Gln Ala Pro
Asn His Val Val Val Ser Arg Lys 580 585 590 Glu Lys Ala Ala Arg Val
Lys Ala Val Leu Thr Ser Gln Glu Thr Leu 595 600 605 Phe Gly Gly Ser
Asp Cys Thr Gly Asn Phe Cys Leu Phe Lys Ser Thr 610 615 620 Thr Lys
Asp Leu Leu Phe Arg Asp Asp Thr Lys Cys Phe Val Lys Leu 625 630 635
640 Pro Glu Gly Thr Thr Pro Glu Lys Tyr Leu Gly Ala Glu Tyr Met Gln
645 650 655 Ser Val Gly Asn Met Arg Lys Cys Ser Thr Ser Arg Leu Leu
Glu Ala 660 665 670 Cys Thr Phe His Lys 675 87 688 PRT Equus
caballus 87 Ala Glu Gln Thr Val Arg Trp Cys Thr Val Ser Asn His Glu
Val Ser 1 5 10 15 Lys Cys Ala Ser Phe Arg Asp Ser Met Lys Ser Ile
Val Pro Ala Pro 20 25 30 Pro Leu Val Ala Cys Val Lys Arg Thr Ser
Tyr Leu Glu Cys Ile Lys 35 40 45 Ala Ile Ala Asp Asn Glu Ala Asp
Ala Val Thr Leu Asp Ala Gly Leu 50 55 60 Val Phe Glu Ala Gly Leu
Ser Pro Tyr Asn Leu Lys Pro Val Val Ala 65 70 75 80 Glu Phe Tyr Gly
Ser Lys Thr Glu Pro Gln Thr His Tyr Tyr Ala Val 85 90 95 Ala Val
Val Lys Lys Asn Ser Asn Phe Gln Leu Asn Gln Leu Gln Gly 100 105 110
Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp Asn Ile 115
120 125 Pro Ile Gly Leu Leu Tyr Trp Gln Leu Pro Glu Pro Arg Glu Ser
Leu 130 135 140 Gln Lys Ala Val Ser Asn Phe Phe Ala Gly Ser Cys Val
Pro Cys Ala 145 150 155 160 Asp Arg Thr Ala Val Pro Asn Leu Cys Gln
Leu Cys Val Gly Lys Gly 165 170 175 Thr Asp Lys Cys Ala Cys Ser Asn
His Glu Pro Tyr Phe Gly Tyr Ser 180 185 190 Gly Ala Phe Lys Cys Leu
Ala Asp Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr
Val Leu Glu Asn Leu Pro Gln Glu Ala Asp Arg Asp 210 215 220 Glu Tyr
Gln Leu Leu Cys Arg Asp Asn Thr Arg Lys Ser Val Asp Glu 225 230 235
240 Tyr Lys Asp Cys Tyr Leu Ala Ser Ile Pro Ser His Ala Val Val Ala
245 250 255 Arg Ser Val Asp Gly Lys Glu Asp Leu Ile Trp Gly Leu Leu
Asn Gln 260 265 270 Ala Gln Glu His Phe Gly Thr Glu Lys Ser Lys Asp
Phe His Leu Phe 275 280 285 Ser Ser Pro His Gly Lys Asp Leu Leu Phe
Lys Asp Ser Ala Leu Gly 290 295 300 Phe Leu Arg Ile Pro Pro Ala Met
Asp Thr Trp Leu Tyr Leu Gly Tyr 305 310 315 320 Glu Tyr Val Thr Ala
Ile Arg Asn Leu Arg Glu Asp Ile Arg Pro Glu 325 330 335 Val Pro Lys
Asp Glu Cys Lys Lys Val Lys Trp Cys Ala Ile Gly His 340 345 350 His
Glu Lys Val Lys Cys Asp Glu Trp Ser Val Asn Ser Gly Gly Asn 355 360
365 Ile Glu Cys Glu Ser Ala Gln Ser Thr Glu Asp Cys Ile Ala Lys Ile
370 375 380 Val Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe
Ile Tyr 385 390 395 400 Ile Ala Gly Lys Cys Gly Leu Val Pro Val Leu
Ala Glu Asn Tyr Glu 405 410 415 Thr Arg Ser Gly Ser Ala Cys Val Asp
Thr Pro Glu Glu Gly Tyr His 420 425 430 Ala Val Ala Val Val Lys Ser
Ser Ser Asp Pro Asp Leu Thr Trp Asn 435 440 445 Ser Leu Lys Gly Lys
Lys Ser Cys His Thr Gly Val Asp Arg Thr Ala 450 455 460 Gly Trp Asn
Ile Pro Met Gly Leu Leu Tyr Ser Glu Ile Lys His Cys 465 470 475 480
Glu Phe Asp Lys Phe Phe Arg Glu Gly Cys Ala Pro Gly Tyr Arg Arg 485
490 495 Asn Ser Thr Leu Cys Asn Leu Cys Ile Gly Ser Ala Ser Gly Pro
Gly 500 505 510 Arg Glu Cys Glu Pro Asn Asn His Glu Arg Tyr Tyr Gly
Tyr Thr Gly 515 520 525 Ala Phe Arg Cys Leu Val Glu Lys Gly Asp Val
Ala Phe Val Lys His 530 535 540 Gln Thr Val Glu Gln Asn Thr Asp Gly
Arg Asn Pro Asp Asp Trp Ala 545 550 555 560 Lys Asp Leu Lys Ser Glu
Asn Phe Lys Leu Leu Cys Pro Asp Gly Thr 565 570 575 Arg Lys Ser Val
Thr Glu Phe Lys Ser Cys Tyr Leu Ala Arg Ala Pro 580 585 590 Asn His
Ala Val Val Ser Arg Lys Glu Lys Ala Ala Cys Val Cys Gln 595 600 605
Glu Leu His Asn Gln Gln Ala Ser Tyr Gly Lys Asn Gly Ser His Cys 610
615 620 Pro Asp Lys Phe Cys Leu Phe Gln Ser Ala Thr Lys Asp Leu Leu
Phe 625 630 635 640 Arg Asp Asp Thr Gln Cys Leu Ala Asn Leu Gln Pro
Thr Thr Thr Tyr 645 650 655 Lys Thr Tyr Leu Gly Glu Lys Tyr Leu Thr
Ala Val Ala Asn Leu Arg 660 665 670 Gln Cys Ser Thr Ser Arg Leu Leu
Glu Ala Cys Thr Phe His Arg Val 675 680 685 88 685 PRT Bos taurus
88 Asp Pro Glu Arg Thr Val Arg Trp Cys Thr Ile Ser Thr His Glu Ala
1 5 10 15 Asn Lys Cys Ala Ser Phe Arg Glu Asn Val Leu Arg Ile Leu
Glu Ser 20 25 30 Gly Pro Phe Val Ser Cys Val Lys Lys Thr Ser His
Met Asp Cys Ile 35 40 45 Lys Ala Ile Ser Asn Asn Glu Ala Asp Ala
Val Thr Leu Asp Gly Gly 50 55 60 Leu Val Tyr Glu Ala Gly Leu Lys
Pro Asn Asn Leu Lys Pro Val Val 65 70 75 80 Ala Glu Phe His Gly Thr
Lys Asp Asn Pro Gln Thr His Tyr Tyr Ala 85 90 95 Val Ala Val Val
Lys Lys Asp Thr Asp Phe Lys Leu Asn Glu Leu Arg 100 105 110 Gly Lys
Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp Asn 115 120 125
Ile Pro Met Ala Lys Leu Tyr Lys Glu Leu Pro Asp Pro Gln Glu Ser 130
135 140 Ile Gln Arg Ala Ala Ala Asn Phe Phe Ser Ala Ser Cys Val Pro
Cys 145 150 155 160 Ala Asp Gln Ser Ser Phe Pro Lys Leu Cys Gln Leu
Cys Ala Gly Lys 165 170 175 Gly Thr Asp Lys Cys Ala Cys Ser Asn His
Glu Pro Tyr Phe Gly Tyr 180 185 190 Ser Gly Ala Phe Lys Cys Leu Met
Glu Gly Ala Gly Asp Val Ala Phe 195 200 205 Val Lys His Ser Thr Val
Phe Asp Asn Leu Pro Asn Pro Glu Asp Arg 210 215 220 Lys Asn Tyr Glu
Leu Leu Cys Gly Asp Asn Thr Arg Lys Ser Val Asp 225 230 235 240 Asp
Tyr Gln Glu Cys Tyr Leu Ala Met Val Pro Ser His Ala Val Val 245 250
255 Ala Arg Thr Val Gly Gly Lys Glu Asp Val Ile Trp Glu Leu Leu Asn
260 265 270 His Ala Gln Glu His Phe Gly Lys Asp Lys Pro Asp Asn Phe
Gln Leu 275 280 285 Phe Gln Ser Pro His Gly Lys Asp Leu Leu Phe Lys
Asp Ser Ala Asp 290 295 300 Gly Phe Leu Lys Ile Pro Ser Lys Met Asp
Phe Glu Leu Tyr Leu Gly 305 310 315 320 Tyr Glu Tyr Val Thr Ala Leu
Gln Asn Leu Arg Glu Ser Lys Pro Pro 325 330 335 Asp Ser Ser Lys Asp
Glu Cys Met Val Lys Trp Cys Ala Ile Gly His 340 345 350 Gln Glu Arg
Thr Lys Cys Asp Arg Trp Ser Gly Phe Ser Gly Gly Ala 355 360 365 Ile
Glu Cys Glu Thr Ala Glu Asn Thr Glu Glu Cys Ile Ala Lys Ile 370 375
380 Met Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Tyr Leu Tyr
385 390 395 400 Ile Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu
Asn Tyr Lys 405 410 415 Thr Glu Gly Glu Ser Cys Lys Asn Thr Pro Glu
Lys Gly Tyr Leu Ala 420 425 430 Val Ala Val Val Lys Thr Ser Asp Ala
Asn Ile Asn Trp Asn Asn Leu 435 440 445 Lys Asp Lys Lys Ser Cys His
Thr Ala Val Asp Arg Thr Ala Gly Trp 450 455 460 Asn Ile Pro Met Gly
Leu Leu Tyr Ser Lys Ile Asn Asn Cys Lys Phe 465 470 475 480 Asp Glu
Phe Phe Ser Ala Gly Cys Ala Pro Gly Ser Pro Arg Asn Ser 485 490 495
Ser Leu Cys Ala Leu Cys Ile Gly Ser Glu Lys Gly Thr Gly Lys Glu 500
505 510 Cys Val Pro Asn Ser Asn Glu Arg Tyr Tyr Gly Tyr Thr Gly Ala
Phe 515 520 525 Arg Cys Leu Val Glu Lys Gly Asp Val Ala Phe Val Lys
Asp Gln Thr 530 535 540 Val Ile Gln Asn Thr Asp Gly Asn Asn Asn Glu
Ala Trp Ala Lys Asn 545 550 555 560 Leu Lys Lys Glu Asn Phe Glu Val
Leu Cys Lys Asp Gly Thr Arg Lys 565 570 575 Pro Val Thr Asp Ala Glu
Asn Cys His Leu Ala Arg Gly Pro Asn His 580 585 590 Ala Val Val Ser
Arg Lys Asp Lys Ala Thr Cys Val Glu Lys Ile Leu 595 600 605 Asn Lys
Gln Gln Asp Asp Phe Gly Lys Ser Val Thr Asp Cys Thr Ser 610 615 620
Asn Phe Cys Leu Phe Gln Ser Asn Ser Lys Asp Leu Leu Phe Arg Asp 625
630 635 640 Asp Thr Lys Cys Leu Ala Ser Ile Ala Lys Lys Thr Tyr Asp
Ser Tyr 645 650 655 Leu Gly Asp Asp Tyr Val Arg Ala Met Thr Asn Leu
Arg Gln Cys Ser 660 665 670 Thr Ser Lys Leu Leu Glu Ala Cys Thr Phe
His Lys Pro 675 680 685 89 696 PRT Sus scrofa misc_feature
(308)..(308) Xaa can be any naturally occurring amino acid 89 Val
Ala Gln Lys Thr Val Arg Trp Cys Thr Ile Ser Asn Gln Glu Ala 1 5 10
15 Asn Lys Cys Ser Ser Phe Arg Glu Asn Met Ser Lys Ala Val Lys Asn
20 25 30 Gly Pro Leu Val Ser Cys Val Lys Lys Ser Ser Tyr Leu Asp
Cys Ile 35 40 45 Lys Ala Ile Arg Asp Lys Glu Ala Asp Ala Val Thr
Leu Asp Ala Gly 50 55 60 Leu Val Phe Glu Ala Gly Leu Ala Pro Tyr
Asn Leu Lys Pro Val Val 65 70 75 80 Ala Glu Phe Tyr Gly Gln Lys Asp
Asn Pro Gln Thr His Tyr Tyr Ala 85 90 95 Val Ala Val Val Lys Lys
Gly Ser Asn Phe Gln Trp Asn Gln Leu Gln 100 105 110 Gly Lys Arg Ser
Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp Ile 115 120 125 Ile Pro
Met Gly Leu Leu Tyr Asp Gln Leu Pro Glu Pro Arg Lys Pro 130 135 140
Ile Glu Lys Ala Val Ala Ser Phe Phe Ser Ser Ser Cys Val Pro Cys 145
150 155 160 Ala Asp Pro Val Asn Phe Pro Lys Leu Cys Gln Gln Cys Ala
Gly Lys 165 170 175 Gly Ala Glu Lys Cys Ala Cys Ser Asn His Glu Pro
Tyr Phe Gly Tyr 180 185 190 Ala Gly Ala Phe Asn Cys Leu Lys Glu Asp
Ala Gly Asp Val Ala Phe 195 200 205 Val Lys His Ser Thr Val Leu Glu
Asn Leu Pro Asp Lys Ala Asp Arg 210 215 220 Asp Gln Tyr Glu Leu Leu
Cys Arg Asp Asn Thr Arg Arg Pro Val Asp 225 230 235 240 Asp Tyr Glu
Asn Cys Tyr Leu Ala Gln Val Pro Ser His Ala Val Val 245 250 255 Ala
Arg Ser Val Asp Gly Gln Glu Asp Ser Ile Trp Glu Leu Leu Asn 260 265
270 Gln Ala Gln Glu His Phe Gly Arg Asp Lys Ser Pro Asp Phe Gln Leu
275 280 285 Phe Ser Ser Ser His Gly Lys Asp Leu Leu Phe Lys Asp Ser
Ala Asn 290 295 300 Gly Phe Leu Xaa Ile Pro Ser Lys Met Asp Ser Ser
Leu Tyr Leu Gly 305 310 315 320 Tyr Gln Tyr Val Thr Ala Leu Arg Asn
Leu Arg Glu Glu Ile Ser Pro 325 330 335 Asp Ser Ser Lys Asn Glu Cys
Lys Lys Val Arg Trp Cys Ala Ile Gly 340 345 350 His Glu Glu Thr Gln
Lys Cys Asp Ala Trp Ser Ile Asn Ser Gly Gly 355 360 365 Lys Ile Glu
Cys Val Ser Ala Glu Asn Thr Glu Asp Cys Ile Ala Lys 370 375 380 Ile
Val Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Tyr Ile 385 390
395 400 Tyr Ile Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn
Tyr 405 410 415 Lys Thr Glu Gly Glu Asn Cys Val Asn Thr Pro Glu Lys
Gly Tyr Leu 420 425 430 Ala Val Ala Val Val Lys Lys Ser Ser Gly Pro
Asp Leu Asn Trp Asn 435 440
445 Asn Leu Lys Gly Lys Lys Ser Cys His Thr Ala Val Asp Arg Thr Ala
450 455 460 Gly Trp Asn Ile Pro Met Gly Leu Leu Tyr Asn Lys Ile Asn
Ser Cys 465 470 475 480 Lys Phe Asp Gln Phe Phe Gly Glu Gly Cys Ala
Pro Gly Ser Gln Arg 485 490 495 Asn Ser Ser Leu Cys Ala Leu Cys Ile
Gly Ser Glu Arg Ala Pro Gly 500 505 510 Arg Glu Cys Leu Ala Asn Asn
His Glu Arg Tyr Tyr Gly Tyr Thr Gly 515 520 525 Ala Phe Arg Cys Leu
Val Glu Lys Gly Asp Val Ala Phe Val Lys Asp 530 535 540 Gln Val Val
Gln Gln Asn Thr Asp Gly Lys Asn Lys Asp Asp Trp Ala 545 550 555 560
Lys Asp Leu Lys Gln Met Asp Phe Glu Leu Leu Cys Gln Asn Gly Ala 565
570 575 Arg Glu Pro Val Asp Asn Ala Glu Asn Cys His Leu Ala Arg Ala
Pro 580 585 590 Asn His Ala Val Val Ala Arg Asp Asp Lys Val Thr Cys
Val Ala Glu 595 600 605 Glu Leu Leu Lys Gln Gln Ala Gln Phe Gly Arg
His Val Thr Asp Cys 610 615 620 Ser Ser Ser Phe Cys Met Phe Lys Ser
Asn Thr Lys Asp Leu Leu Phe 625 630 635 640 Arg Asp Asp Thr Gln Cys
Leu Ala Arg Val Gly Lys Thr Thr Tyr Glu 645 650 655 Ser Tyr Leu Gly
Ala Asp Tyr Ile Thr Ala Val Ala Asn Leu Arg Lys 660 665 670 Cys Ser
Thr Ser Lys Leu Leu Glu Ala Cys Thr Phe His Ser Ala Lys 675 680 685
Asn Pro Arg Val Glu Thr Thr Thr 690 695 90 686 PRT Gallus gallus 90
Ala Pro Pro Lys Ser Val Ile Arg Trp Cys Thr Ile Ser Ser Pro Glu 1 5
10 15 Glu Lys Lys Cys Asn Asn Leu Arg Asp Leu Thr Gln Gln Glu Arg
Ile 20 25 30 Ser Leu Thr Cys Val Gln Lys Ala Thr Tyr Leu Asp Cys
Ile Lys Ala 35 40 45 Ile Ala Asn Asn Glu Ala Asp Ala Ile Ser Leu
Asp Gly Gly Gln Ala 50 55 60 Phe Glu Ala Gly Leu Ala Pro Tyr Lys
Leu Lys Pro Ile Ala Ala Glu 65 70 75 80 Val Tyr Glu His Thr Glu Gly
Ser Thr Thr Ser Tyr Tyr Ala Val Ala 85 90 95 Val Val Lys Lys Gly
Thr Glu Phe Thr Val Asn Asp Leu Gln Gly Lys 100 105 110 Thr Ser Cys
His Thr Gly Leu Gly Arg Ser Ala Gly Trp Asn Ile Pro 115 120 125 Ile
Gly Thr Leu Leu His Arg Gly Ala Ile Glu Trp Glu Gly Ile Glu 130 135
140 Ser Gly Ser Val Glu Gln Ala Val Ala Lys Phe Phe Ser Ala Ser Cys
145 150 155 160 Val Pro Gly Ala Thr Ile Glu Gln Lys Leu Cys Arg Gln
Cys Lys Gly 165 170 175 Asp Pro Lys Thr Lys Cys Ala Arg Asn Ala Pro
Tyr Ser Gly Tyr Ser 180 185 190 Gly Ala Phe His Cys Leu Lys Asp Gly
Lys Gly Asp Val Ala Phe Val 195 200 205 Lys His Thr Thr Val Asn Glu
Asn Ala Pro Asp Gln Lys Asp Glu Tyr 210 215 220 Glu Leu Leu Cys Leu
Asp Gly Ser Arg Gln Pro Val Asp Asn Tyr Lys 225 230 235 240 Thr Cys
Asn Trp Ala Arg Val Ala Ala His Ala Val Val Ala Arg Asp 245 250 255
Asp Asn Lys Val Glu Asp Ile Trp Ser Phe Leu Ser Lys Ala Gln Ser 260
265 270 Asp Phe Gly Val Asp Thr Lys Ser Asp Phe His Leu Phe Gly Pro
Pro 275 280 285 Gly Lys Lys Asp Pro Val Leu Lys Asp Leu Leu Phe Lys
Asp Ser Ala 290 295 300 Ile Met Leu Lys Arg Val Pro Ser Leu Met Asp
Ser Gln Leu Tyr Leu 305 310 315 320 Gly Phe Glu Tyr Tyr Ser Ala Ile
Gln Ser Met Arg Lys Asp Gln Leu 325 330 335 Thr Pro Ser Pro Arg Glu
Asn Arg Ile Gln Trp Cys Ala Val Gly Lys 340 345 350 Asp Glu Lys Ser
Lys Cys Asp Arg Trp Ser Val Val Ser Asn Gly Asp 355 360 365 Val Glu
Cys Thr Val Val Asp Glu Thr Lys Asp Cys Ile Ile Lys Ile 370 375 380
Met Lys Gly Glu Ala Asp Ala Val Ala Leu Asp Gly Gly Leu Val Tyr 385
390 395 400 Thr Ala Gly Val Cys Gly Leu Val Pro Val Met Ala Glu Arg
Tyr Asp 405 410 415 Asp Glu Ser Gln Cys Ser Lys Thr Asp Glu Arg Pro
Ala Ser Tyr Phe 420 425 430 Ala Val Ala Val Ala Arg Lys Asp Ser Asn
Val Asn Trp Asn Asn Leu 435 440 445 Lys Gly Lys Lys Ser Cys His Thr
Ala Val Gly Arg Thr Ala Gly Trp 450 455 460 Val Ile Pro Met Gly Leu
Ile His Asn Arg Thr Gly Thr Cys Asn Phe 465 470 475 480 Asp Glu Tyr
Phe Ser Glu Gly Cys Ala Pro Gly Ser Pro Pro Asn Ser 485 490 495 Arg
Leu Cys Gln Leu Cys Gln Gly Ser Gly Gly Ile Pro Pro Glu Lys 500 505
510 Cys Val Ala Ser Ser His Glu Lys Tyr Phe Gly Tyr Thr Gly Ala Leu
515 520 525 Arg Cys Leu Val Glu Lys Gly Asp Val Ala Phe Ile Gln His
Ser Thr 530 535 540 Val Glu Glu Asn Thr Gly Gly Lys Asn Lys Ala Asp
Trp Ala Lys Asn 545 550 555 560 Leu Gln Met Asp Asp Phe Glu Leu Leu
Cys Thr Asp Gly Arg Arg Ala 565 570 575 Asn Val Met Asp Tyr Arg Glu
Cys Asn Leu Ala Glu Val Pro Thr His 580 585 590 Ala Val Val Val Arg
Pro Glu Lys Ala Asn Lys Ile Arg Asp Leu Leu 595 600 605 Glu Arg Gln
Glu Lys Arg Phe Gly Val Asn Gly Ser Glu Lys Ser Lys 610 615 620 Phe
Met Met Phe Glu Ser Gln Asn Lys Asp Leu Leu Phe Lys Asp Leu 625 630
635 640 Thr Lys Cys Leu Phe Lys Val Arg Glu Gly Thr Thr Tyr Lys Glu
Phe 645 650 655 Leu Gly Asp Lys Phe Tyr Thr Val Ile Ser Ser Leu Lys
Thr Cys Asn 660 665 670 Pro Ser Asp Ile Leu Gln Met Cys Ser Phe Leu
Glu Gly Lys 675 680 685
* * * * *
References