U.S. patent application number 17/307240 was filed with the patent office on 2021-08-19 for vegf antagonist formulations.
This patent application is currently assigned to REGENERON PHARMACEUTICALS, INC.. The applicant listed for this patent is REGENERON PHARMACEUTICALS, INC.. Invention is credited to Daniel B. DIX, Kelly FRYE, Susan KAUTZ.
Application Number | 20210252147 17/307240 |
Document ID | / |
Family ID | 1000005568393 |
Filed Date | 2021-08-19 |
United States Patent
Application |
20210252147 |
Kind Code |
A1 |
DIX; Daniel B. ; et
al. |
August 19, 2021 |
VEGF Antagonist Formulations
Abstract
Formulations of a vascular endothelial growth factor
(VEGF)-specific fusion protein antagonist are provided including a
pre-lyophilized formulation, a reconstituted lyophilized
formulation, and a stable liquid formulation. Preferably, the
fusion protein has the sequence of SEQ ID NO:4.
Inventors: |
DIX; Daniel B.;
(LaGrangeville, NY) ; FRYE; Kelly; (Mendham,
NJ) ; KAUTZ; Susan; (Albany, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REGENERON PHARMACEUTICALS, INC. |
Tarrytown |
NY |
US |
|
|
Assignee: |
REGENERON PHARMACEUTICALS,
INC.
Tarrytown
NY
|
Family ID: |
1000005568393 |
Appl. No.: |
17/307240 |
Filed: |
May 4, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16950584 |
Nov 17, 2020 |
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17307240 |
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16535610 |
Aug 8, 2019 |
10857231 |
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16950584 |
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15692893 |
Aug 31, 2017 |
10406226 |
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16535610 |
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15342989 |
Nov 3, 2016 |
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15692893 |
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15064343 |
Mar 8, 2016 |
9511140 |
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15342989 |
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14550385 |
Nov 21, 2014 |
9416167 |
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15064343 |
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13909745 |
Jun 4, 2013 |
8921316 |
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14550385 |
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13428510 |
Mar 23, 2012 |
8710004 |
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13909745 |
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13343214 |
Jan 4, 2012 |
8404638 |
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13428510 |
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12835065 |
Jul 13, 2010 |
8110546 |
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13343214 |
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11387256 |
Mar 22, 2006 |
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12835065 |
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60665125 |
Mar 25, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2318/20 20130101;
A61K 38/16 20130101; A61K 39/39591 20130101; A61K 9/0019 20130101;
A61K 47/22 20130101; C07K 16/22 20130101; A61K 9/19 20130101; A61K
47/10 20130101; A61K 47/183 20130101; A61K 47/12 20130101; A61K
38/179 20130101; A61K 31/7012 20130101; A61K 31/4172 20130101; A61K
47/26 20130101; C07K 2319/30 20130101; A61K 9/08 20130101; A61K
47/02 20130101; C07K 14/71 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 38/17 20060101 A61K038/17; A61K 9/00 20060101
A61K009/00; A61K 9/08 20060101 A61K009/08; A61K 9/19 20060101
A61K009/19; A61K 31/4172 20060101 A61K031/4172; A61K 31/7012
20060101 A61K031/7012; A61K 47/02 20060101 A61K047/02; A61K 47/26
20060101 A61K047/26; A61K 38/16 20060101 A61K038/16; A61K 47/10
20060101 A61K047/10; A61K 47/18 20060101 A61K047/18; A61K 47/22
20060101 A61K047/22; C07K 14/71 20060101 C07K014/71; C07K 16/22
20060101 C07K016/22; A61K 47/12 20060101 A61K047/12 |
Claims
1.-20. (canceled)
21. A vial comprising a formulation comprising: 10-50 mg/ml of a
vascular endothelial growth factor (VEGF) antagonist fusion protein
comprising amino acids 27-457 of SEQ ID NO: 4, a buffer comprising
histidine, an organic co-solvent comprising polysorbate, and a
stabilizing agent comprising a sugar, an amino acid or both.
22. The vial of claim 21, wherein said cosolvent comprises
polysorbate 20.
23. The vial of claim 21, wherein said buffer comprises 5-50 mM
histidine.
24. The vial of claim 23, wherein said sugar comprises a sugar
selected from group consisting of dextrose, ribose, fructose,
mannitol, inositol, sorbitol, trehalose, sucrose, and lactose.
25. The vial of claim 23, wherein said sugar comprises sucrose,
trehalose, or mannitol.
26. The vial of claim 25, wherein said sugar comprises sucrose.
27. The vial of claim 26, wherein said sugar comprises 5-30%
sucrose.
28. The vial of claim 26, wherein said sugar comprises 5-20%
sucrose.
29. The vial of claim 28, wherein said formulation further
comprises a tonicity agent.
30. The vial of claim 29, wherein said tonicity agent is sodium
chloride.
31. The vial of claim 29, wherein said tonicity agent is 40 mM
sodium chloride.
32. The vial of claim 22, wherein said formulation has a pH of
6.0-6.5.
33. The vial of claim 32, wherein said formulation has a pH of
6.2.
34. The vial of claim 32, wherein said formulation has a pH of
6.5.
35. The vial of claim 32, wherein said sugar comprises
trehalose.
36. The vial of claim 32, wherein said buffer comprises 10 mM
histidine.
37. The vial of claim 21, wherein said formulation comprises 40
mg/ml of said VEGF antagonist fusion protein.
38. The vial of claim 37, wherein said cosolvent comprises
polysorbate 20.
39. The vial of claim 38, wherein said buffer comprises 5-50 mM
histidine.
40. The vial of claim 39, wherein said sugar comprises a sugar
selected from group consisting of dextrose, ribose, fructose,
mannitol, inositol, sorbitol, trehalose, sucrose, and lactose.
41. The vial of claim 39, wherein said sugar comprises sucrose,
trehalose, or mannitol.
42. The vial of claim 39, wherein said sugar comprises sucrose.
43. The vial of claim 42, wherein said sugar comprises 5-30%
sucrose.
44. The vial of claim 42, wherein said sugar comprises 5-20%
sucrose.
45. The vial of claim 44, wherein said formulation further
comprises a tonicity agent.
46. The vial of claim 45, wherein said tonicity agent is sodium
chloride.
47. The vial of claim 45, wherein said tonicity agent is 40 mM
sodium chloride.
48. The vial of claim 41, wherein said formulation has a pH of
6.0-6.5.
49. The vial of claim 48, wherein said formulation has a pH of
6.2.
50. The vial of claim 48, wherein said formulation has a pH of
6.5.
51. The vial of claim 48, wherein said sugar comprises
trehalose.
52. The vial of claim 49 wherein said buffer comprises 10 mM
histidine.
53. A pre-filled syringe comprising a formulation comprising: 10-50
mg/ml of a vascular endothelial growth factor (VEGF) antagonist
fusion protein comprising amino acids 27-457 of SEQ ID NO: 4, a
buffer comprising histidine, an organic co-solvent comprising
polysorbate, and a stabilizing agent comprising a sugar, an amino
acid or both.
54. The pre-filled syringe of claim 53, wherein said cosolvent
comprises polysorbate 20.
55. The pre-filled syringe of claim 53, wherein said buffer
comprises 5-50 mM histidine.
56. The pre-filled syringe of claim 55, wherein said sugar
comprises a sugar selected from group consisting of dextrose,
ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose,
and lactose.
57. The pre-filled syringe of claim 55, wherein said sugar
comprises sucrose, trehalose, or mannitol.
58. The pre-filled syringe of claim 57, wherein said sugar
comprises sucrose.
59. The pre-filled syringe of claim 58, wherein said sugar
comprises 5-30% sucrose.
60. The pre-filled syringe of claim 58, wherein said sugar
comprises 5-20% sucrose.
61. The pre-filled syringe of claim 60, wherein said formulation
further comprises a tonicity agent.
62. The pre-filled syringe of claim 61, wherein said tonicity agent
is sodium chloride.
63. The pre-filled syringe of claim 61, wherein said tonicity agent
is 40 mM sodium chloride.
64. The pre-filled syringe of claim 54, wherein said formulation
has a pH of 6.0-6.5.
65. The pre-filled syringe of claim 64, wherein said formulation
has a pH of 6.2.
66. The pre-filled syringe of claim 64, wherein said formulation
has a pH of 6.5.
67. The pre-filled syringe of claim 64, wherein said sugar
comprises trehalose.
68. The pre-filled syringe of claim 64, wherein said buffer
comprises 10 mM histidine.
69. The pre-filled syringe of claim 53, wherein said formulation
comprises 40 mg/ml of said VEGF antagonist fusion protein.
70. The pre-filled syringe of claim 69, wherein said cosolvent
comprises polysorbate 20.
71. The pre-filled syringe of claim 70, wherein said buffer
comprises 5-50 mM histidine.
72. The pre-filled syringe of claim 71, wherein said sugar
comprises a sugar selected from group consisting of dextrose,
ribose, fructose, mannitol, inositol, sorbitol, trehalose, sucrose,
and lactose.
73. The pre-filled syringe of claim 71, wherein said sugar
comprises sucrose, trehalose, or mannitol.
74. The pre-filled syringe of claim 71, wherein said sugar
comprises sucrose.
75. The pre-filled syringe of claim 74, wherein said sugar
comprises 5-30% sucrose.
76. The pre-filled syringe of claim 74, wherein said sugar
comprises 5-20% sucrose.
77. The pre-filled syringe of claim 76, wherein said formulation
further comprises a tonicity agent.
78. The pre-filled syringe of claim 77, wherein said tonicity agent
is sodium chloride.
79. The pre-filled syringe of claim 77, wherein said tonicity agent
is 40 mM sodium chloride.
80. The pre-filled syringe of claim 73, wherein said formulation
has a pH of 6.0-6.5.
81. The pre-filled syringe of claim 80, wherein said formulation
has a pH of 6.2.
82. The pre-filled syringe of claim 80, wherein said formulation
has a pH of 6.5.
83. The pre-filled syringe of claim 80, wherein said sugar
comprises trehalose.
84. The pre-filled syringe of claim 81, wherein said buffer
comprises 10 mM histidine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/342,989, filed on Nov. 3, 2016, which is a
continuation of U.S. patent application Ser. No. 15/064,343, filed
on Mar. 8, 2016 and granted on Dec. 6, 2016 as U.S. Pat. No.
9,511,140, which is a continuation of U.S. patent application Ser.
No. 14/550,385, filed on Nov. 21, 2014 and granted on Aug. 16, 2016
as U.S. Pat. No. 9,416,167, which is a continuation of U.S. patent
application Ser. No. 13/909,745, filed on Jun. 4, 2013 and granted
on Dec. 30, 2014 as U.S. Pat. No. 8,921,316, which is a
continuation of U.S. patent application Ser. No. 13/428,510, filed
on Mar. 23, 2012 and granted on Apr. 29, 2014 as U.S. Pat. No.
8,710,004, which is a continuation of U.S. patent application Ser.
No. 13/343,214, filed on Jan. 4, 2012 and granted on Mar. 26, 2013
as U.S. Pat. No. 8,404,638, which is a division of U.S. patent
application Ser. No. 12/835,065, filed on Jul. 13, 2010 and granted
on Feb. 7, 2012 as U.S. Pat. No. 8,110,546, which is a continuation
of U.S. patent application Ser. No. 11/387,256, filed on Mar. 22,
2006, which claims the benefit of priority under 35 USC .sctn.
119(e) of U.S. Provisional Application No. 60/665,125, filed on
Mar. 25, 2005, all of which are herein specifically incorporated by
reference in their entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention is directed to pharmaceutical
formulations comprising agents capable of inhibiting vascular
endothelial growth factor (VEGF), and to methods for making and
using such formulations. The invention includes pharmaceutical
formulations having increased stability.
Statement of Related Art
[0003] Vascular endothelial growth factor (VEGF) expression is
nearly ubiquitous in human cancer, consistent with its role as a
key mediator of tumor neoangiogenesis. Blockade of VEGF function,
by binding to the molecule or its VEGFR-2 receptor, inhibits growth
of implanted tumor cells in multiple different xenograft models
(see, for example, Gerber et al. (2000) Cancer Res. 60:6253-6258).
A soluble VEGF-specific fusion protein antagonist, termed a "VEGF
trap" has been described (Kim et al. (2002) Proc. Natl. Acad. Sci.
USA 99:11399-404; Holash et al. (2002) Proc. Natl. Acad. Sci. USA
99:11393-8), which references are specifically incorporated by
reference in their entirety.
[0004] Lyophilization (freeze drying under controlled conditions)
is commonly used for long term storage of proteins. The lyophilized
protein is substantially resistant to degradation, aggregation,
oxidation, and other degenerative processes while in the
freeze-dried state (see, for example, U.S. Pat. No. 6,436,897).
BRIEF SUMMARY OF THE INVENTION
[0005] Stable formulations of a VEGF-specific fusion protein
antagonist are herein provided. The pharmaceutically acceptable
formulations of the invention comprise the VEGF "trap" antagonist
with a pharmaceutically acceptable carrier. In specific
embodiments, liquid and freeze-dried, or lyophilized formulations
are provided.
[0006] In a first aspect, the invention features a stable liquid
formulation of a VEGF-specific fusion protein antagonist,
comprising a fusion protein comprising a receptor component
consisting essentially of an immunoglobulin-like (Ig) domain 2 of a
first VEGF receptor and Ig domain 3 of a second VEGF receptor, and
a multimerizing component, one or more buffers, and one or more
thermal stabilizers. In a specific embodiment of the VEGF-specific
fusion protein antagonist, the first VEGF receptor is Flt1 and the
second VEGF receptor is Flk1 or Flt4. In a more specific embodiment
the fusion protein has the amino acid sequence of SEQ ID NO:2 or
SEQ ID NO:4. In one embodiment, the buffer is a phosphate buffer
and/or citrate. More preferably, the buffers are phosphate and
citrate. In one embodiment, the thermal stabilizers are NaCl and/or
sucrose. More preferably, the thermal stabilizers are both NaCl and
sucrose.
[0007] In a specific embodiment, the stable liquid formulation of a
VEGF-specific fusion protein antagonist comprises 1-10 mM phosphate
buffer, 1-10 mM citrate, 25-150 mM NaCl, 5-30% sucrose, 10-50 mg/ml
of the fusion protein, at a pH of about 6-6.5. In a more specific
embodiment, the stable liquid formulation comprises 5 mM phosphate
buffer, 5 mM citrate buffer, 100 mM NaCl, 20% sucrose, 25 mg/ml of
the fusion protein, at a pH of about 6.0. Additionally, polysorbate
may be present, for example 0.05-0.15% polysorbate 20. The stable
liquid formulation of the VEGF-specific fusion protein antagonist
of the invention exhibits little or no precipitation after storage
of a 25 mg/ml VEGF formulation for about 6 months at -80.degree. C.
and little or no precipitation after storage for 6 months at
5.degree. C.
[0008] In a second aspect, the invention features a high
concentration stable liquid formulation of a VEGF antagonist
comprising 1-50 mM histidine, 25-150 mM NaCl, 5-30% sucrose, 50-100
mg/ml of the fusion protein, at a pH of about 6-6.5, and either
0.1-0.5% polysorbate or 1-5% PEG. In a more specific embodiment,
the high concentration stable liquid formulation comprises 10 mM
histidine, 50 mM NaCl, 5-20% sucrose, 50-100 mg/ml of the fusion
protein, at a pH of about 6.0-6.5, with either 0.1% polysorbate
(e.g., polysorbate 20) or 3% PEG (e.g., PEG 3350). The high
concentration stable liquid formulation of the VEGF-specific fusion
protein antagonist of the invention exhibits less than about 3%
degradation after 15 months of storage at 5.degree. C. (75 or 100
mg/ml VEGF trap protein) or less than about 1.5% degradation after
24 months (50 mg/ml).
[0009] In a third aspect, the invention features a pre-lyophilized
formulation of a vascular endothelial growth factor (VEGF)-specific
fusion protein antagonist, comprising a (i) fusion protein
comprising a receptor component consisting essentially of an
immunoglobulin-like (Ig) domain 2 of a first VEGF receptor and Ig
domain 3 of a second VEGF receptor, and a multimerizing component,
(ii) a buffer, (iii) an organic co-solvent or bulking agent, and
(iv) one or more lyoprotectants. In various embodiments, the buffer
is histidine, the organic co-solvent or bulking agent is PEG, and
the lyoprotectant(s) is at least one of glycine and sucrose. In one
embodiment, the pre-lyophilized formulation of the invention does
not contain a preservative.
[0010] In one embodiment of the pre-lyophilized formulation of the
invention, the formulation comprises 5-50 mM histidine, 0.1-3.0%
PEG, 0.25-3.0% glycine, 0.5-6.0% sucrose, and 5-75 mg/ml of the
fusion protein, at a pH of about 6.0-6.5. In any embodiment, the
pre-lyophilized formulation may further comprise up to 0.05 mM
citrate and/or 0.003-0.005% polysorbate. The polysorbate present
may be, for example, polysorbate 20.
[0011] In a more specific embodiment, the pre-lyophilized
formulation comprises about 10 mM histidine, about 1.5% PEG 3350,
about 0.75% glycine, about 2.5% sucrose, and about 12.5 to 75 mg/ml
VEGF-specific fusion protein, at a pH of about 6.25. In specific
embodiments, the fusion protein comprises the protein sequence of
SEQ ID NO:4, present as a multimer, e.g., a dimer. In separate
embodiments, the reconstituted formulation is 2 times the
concentration of the pre-lyophilized formulation, e.g., a 20 mg
fusion protein/ml pre-lyophilized formulation is reconstituted to a
final formulation of 60 mg fusion protein/mi. Generally, the
lyophilized formulation is reconstituted with sterile water
suitable for injection. In one embodiment, the reconstitution
liquid may be bacteriostatic water.
[0012] In a preferred embodiment, the pre-lyophilized formulation
consists essentially of about 10 mM histidine, about 1.5% PEG 3350,
about 0.75% glycine, about 2.5% sucrose, and about 50 mg/ml of the
fusion protein having the sequence of SEQ ID NO:4 as a dimer, at a
pH of about 6.25. Citrate (less than or equal to about 0.02 mM)
and/or polysorbate (less than or equal to about 0.0005%) may be
present. Optionally, the pre-lyophilized formulation does not
contain a preservative, a phosphate buffer, and/or more than trace
amounts of NaCl. In one embodiment, the pre-lyophilized formulation
consists of about 10 mM histidine, about 1.5% PEG 3350, about 0.75%
glycine, about 2.5% sucrose, and about 50 mg/ml of the VEGF trap
protein (SEQ ID NO:4), pH 6.3, and upon reconstitution contains 20
mM histidine, 3% PEG, 1.5% glycine, about 5% sucrose, and about 100
mg/ml VEGF trap protein.
[0013] In a fourth aspect, the invention features a method of
producing a lyophilized formulation of a VEGF-specific fusion
protein antagonist, comprising subjecting the pre-lyophilized
formulation of the invention to lyophilization to generate a
lyophilized formulation. The lyophilized formulation may be
lyophilized by any method known in the art for lyophilizing a
liquid.
[0014] In a fifth related aspect, the invention features a method
of producing a reconstituted lyophilized formulation of a
VEGF-specific fusion protein antagonist, comprising reconstituting
the lyophilized formulation of the invention to a reconstituted
formulation. In one embodiment, the reconstituted formulation is
twice the concentration of the pre-lyophilized formulation, e.g.,
the method of the invention comprises: (a) producing a
pre-lyophilized formulation of a VEGF-specific fusion protein
antagonist, (b) subjecting the pre-lyophilized formulation of step
(a) to lyophilization; and (c) reconstituting the lyophilized
formulation of step (b).
[0015] In specific embodiments of the method of producing a
reconstituted lyophilized formulation, a pre-lyophilized solution
is present in a vial as a 25 mg VEGF-specific fusion protein
antagonist per ml solution of pre-lyophilized formulation, which is
lyophilized and reconstituted to an 50 mg/ml solution. In another
embodiment, a 30 mg/ml pre-lyophilized solution is lyophilized and
reconstituted to a 60 mg/ml solution. In another embodiment, a 40
mg/ml pre-lyophilized solution is lyophilized and reconstituted to
a 80 mg/ml solution. In another embodiment, a 12.5 mg/ml
pre-lyophilized solution is lyophilized and reconstituted to a 25
mg/ml solution. In another embodiment, a 50 mg/ml pre-lyophilized
solution is lyophilized and reconstituted to a 100 mg/ml solution.
In another embodiment, a 75 mg/ml pre-lyophilized solution is
lyophilized and reconstituted to a 150 mg/ml solution. Preferably,
the reconstituted lyophilized formulation does not contain a
preservative.
[0016] Other objects and advantages will become apparent from a
review of the ensuing detailed description.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention is not limited to particular methods,
and experimental conditions described, as such methods and
conditions may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting unless
indicated, since the scope of the present invention will be limited
only by the appended claims.
[0018] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus for example,
references to "a method" include one or more methods, and/or steps
of the type described herein and/or which will become apparent to
those persons skilled in the art upon reading this disclosure.
[0019] Unless stated otherwise, all technical and scientific terms
and phrases used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which the
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 now described. All publications mentioned herein are
incorporated herein by reference.
General Description
[0020] Safe handling and administration of formulations comprising
proteins represent significant challenges to pharmaceutical
formulators. Proteins possess unique chemical and physical
properties that present stability problems: a variety of
degradation pathways exist for proteins, implicating both chemical
and physical instability. Chemical instability includes
deamination, aggregation, clipping of the peptide backbone, and
oxidation of methionine residues. Physical instability encompasses
many phenomena, including, for example, aggregation.
[0021] Chemical and physical stability can be promoted by removing
water from the protein. Lyophilization (freeze-drying under
controlled conditions) is commonly used for long-term storage of
proteins. The lyophilized protein is substantially resistant to
degradation, aggregation, oxidation, and other degenerative
processes while in the freeze-dried state. The lyophilized protein
is normally reconstituted with water optionally containing a
bacteriostatic preservative (e.g., benzyl alcohol) prior to
administration.
Definitions
[0022] The term "carrier" includes a diluent, adjuvant, excipient,
or vehicle with which a composition is administered. Carriers can
include sterile liquids, such as, for example, water and oils,
including oils of petroleum, animal, vegetable or synthetic origin,
such as, for example, peanut oil, soybean oil, mineral oil, sesame
oil and the like.
[0023] The term "excipient" includes a non-therapeutic agent added
to a pharmaceutical composition to provide a desired consistency or
stabilizing effect. Suitable pharmaceutical excipients include, for
example, starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica gel, sodium stearate, glycerol monostearate,
talc, sodium chloride, dried skim milk, glycerol, propylene,
glycol, water, ethanol and the like.
[0024] The term "lyophilized" or "freeze-dried" includes a state of
a substance that has been subjected to a drying procedure such as
lyophilization, where at least 50% of moisture has been
removed.
[0025] The phrase "bulking agent" includes a compound that is
pharmaceutically acceptable and that adds bulk to a lyo cake.
Generally, acceptable bulking agents known to the art include, for
example, carbohydrates, including simple sugars such as dextrose,
ribose, fructose and the like, alcohol sugars such as mannitol,
inositol and sorbitol, disaccharides including trehalose, sucrose
and lactose, naturally occurring polymers such as starch, dextrans,
chitosan, hyaluronate, proteins (e.g., gelatin and serum albumin),
glycogen, and synthetic monomers and polymers. In the formulations
of the invention, PEG 3350 is an organic co-solvent which is used
to stabilize the fusion protein when agitated, mixed, or handled,
and as a bulking agent to help produce an acceptable bulk.
[0026] The term "lyoprotectant" includes a substance that may be
added to a freeze-dried or lyophilized formulation to help maintain
protein structure when freeze-dried or lyophilized.
[0027] A "preservative" includes a bacteriostatic, bacteriocidal,
fungistatic or fungicidal compound that is generally added to
formulations to retard or eliminate growth of bacteria or other
contaminating microorganisms in the formulations. Preservatives
include, for example, benzyl alcohol, phenol, benzalkonium
chloride, m-cresol, thimerosol, chlorobutanol, methylparaben,
propylparaben and the like. Other examples of pharmaceutically
acceptable preservatives can be found in the USP.
VEGF Antagonists
[0028] An VEGF antagonist is a compound capable of blocking or
inhibiting the biological action of vascular endothelial growth
factor (VEGF), and includes fusion proteins capable of trapping
VEGF. In a preferred embodiment, the VEGF antagonist is the fusion
protein of SEQ ID NO:2 or 4; more preferably, SEQ ID NO:4. In
specific embodiments, the VEGF antagonist is expressed in a
mammalian cell line such as a CHO cell and may be modified
posttranslationally. In a specific embodiment, the fusion protein
comprises amino acids 27-457 of SEQ ID NO:4 and is glycosylated at
Asn residues 62, 94, 149, 222 and 308.
[0029] The VEGF antagonist of the methods and formulations of the
invention can be prepared by any suitable method known in the art,
or that comes to be known. The VEGF antagonist is preferably
substantially free of protein contaminants at the time it is used
to prepare the pharmaceutically acceptable formulation. By
"substantially free of protein contaminants" is meant, preferably,
that at least 90% of the weight of protein of the VEGF-specific
fusion protein antagonist preparation used for making a formulation
is VEGF fusion protein antagonist protein, more preferably at least
95%, most preferably at least 99%. The fusion protein is preferably
substantially free of aggregates. "Substantially free of
aggregates" means that at least 90% of the weight of fusion protein
is not present in an aggregate at the time the fusion protein is
used to prepare the pharmaceutically effective formulation. The
fusion protein of the methods and formulations of the invention may
contain low or trace amounts of compounds as a results of the
purification process, for example, low or trace amounts of citrate
and/or polysorbate. In one embodiment of the pre-lyophilized
formulation of the invention containing about 50 mg of fusion
protein/ml, citrate may be present at a concentration of about 0.02
mM and/or polysorbate may be present at a concentration of about
0.0005%. If the pre-lyophilized formulation is reconstituted after
lyophilization to half of the original volume (e.g., 100 mg/ml of
fusion protein), the resulting concentrations may be 0.04 mM
citrate and/or 0.001% polysorbate.
Lyophilization and Lyophilized Formulations
[0030] In one aspect of the invention, a pharmaceutically
acceptable formulation comprising a VEGF-specific fusion protein
antagonist is provided, wherein the formulation is a freeze-dried
or lyophilized formulation. Lyophilized formulations can be
reconstituted into solutions, suspensions, emulsions, or any other
suitable form for administration or use. Lyophilized formulations
are typically first prepared as liquids, then frozen and
lyophilized. The total liquid volume before lyophilization can be
less, equal to, or more than, the final reconstituted volume of the
lyophilized formulation. The lyophilization process is well known
to those of ordinary skill in the art, and typically includes
sublimation of water from a frozen formulation under controlled
conditions.
[0031] Lyophilized formulations can be stored at a wide range of
temperatures. Lyophilized formulations may be stored below
25.degree. C., for example, refrigerated at 4.degree. C., or at
room temperature (e.g., approximately 25.degree. C.). Preferably,
lyophilized formulations are stored below about 25.degree. C., more
preferably, at about 4-20.degree. C.; below about 4.degree. C.;
below about -20.degree. C.; about -40.degree. C.; about -70.degree.
C., or about -80.degree. C.
[0032] Lyophilized formulations are typically reconstituted for use
by addition of an aqueous solution to dissolve the lyophilized
formulation. A wide variety of aqueous solutions can be used to
reconstitute a lyophilized formulation. Preferably, lyophilized
formulations are reconstituted using water. Lyophilized
formulations are preferably reconstituted with a solution
consisting essentially of water (e.g., USP WFI, or water for
injection) or bacteriostatic water (e.g., USP WFI with 0.9% benzyl
alcohol). However, solutions comprising buffers and/or excipients
and/or one or more pharmaceutically acceptable carries can also be
used.
[0033] Freeze-dried or lyophilized formulations are typically
prepared from liquids, that is, from solutions, suspensions,
emulsions, and the like. Thus, the liquid that is to undergo
freeze-drying or lyophilization preferably comprises all components
desired in a final reconstituted liquid formulation. As a result,
when reconstituted, the freeze-dried or lyophilized formulation
will render a desired liquid formulation upon reconstitution. A
preferred liquid formulation used to generate a freeze-dried or
lyophilized formulation comprises a VEGF-specific fusion protein
antagonist in a pharmaceutically effective amount, a buffer, a
stabilizer, and a bulking agent. Freeze-dried or lyophilized
formulations preferably comprise histidine, since histidine, in
comparison to phosphate, is more effective at stabilizing the
fusion protein when the fusion protein is lyophilized. Organic
co-solvents, such as PEG 3350, are used to stabilize the fusion
protein when agitated, mixed, or handled. A lyoprotectant is
preferably used in freeze-dried or lyophilized formulations.
Lyoprotectants help to maintain the secondary structure of proteins
when freeze-dried or lyophilized. Two preferred example
lyoprotectants are glycine and sucrose, which are preferably used
together.
Stable Liquid Formulations
[0034] In one aspect, the invention provides a stable
pharmaceutically acceptable formulation comprising a VEGF-specific
fusion protein antagonist, wherein the formulation is a liquid
formulation. Preferably, the liquid formulation comprises a
pharmaceutically effective amount of the fusion protein. The
formulation can also comprise one or more pharmaceutically
acceptable carriers, buffers, bulking agents, stabilizers,
preservatives, and/or excipients. An example of a pharmaceutically
acceptable liquid formulation comprises a VEGF-specific fusion
protein antagonist in a pharmaceutically effective amount, a
buffer, a co-solvent, and one or more stabilizers.
[0035] A preferred liquid formulation comprises phosphate buffer,
an organic co-solvent, and one or more thermal stabilizers to
minimize formation of aggregates and low molecular weight products
when stored, and about 10 mg/ml to about 50 mg/ml fusion protein,
wherein the formulation is from about pH 6.0-6.5. A preferred
liquid formulation comprises about 5 mM phosphate buffer, about 5
mM citrate, about 100 mM NaCl, about 25% sucrose, and about 1050
mg/ml fusion protein, wherein the formulation is at a pH of about
6.0; optionally polysorbate may be present (e.g., 0.1% polysorbate
20). Although either NaCl or sucrose can be used as a stabilizer, a
combination of NaCl and sucrose has been established to stabilize
the fusion protein more effectively than either individual
stabilizer alone.
[0036] Stability is determined in a number of ways at specified
time points, including determination of pH, visual inspection of
color and appearance, determination of total protein content by
methods known in the art, e.g., UV spectroscopy, SDS-PAGE,
size-exclusion HPLC, bioassay determination of activity,
isoelectric focusing, and isoaspartate quantification. In one
example of a bioassay useful for determining VEGF antagonist
activity, a BAF/3 VEGFR1/EPOR cell line is used to determine
VEGF165 binding by the VEGF-specific fusion protein antagonist of
the invention.
[0037] Formulations, whether liquid or freeze-dried and
lyophilized, can be stored in an oxygen-deprived environment.
Oxygen-deprived environments can be generated by storing the
formulations under an inert gas such as, for example, argon,
nitrogen, or helium.
EXAMPLES
[0038] Before the present methods are described, it is to be
understood that this invention is not limited to particular
methods, and experimental conditions described, as such methods and
conditions may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only to the appended
claims.
[0039] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus for example, a
reference to "a method" includes one or more methods, and/or steps
of the type described herein and/or which will become apparent to
those persons skilled in the art upon reading this disclosure and
so forth.
[0040] 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 now
described.
Example 1. Stability of a 50 mg/ml Liquid Formulation of VEGF
Trap
[0041] A liquid formulation containing 10 mM phosphate, 50 mM NaCl,
0.1% polysorbate 20, 20% sucrose, and 50 mg/ml VEGF trap (SEQ ID
NO:4), pH 6.25, was stored at 5.degree. C. and samples tested at 3,
6, 9, 12, 18 and 24 months. Stability was determined by SE-HPLC.
The results, shown in Table 1, show that 98.6% and 98.3% of VEGF
trap protein remained intact (non-degraded) at 12 and 24 months,
respectively. Turbidity was measured at OD.sub.405 nm; and percent
recovered protein by size exclusion HPLC.
TABLE-US-00001 TABLE 1 Stability of 50 mg/ml VEGF Trap Protein When
Stored at 5.degree. C. (VGFT-55065) % VEGF % VEGF Visual Trap Trap
Native Months Appearance Turbidity pH Recovered Configuration 0
Pass 0.00 6.2 100 99.0 3 Pass 0.00 6.2 102 98.8 6 Pass 0.01 6.2 103
98.7 9 Pass 0.01 6.3 102 98.2 12 Pass 0.01 6.3 106 98.6 18 Pass
0.00 6.3 103 98.4 24 Pass 0.00 6.2 93 98.3
[0042] A liquid formulation containing 10 mM phosphate, 50 mM NaCl,
3% PEG 3350, 20% sucrose, and 50 mg/ml VEGF trap (SEQ ID NO:4), pH
6.25, was stored at 5.degree. C. and samples tested at 3, 6, 9, 12,
18 and 24 months. Stability results are shown in Table 2.
TABLE-US-00002 TABLE 2 Stability of 50 mg/ml VEGF Trap Protein When
Stored at 5.degree. C. (VGFT-55065) % VEGF % VEGF Visual Trap Trap
Native Months Appearance Turbidity pH Recovered Configuration 0
Pass 0.00 6.2 100 99.0 3 Pass 0.00 6.2 100 98.8 6 Pass 0.01 6.3 103
98.5 9 Pass 0.00 6.3 103 98.3 12 Pass 0.01 6.3 110 98.3 18 Pass
0.00 6.3 113 98.0 24 Pass 0.01 6.2 90 97.8
Example 2. Stability of a 75 mg/ml Liquid Formulation of VEGF
Trap
[0043] A liquid formulation containing 10 mM phosphate, 50 mM NaCl,
0.1% polysorbate 20, 20% sucrose, and 75 mg/ml VEGF trap (SEQ ID
NO:4), pH 6.25, was stored at 5.degree. C. and samples tested at 0,
1, 2.3, 3, 9, 12 and 15 months. Stability results are shown in
Table 3.
TABLE-US-00003 TABLE 3 Stability of 75 mg/ml VEGF Trap Protein When
Stored at 5.degree. C. (VGFT-SS101) % VEGF % VEGF Visual Trap Trap
Native Months Appearance Turbidity pH Recovered Configuration 0
Pass 0.00 6.2 100 97.1 1 Pass 0.00 6.2 96 97.0 2.3 Pass 0.00 6.2 98
96.7 3 Pass 0.00 6.2 97 96.1 9 Pass -0.01 6.0 101 96.0 12 Pass 0.00
6.3 110 94.5 15 Pass 0.00 6.3 92 95.6
[0044] A liquid formulation containing 10 mM phosphate, 50 mM NaCl,
3% PEG 3350, 20% sucrose, and 75 mg/ml VEGF trap (SEQ ID NO:4), pH
6.25, was stored at 5.degree. C. and samples tested at 0, 1, 2.3,
3, 9, 12 and 15 months. Stability results are shown in Table 4.
TABLE-US-00004 TABLE 4 Stability of 75 mg/ml VEGF Trap Protein When
Stored at 5.degree. C. (VGFT-SS101) % VEGF % VEGF Visual Trap Trap
Native Months Appearance Turbidity pH Recovered Configuration 0
Pass 0.00 6.2 100 96.8 1 Pass 0.00 6.2 99 96.7 2.3 Pass 0.00 6.2 97
96.3 3 Pass 0.00 6.2 89 95.6 9 Pass -0.01 6.2 98 95.4 12 Pass -0.01
6.3 112 94.1 15 Pass 0.00 6.3 98 94.8
Example 3. Stability of a 100 mg/ml Liquid Formulation of VEGF
Trap
[0045] A liquid formulation containing 10 mM phosphate, 50 mM NaCl,
0.1% polysorbate 20, 20% sucrose, and 100 mg/ml VEGF trap (SEQ ID
NO:4), pH 6.25, was stored at 5.degree. C. and samples tested at 0,
1, 2.3, 3, 9, 12 and 15 months. Stability results are shown in
Table 5.
TABLE-US-00005 TABLE 5 Stability of 100 mg/ml VEGF Trap Protein
Stored at 5.degree. C. (VGFT-SS101) % VEGF % VEGF Visual Trap Trap
Native Months Appearance Turbidity pH Recovered Configuration 0
Pass 0.00 6.3 100 96.7 1 Pass 0.00 6.2 92 96.6 2.3 Pass 0.00 6.2 92
96.2 6 Pass 0.00 6.2 99 95.5 9 Pass -0.01 6.2 92 95.5 12 Pass -0.01
6.2 110 93.9 15 Pass 0.00 6.3 108 94.8
[0046] A liquid formulation containing 10 mM phosphate, 50 mM NaCl,
3% PEG 3350, 20% sucrose, and 100 mg/ml VEGF trap (SEQ ID NO:4), pH
6.25, was stored at 5.degree. C. and samples tested at 0, 1, 2.3,
3, 9, 12 and 15 months. Stability results are shown in Table 6.
TABLE-US-00006 TABLE 6 Stability of 100 mg/ml VEGF Trap Protein
Stored at 5.degree. C. (VGFT-SS101) % VEGF % VEGF Visual Trap Trap
Native Months Appearance Turbidity pH Recovered Configuration 0
Pass 0.00 6.3 100 96.5 1 Pass 0.01 6.2 94 96.2 2.3 Pass 0.01 6.2 93
95.7 6 Pass 0.01 6.2 102 94.6 9 Pass 0.00 6.2 95 94.6 12 Pass 0.00
6.3 96 92.8 15 Pass 0.01 6.3 102 93.9
Example 4. Further Embodiments of Stable VEGF Trap Formulations
[0047] In one embodiment, the invention provides a stable liquid
VEGF-binding fusion protein (VEGF trap) formulations comprising 5
mM phosphate, 5 mM citrate, 100 mM NaCl, 0.1% Polysorbate 20, 20%
sucrose, 25 mg/ml VEGF trap protein, pH 6.0. This formulation can
either be delivered subcutaneously or diluted and delivered by
intravenous infusion. Due to the high osmolality of this
formulation, it is diluted 3-fold to achieve an iso-osmolar
solution for intravenous administration. Stability studies showed
less than about 1% degradation was detected after 3 years of
storage at 2-8.degree. C.
[0048] In one embodiment, the invention features a lyophilized
formulation which is preferably concentrated two-fold from the
pre-lyophilized to the post-lyophilized formulation, e.g., 50 to
100 mg/ml; 75 to 150 mg/ml, or 100 to 200 mg/ml VEGF trap protein.
In one specific embodiment, the pre-lyophilized formulation
comprises 10 mM histidine, 1.5% PEG 3350, 0.75% glycine, 2.5%
sucrose, 50 mg/ml VEGF trap protein, pH 6.3, and is reconstituted
to a formulation comprising 20 mM histidine, 3% PEG 3350, 1.5%
glycine, 5% sucrose, 100 mg/ml VEGF trap protein, pH 6.3. Stability
studied showed no degradation of the VEGF trap was detected after 6
months of storage at 2-8.degree. C.
[0049] In one embodiment of a liquid formulation, the formulation
comprises 10 mM histidine, 50 mM NaCl, 5-20% sucrose, 50-100 mg/ml
VEGF trap, and one of 0.1% polysorbate 20 or TY.degree. PEG 3350.
One advantage of this liquid formulation is that it provides a
higher concentration of VEGF trap without requiring the manufacture
of a lyophilized product. Thus, this formulation provides ease for
subcutaneous delivery, for example, by allowing provision of a
liquid pre-filled syringe at a concentration higher than that
delivered by IV infusion. Also, this formulation could
advantageously be used to provide lower infusion volumes and
shorter infusion times. The amount of degradation determined by
SE-HPLC following incubation at 5.degree. C. for up to 15 or 24
months is summarized in Table 7.
TABLE-US-00007 TABLE 7 Stability of Liquid Formulation with 50-100
mg/ml VEGF Trap (VGFT-SS101) Incubation VEGF Trap % % PEG %
(months) (mg/ml) Polysorbate 20 3350 Degradation 24 50 0.1 -- 0.7
24 50 -- 3 1.3 15 75 0.1 -- 1.5 15 75 -- 3 2.0 15 100 0.1 -- 1.9 15
100 -- 3 2.6
Example 5. Stability and Activity of Lyophilized and Liquid
[0050] The stability of a reconstituted lyophilized formulation was
determined over a 6 month period. The pre-lyophilized formulation
contained 10 mM histidine, 1.5% PEG 3350, 2.5% sucrose, 0.75%
glycine and 50 mg/ml VEGF trap protein. After lyophilization, the
reconstituted formulation contained 20 mM histidine, 3% PEG 3350,
5% sucrose, 1.5% glycine, and 100 mg/ml VEGF trap protein (SEQ ID
NO:4). The results are shown in Table 8. Activity was determined in
a cell based bioassay which directly measures the ability of the
VEGF trap to inhibit the biological effects of human VEGF on a
mouse Baf/3 VEGFR1/EpoR cell line. Therefore, this bioassay
directly measures the biological activity of the protein. The
results are expresses as percent relative potency (test sample
IC.sub.50/reference VEGF IC.sub.5O standard.times.100). The binding
affinity of VEGF to the VEGF trap is measured using a sensitive
ELISA that specifically measures free VEGF in equilibrated mixtures
containing VEGF and various concentrations of the VEGF trap.
Results are expressed as percent relative binding (IC.sub.50 of
test sample/ICH, of reference.times.100). Measured pH ranged
between 6.3-6.5. All solutions where visually clear. The
concentration of VEGF trap recovered was determined with a UV
spectrophotometer as mg/ml at A280 nm. The percent VEGF trap
recovered in the native configuration (main peak purity) was
determined with SE-HPLC.
TABLE-US-00008 TABLE 8 Stability of VEGF Trap Lyophilized
Formulation Stored at 5.degree. C. (VGT-R5475) Binding % % Native
Months Bioassay Assay Recovered Configuration 0 120 126 97.9 98.7 1
117 74 97.9 98.6 1 + 24 hr 126 72 99.0 98.5 1 + 4 hr 94 81 101.5
98.2 3 101 98 98.1 98.6 3 + 24 hr 65 94 98.1 98.2 6 + 4 hr 96.9
98.7 6 + 24 hr 98.8 98.6
[0051] A formulation containing about 5 mM phosphate, 5 mM citrate,
100 mM NaCl, 0.1% polysorbate 20, 20% sucrose, and 25 mg/ml VEGF
trap protein was tested for stability and activity over 36 months
when stored at 5.degree. C. The results are shown in Table 9. All
samples were clear and colorless as determined by visual
inspection. pH ranged from 6.0-6.1. *Binding assay results for two
measurements (1 and 2 months) are expressed directly and not as a
percent of the standard.
TABLE-US-00009 TABLE 9 Stability and Activity of Liquid Formulation
(VGT-FS405) % Native Binding Protein Content Months Configuration
Bioassay Assay mg/ml 0 99.7 106 72 25.0 1 99.9 119 4.4 .rho.M* 25.2
2 99.6 102 5.4 .rho.M* 25.1 3 99.6 97 88 25.1 6 99.6 101 106 25.0 9
99.4 89 126 25.4 12 99.5 85 95 25.2 18 99.4 99 81 25.5 24 99.3 75
95 25.6 36 98.8 109 79 25.6
Sequence CWU 1
1
411453DNAArtificial sequenceSynthetic 1aagcttgggc tgcaggtcga
tcgactctag aggatcgatc cccgggcgag ctcgaattcg 60caaccaccat ggtcagctac
tgggacaccg gggtcctgct gtgcgcgctg ctcagctgtc 120tgcttctcac
aggatctagt tccggaggta gacctttcgt agagatgtac agtgaaatcc
180ccgaaattat acacatgact gaaggaaggg agctcgtcat tccctgccgg
gttacgtcac 240ctaacatcac tgttacttta aaaaagtttc cacttgacac
tttgatccct gatggaaaac 300gcataatctg ggacagtaga aagggcttca
tcatatcaaa tgcaacgtac aaagaaatag 360ggcttctgac ctgtgaagca
acagtcaatg ggcatttgta taagacaaac tatctcacac 420atcgacaaac
caatacaatc atagatgtgg ttctgagtcc gtctcatgga attgaactat
480ctgttggaga aaagcttgtc ttaaattgta cagcaagaac tgaactaaat
gtggggattg 540acttcaactg ggaataccct tcttcgaagc atcagcataa
gaaacttgta aaccgagacc 600taaaaaccca gtctgggagt gagatgaaga
aatttttgag caccttaact atagatggtg 660taacccggag tgaccaagga
ttgtacacct gtgcagcatc cagtgggctg atgaccaaga 720agaacagcac
atttgtcagg gtccatgaaa agggcccggg cgacaaaact cacacatgcc
780caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc
cccccaaaac 840ccaaggacac cctcatgatc tcccggaccc ctgaggtcac
atgcgtggtg gtggacgtga 900gccacgaaga ccctgaggtc aagttcaact
ggtacgtgga cggcgtggag gtgcataatg 960ccaagacaaa gccgcgggag
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca 1020ccgtcctgca
ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag
1080ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc
cgagaaccac 1140aggtgtacac cctgccccca tcccgggatg agctgaccaa
gaaccaggtc agcctgacct 1200gcctggtcaa aggcttctat cccagcgaca
tcgccgtgga gtgggagagc aatgggcagc 1260cggagaacaa ctacaagacc
acgcctcccg tgctggactc cgacggctcc ttcttcctct 1320atagcaagct
caccgtggac aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg
1380tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg
tctccgggta 1440aatgagcggc cgc 14532458PRTArtificial
sequencesynthetic 2Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys
Ala Leu Leu Ser1 5 10 15Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Gly
Arg Pro Phe Val Glu 20 25 30Met Tyr Ser Glu Ile Pro Glu Ile Ile His
Met Thr Glu Gly Arg Glu 35 40 45Leu Val Ile Pro Cys Arg Val Thr Ser
Pro Asn Ile Thr Val Thr Leu 50 55 60Lys Lys Phe Pro Leu Asp Thr Leu
Ile Pro Asp Gly Lys Arg Ile Ile65 70 75 80Trp Asp Ser Arg Lys Gly
Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu 85 90 95Ile Gly Leu Leu Thr
Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys 100 105 110Thr Asn Tyr
Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Val 115 120 125Leu
Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val 130 135
140Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe
Asn145 150 155 160Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys
Leu Val Asn Arg 165 170 175Asp Leu Lys Thr Gln Ser Gly Ser Glu Met
Lys Lys Phe Leu Ser Thr 180 185 190Leu Thr Ile Asp Gly Val Thr Arg
Ser Asp Gln Gly Leu Tyr Thr Cys 195 200 205Ala Ala Ser Ser Gly Leu
Met Thr Lys Lys Asn Ser Thr Phe Val Arg 210 215 220Val His Glu Lys
Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro Cys225 230 235 240Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 245 250
255Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
260 265 270Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp 275 280 285Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 290 295 300Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu305 310 315 320His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 325 330 335Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 340 345 350Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 355 360 365Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 370 375
380Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn385 390 395 400Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 405 410 415Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 420 425 430Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr 435 440 445Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 450 45531377DNAArtificial sequencesynthetic
3atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc
60acaggatcta gttccggaag tgataccggt agacctttcg tagagatgta cagtgaaatc
120cccgaaatta tacacatgac tgaaggaagg gagctcgtca ttccctgccg
ggttacgtca 180cctaacatca ctgttacttt aaaaaagttt ccacttgaca
ctttgatccc tgatggaaaa 240cgcataatct gggacagtag aaagggcttc
atcatatcaa atgcaacgta caaagaaata 300gggcttctga cctgtgaagc
aacagtcaat gggcatttgt ataagacaaa ctatctcaca 360catcgacaaa
ccaatacaat catagatgtg gttctgagtc cgtctcatgg aattgaacta
420tctgttggag aaaagcttgt cttaaattgt acagcaagaa ctgaactaaa
tgtggggatt 480gacttcaact gggaataccc ttcttcgaag catcagcata
agaaacttgt aaaccgagac 540ctaaaaaccc agtctgggag tgagatgaag
aaatttttga gcaccttaac tatagatggt 600gtaacccgga gtgaccaagg
attgtacacc tgtgcagcat ccagtgggct gatgaccaag 660aagaacagca
catttgtcag ggtccatgaa aaggacaaaa ctcacacatg cccaccgtgc
720ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa
acccaaggac 780accctcatga tctcccggac ccctgaggtc acatgcgtgg
tggtggacgt gagccacgaa 840gaccctgagg tcaagttcaa ctggtacgtg
gacggcgtgg aggtgcataa tgccaagaca 900aagccgcggg aggagcagta
caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 960caccaggact
ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca
1020gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc
acaggtgtac 1080accctgcccc catcccggga tgagctgacc aagaaccagg
tcagcctgac ctgcctggtc 1140aaaggcttct atcccagcga catcgccgtg
gagtgggaga gcaatgggca gccggagaac 1200aactacaaga ccacgcctcc
cgtgctggac tccgacggct ccttcttcct ctacagcaag 1260ctcaccgtgg
acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat
1320gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga
13774458PRTArtificial sequencesynthetic 4Met Val Ser Tyr Trp Asp
Thr Gly Val Leu Leu Cys Ala Leu Leu Ser1 5 10 15Cys Leu Leu Leu Thr
Gly Ser Ser Ser Gly Ser Asp Thr Gly Arg Pro 20 25 30Phe Val Glu Met
Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu 35 40 45Gly Arg Glu
Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr 50 55 60Val Thr
Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys65 70 75
80Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr
85 90 95Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly
His 100 105 110Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn
Thr Ile Ile 115 120 125Asp Val Val Leu Ser Pro Ser His Gly Ile Glu
Leu Ser Val Gly Glu 130 135 140Lys Leu Val Leu Asn Cys Thr Ala Arg
Thr Glu Leu Asn Val Gly Ile145 150 155 160Asp Phe Asn Trp Glu Tyr
Pro Ser Ser Lys His Gln His Lys Lys Leu 165 170 175Val Asn Arg Asp
Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe 180 185 190Leu Ser
Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu 195 200
205Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr
210 215 220Phe Val Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro
Pro Cys225 230 235 240Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro 245 250 255Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 260 265 270Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 275 280 285Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu305 310 315
320His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
325 330 335Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 340 345 350Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu 355 360 365Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr 370 375 380Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn385 390 395 400Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 405 410 415Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 420 425 430Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440
445Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455
* * * * *