U.S. patent application number 11/387256 was filed with the patent office on 2006-09-28 for vegf antagonist formulations.
Invention is credited to Daniel Dix, Kelly Frye, Susan Kautz.
Application Number | 20060217311 11/387256 |
Document ID | / |
Family ID | 36678320 |
Filed Date | 2006-09-28 |
United States Patent
Application |
20060217311 |
Kind Code |
A1 |
Dix; Daniel ; et
al. |
September 28, 2006 |
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; (LaGrangeville,
NY) ; Frye; Kelly; (Pomona, NY) ; Kautz;
Susan; (Albany, NY) |
Correspondence
Address: |
REGENERON PHARMACEUTICALS, INC
777 OLD SAW MILL RIVER ROAD
TARRYTOWN
NY
10591
US
|
Family ID: |
36678320 |
Appl. No.: |
11/387256 |
Filed: |
March 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60665125 |
Mar 25, 2005 |
|
|
|
Current U.S.
Class: |
514/8.1 ;
514/400; 514/53 |
Current CPC
Class: |
A61K 39/39591 20130101;
A61K 38/16 20130101; A61P 35/00 20180101; A61K 31/7012 20130101;
A61K 9/08 20130101; A61K 9/19 20130101; C07K 14/71 20130101; A61K
9/0019 20130101; A61K 47/10 20130101; A61K 47/22 20130101; A61P
9/00 20180101; A61K 47/12 20130101; A61K 47/02 20130101; C07K
2318/20 20130101; C07K 2319/30 20130101; A61K 47/26 20130101; A61P
7/00 20180101; C07K 16/22 20130101; A61K 38/179 20130101; A61K
47/183 20130101; A61P 43/00 20180101; A61K 31/4172 20130101 |
Class at
Publication: |
514/012 ;
514/053; 514/400 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 31/7012 20060101 A61K031/7012; A61K 31/4172
20060101 A61K031/4172 |
Claims
1. A stable liquid formulation of a vascular endothelial growth
factor (VEGF)-specific fusion protein antagonist, comprising a
fusion protein having a receptor component consisting 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.
2. The stable liquid formulation of claim 1, wherein the first VEGF
receptor is Flt1 and the second VEGF receptor is Flk1 or Flt4.
3. The stable liquid formulation of claim 2, wherein the fusion
protein has the amino acid sequence of SEQ ID NO:4.
4. The stable liquid formulation of claim 1, wherein the buffer is
at least one of phosphate buffer and citrate buffer.
5. The stable liquid formulation of claim 1, wherein the thermal
stabilizer are at least one of NaCl and sucrose.
6 The stable liquid formulation of claim 1, comprising 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,
optionally further comprising 0.05-0.10% polysorbate.
7. The stable liquid formulation of claim 6, comprising 5 mM
phosphate buffer, 5 mM citrate buffer, 100 mM NaCl, 25% sucrose, 25
mg/ml of the fusion protein of SEQ ID NO:4, at a pH of about
6.0.
8. A pre-lyophilized formulation, comprising (i) a vascular
endothelial growth factor (VEGF)-specific fusion protein
antagonist, (ii) a buffer, (iii) an organic co-solvent or bulking
agent, and (iv) one or more lyoprotectants, wherein the fusion
protein has the amino acid sequence of SEQ ID NO:4.
9. The pre-lyophilized formulation of claim 8, wherein the buffer
is histidine.
10. The pre-lyophilized formulation of claim 8, wherein the organic
co-solvent or bulking agent is PEG.
11. The pre-lyophilized formulation of claim 8, wherein the
lyoprotectant(s) is at least one of glycine and sucrose.
12. The pre-lyophilized formulation of claim 8, comprising 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.
13. The pre-lyophilized formulation of claim 12, comprising 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.
14. The pre-lyophilized formulation of claim 13, wherein the fusion
protein is 50 mg/ml.
15. A liquid stable formulation of a VEGF antagonist, comprising
1-50 mM histidine, 25-150 mM NaCl, 5-30% sucrose, and 50-100 mg/ml
of the fusion protein, at a pH of about 6.0-6.5, optionally
comprising 0.01-0.5% polysorbate, and optionally further comprising
0.1-5% PEG.
16. The liquid stable formulation of claim 15, comprising about 10
mM histidine, about 50 mM NaCl, 5-20% sucrose, and 50-100 mg/ml of
the fusion protein, at a pH of about 6.0-6.5, optionally comprising
0.1% polysorbate 20, and optionally further comprising 3% PEG
3350.
17. A method of producing a lyophilized formulation of a
VEGF-specific fusion protein antagonist, comprising subjecting the
pre-lyophilized formulation of claim 14 to lyophilization to
generate a lyophilized formulation.
18. A method of producing a reconstituted lyophilized formulation
of a VEGF-specific fusion protein antagonist, comprising
reconstituting the lyophilized formulation claim 17 with liquid,
wherein a reconstituted lyophilized formulation is generated.
19. The method of claim 18, wherein the liquid is sterile water or
bacteriostatic water.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 USC .sctn.
119(e) of U.S. Provisional 60/665,125 filed 25 Mar. 2005, which
application is herein specifically incorporated by reference in its
entirety.
BACKGROUND OF INVENTION
[0002] 1. Field of the Invention
[0003] 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.
[0004] 2. Statement of Related Art
[0005] 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 applications are specifically incorporated by
reference in their entirety.
[0006] 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
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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).
[0011] 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.
[0012] 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.
[0013] 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/ml. Generally, the
lyophilized formulation is reconstituted with sterile water
suitable for injection. In one embodiment, the reconstitution
liquid may be bacteriostatic water.
[0014] 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.
[0015] 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.
[0016] 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).
[0017] 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.
[0018] Other objects and advantages will become apparent from a
review of the ensuing detailed description.
DETAILED DESCRIPTION OF THE INVENTION
[0019] 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.
[0020] 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.
[0021] 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
[0022] 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.
[0023] 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
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] A "preservative" includes a bacteriostatic, bacteriocidal,
fungistatic or fungicides 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
[0030] 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
post-translationally. 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.
[0031] 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
[0032] 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.
[0033] 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.
[0034] 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 pharmacetically acceptable carries can also be
used.
[0035] 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
cosolvents, 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
[0036] 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.
[0037] 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 10-50
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.
[0038] 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.
[0039] 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
[0040] 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.
[0041] 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.
[0042] 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.
All publications mentioned herein are incorporated herein by
reference in their entirety.
Example 1
Stability of a 50 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 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 remain undegraded 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-SS065)
% VEGF Trap Visual % VEGF 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
[0044] 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-SS065) % VEGF Trap Visual % VEGF 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
[0045] 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 Trap Visual
% VEGF 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
[0046] 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 Trap Visual % VEGF 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
[0047] 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 Trap Visual %
VEGF 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
[0048] 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 Trap Visual % VEGF 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
[0049] 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.
[0050] 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 comprisisng 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.
[0051] 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 3% 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 concentraion 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 % (months) (mg/ml) % Polysorbate 20 % PEG 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
[0052] 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 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.50 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/IC.sub.50 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 A.sub.280 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-RS475) %
Native Months Bioassay Binding 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
[0053] 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 Protein Content
Months Configuration Bioassay Binding Assay mg/ml 0 99.7 106 72
25.0 1 99.9 119 4.4 pM* 25.2 2 99.6 102 5.4 pM* 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
[0054]
Sequence CWU 1
1
4 1 1453 DNA Artificial Sequence Synthetic 1 aagcttgggc tgcaggtcga
tcgactctag aggatcgatc cccgggcgag ctcgaattcg 60 caaccaccat
ggtcagctac tgggacaccg gggtcctgct gtgcgcgctg ctcagctgtc 120
tgcttctcac aggatctagt tccggaggta gacctttcgt agagatgtac agtgaaatcc
180 ccgaaattat acacatgact gaaggaaggg agctcgtcat tccctgccgg
gttacgtcac 240 ctaacatcac tgttacttta aaaaagtttc cacttgacac
tttgatccct gatggaaaac 300 gcataatctg ggacagtaga aagggcttca
tcatatcaaa tgcaacgtac aaagaaatag 360 ggcttctgac ctgtgaagca
acagtcaatg ggcatttgta taagacaaac tatctcacac 420 atcgacaaac
caatacaatc atagatgtgg ttctgagtcc gtctcatgga attgaactat 480
ctgttggaga aaagcttgtc ttaaattgta cagcaagaac tgaactaaat gtggggattg
540 acttcaactg ggaataccct tcttcgaagc atcagcataa gaaacttgta
aaccgagacc 600 taaaaaccca gtctgggagt gagatgaaga aatttttgag
caccttaact atagatggtg 660 taacccggag tgaccaagga ttgtacacct
gtgcagcatc cagtgggctg atgaccaaga 720 agaacagcac atttgtcagg
gtccatgaaa agggcccggg cgacaaaact cacacatgcc 780 caccgtgccc
agcacctgaa ctcctggggg gaccgtcagt cttcctcttc cccccaaaac 840
ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga
900 gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag
gtgcataatg 960 ccaagacaaa gccgcgggag gagcagtaca acagcacgta
ccgtgtggtc agcgtcctca 1020 ccgtcctgca ccaggactgg ctgaatggca
aggagtacaa gtgcaaggtc tccaacaaag 1080 ccctcccagc ccccatcgag
aaaaccatct ccaaagccaa agggcagccc cgagaaccac 1140 aggtgtacac
cctgccccca tcccgggatg agctgaccaa gaaccaggtc agcctgacct 1200
gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc aatgggcagc
1260 cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc
ttcttcctct 1320 atagcaagct caccgtggac aagagcaggt ggcagcaggg
gaacgtcttc tcatgctccg 1380 tgatgcatga ggctctgcac aaccactaca
cgcagaagag cctctccctg tctccgggta 1440 aatgagcggc cgc 1453 2 458 PRT
Artificial Sequence Synthetic 2 Met Val Ser Tyr Trp Asp Thr Gly Val
Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser
Ser Ser Gly Gly Arg Pro Phe Val Glu 20 25 30 Met Tyr Ser Glu Ile
Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu 35 40 45 Leu Val Ile
Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu 50 55 60 Lys
Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile 65 70
75 80 Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys
Glu 85 90 95 Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His
Leu Tyr Lys 100 105 110 Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr
Ile Ile Asp Val Val 115 120 125 Leu Ser Pro Ser His Gly Ile Glu Leu
Ser Val Gly Glu Lys Leu Val 130 135 140 Leu Asn Cys Thr Ala Arg Thr
Glu Leu Asn Val Gly Ile Asp Phe Asn 145 150 155 160 Trp Glu Tyr Pro
Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg 165 170 175 Asp Leu
Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr 180 185 190
Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr Cys 195
200 205 Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val
Arg 210 215 220 Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys
Pro Pro Cys 225 230 235 240 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 260 265 270 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 275 280 285 Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300 Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 305 310 315
320 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
325 330 335 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 340 345 350 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu 355 360 365 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr 370 375 380 Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn 385 390 395 400 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 405 410 415 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 420 425 430 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440
445 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 3 1377 DNA
Artificial Sequence Synthetic 3 atggtcagct actgggacac cggggtcctg
ctgtgcgcgc tgctcagctg tctgcttctc 60 acaggatcta gttccggaag
tgataccggt agacctttcg tagagatgta cagtgaaatc 120 cccgaaatta
tacacatgac tgaaggaagg gagctcgtca ttccctgccg ggttacgtca 180
cctaacatca ctgttacttt aaaaaagttt ccacttgaca ctttgatccc tgatggaaaa
240 cgcataatct gggacagtag aaagggcttc atcatatcaa atgcaacgta
caaagaaata 300 gggcttctga cctgtgaagc aacagtcaat gggcatttgt
ataagacaaa ctatctcaca 360 catcgacaaa ccaatacaat catagatgtg
gttctgagtc cgtctcatgg aattgaacta 420 tctgttggag aaaagcttgt
cttaaattgt acagcaagaa ctgaactaaa tgtggggatt 480 gacttcaact
gggaataccc ttcttcgaag catcagcata agaaacttgt aaaccgagac 540
ctaaaaaccc agtctgggag tgagatgaag aaatttttga gcaccttaac tatagatggt
600 gtaacccgga gtgaccaagg attgtacacc tgtgcagcat ccagtgggct
gatgaccaag 660 aagaacagca catttgtcag ggtccatgaa aaggacaaaa
ctcacacatg cccaccgtgc 720 ccagcacctg aactcctggg gggaccgtca
gtcttcctct tccccccaaa acccaaggac 780 accctcatga tctcccggac
ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 840 gaccctgagg
tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 900
aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg
960 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa
agccctccca 1020 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc
cccgagaacc acaggtgtac 1080 accctgcccc catcccggga tgagctgacc
aagaaccagg tcagcctgac ctgcctggtc 1140 aaaggcttct atcccagcga
catcgccgtg gagtgggaga gcaatgggca gccggagaac 1200 aactacaaga
ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1260
ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat
1320 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga
1377 4 458 PRT Artificial Sequence Synthetic 4 Met Val Ser Tyr Trp
Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu
Leu Thr Gly Ser Ser Ser Gly Ser Asp Thr Gly Arg Pro 20 25 30 Phe
Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu 35 40
45 Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr
50 55 60 Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp
Gly Lys 65 70 75 80 Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile
Ser Asn Ala Thr 85 90 95 Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
Ala Thr Val Asn Gly His 100 105 110 Leu Tyr Lys Thr Asn Tyr Leu Thr
His Arg Gln Thr Asn Thr Ile Ile 115 120 125 Asp Val Val Leu Ser Pro
Ser His Gly Ile Glu Leu Ser Val Gly Glu 130 135 140 Lys Leu Val Leu
Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile 145 150 155 160 Asp
Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu 165 170
175 Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe
180 185 190 Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln
Gly Leu 195 200 205 Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys
Lys Asn Ser Thr 210 215 220 Phe Val Arg Val His Glu Lys Asp Lys Thr
His Thr Cys Pro Pro Cys 225 230 235 240 Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 260 265 270 Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 275 280 285 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 290 295
300 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
305 310 315 320 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn 325 330 335 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 340 345 350 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu 355 360 365 Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 370 375 380 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 385 390 395 400 Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 405 410 415
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 420
425 430 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 435 440 445 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455
* * * * *