U.S. patent application number 16/034648 was filed with the patent office on 2018-11-01 for fc fusion high affinity ige receptor alpha-chain.
This patent application is currently assigned to Kissei Pharmaceutical Co., Ltd.. The applicant listed for this patent is Kissei Pharmaceutical Co., Ltd.. Invention is credited to Yoichi Inada, Takashi Sakamoto, Kazumasa Yokoyama.
Application Number | 20180312566 16/034648 |
Document ID | / |
Family ID | 56692248 |
Filed Date | 2018-11-01 |
United States Patent
Application |
20180312566 |
Kind Code |
A1 |
Sakamoto; Takashi ; et
al. |
November 1, 2018 |
Fc FUSION HIGH AFFINITY IgE RECEPTOR ALPHA-CHAIN
Abstract
Provided is an Fc fusion high affinity IgE receptor
.alpha.-chain having excellent stability at low pH. An Fc fusion
protein comprising: (i) a high affinity IgE receptor .alpha.-chain;
and (ii) an Fc region of IgG1, wherein a linker fragment region
between the (i) and the (ii) is the amino acid sequence shown in
SEQ ID NO: 2.
Inventors: |
Sakamoto; Takashi;
(Azumino-shi, JP) ; Inada; Yoichi; (Azumino-shi,
JP) ; Yokoyama; Kazumasa; (Azumino-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kissei Pharmaceutical Co., Ltd. |
Matsumoto-shi |
|
JP |
|
|
Assignee: |
Kissei Pharmaceutical Co.,
Ltd.
Matsumoto-shi
JP
|
Family ID: |
56692248 |
Appl. No.: |
16/034648 |
Filed: |
July 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15552065 |
Aug 18, 2017 |
10077297 |
|
|
PCT/JP2016/054854 |
Feb 19, 2016 |
|
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16034648 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 19/00 20130101;
C12N 15/09 20130101; A61K 38/16 20130101; C07K 14/70535 20130101;
C07K 16/18 20130101; C07K 14/705 20130101; C07K 2319/30
20130101 |
International
Class: |
C07K 14/735 20060101
C07K014/735; C12N 15/09 20060101 C12N015/09; A61K 38/16 20060101
A61K038/16; C07K 16/18 20060101 C07K016/18; C07K 14/705 20060101
C07K014/705; C07K 19/00 20060101 C07K019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 2015 |
JP |
2015-032231 |
Dec 24, 2015 |
JP |
2015-252231 |
Claims
1. An Fc fusion protein comprising: (i) a high affinity IgE
receptor .alpha.-chain; and (ii) an Fc region of IgG1, wherein a
linker fragment region between the (i) and the (ii) is the amino
acid sequence shown in SEQ ID NO: 2.
2. The Fc fusion protein according to claim 1, which is a
dimer.
3. The Fc fusion protein according to claim 2, wherein cysteine
residues in the linker fragment region form three disulfide
bonds.
4. A pharmaceutical composition comprising, as an active
ingredient, the Fc fusion protein according to claims 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 15/552,065, which is a National Stage application of
PCT/JP2016/054854, filed Feb. 19, 2016, which claims priority from
Japanese application nos. JP 2015-032231, filed Feb. 20, 2015, and
JP 2015-252231, filed Dec. 24, 2015.
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-WEB and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jul. 10, 2018, is named sequence.txt and is 10 KB.
TECHNICAL FIELD
[0003] The present invention relates to an Fc fusion high affinity
IgE receptor .alpha.-chain that is useful as a pharmaceutical
product.
[0004] More specifically, the present invention relates to an Fc
fusion high affinity IgE receptor .alpha.-chain having excellent
stability at low pH, and a medicinal use thereof.
BACKGROUND ART
[0005] Immunoglobulin E (IgE) is one of an immunoglobulin group,
which plays a role in allergic reactions. The IgE, which is
secreted from B cells or is expressed on the surface of the B
cells, binds to a high affinity IgE receptor (Fc.epsilon.RI) found
on the surface of mast cells, basophils, etc. When an antigenic
protein binds to IgE on a mast cell surface receptor, the IgE
becomes a form in which it crosslinks the antigen. Thereafter,
chemical mediators such as histamine or serotonin, which are stored
in intracellular granules, are released. Consequently, an
inflammatory reaction is induced, and type I allergy symptoms such
as telangiectasis or vascular hyperpermeability are provoked (Non
Patent Literature 1).
[0006] Accordingly, since a compound or a protein, which inhibits
the binding of IgE to Fc.epsilon.RI, inhibits the binding of the
IgE to the Fc.epsilon.RI found on the surface of mast cells,
basophils, etc., such a compound or a protein is expected as a
therapeutic agent for type I allergic diseases such as bronchial
asthma, allergic rhinitis, and allergic conjunctivitis (Non Patent
Literature 2).
[0007] In recent years, not only a conventional pharmaceutical
product comprising, as an active ingredient, a low-molecular-weight
compound, but also a protein pharmaceutical product, which strongly
binds to a specific receptor or the like in a living body and
exhibits excellent therapeutic effects, has been developed. For
example, Etanercept has been known as a therapeutic agent for
rheumatoid arthritis. Such Etanercept is a completely humanized
soluble TNF.alpha./LT.alpha. receptor formulation, which has been
focused because of the role of a soluble receptor of a tumor
necrosis factor (TNF) to suppress the action of TNF in a living
body, and has been then developed.
[0008] The protein pharmaceutical product can be expected to have
high therapeutic effects. On the other hand, it may cause a problem
specific to the protein pharmaceutical product in the production
process thereof.
[0009] In general, when an antibody or an Fc fusion protein is
produced in the form of a pharmaceutical product, a purification
method of using protein A is applied. In this method, a buffer with
a low pH value is used to elute a protein of interest that binds to
the protein A. Moreover, in order to inactivate virus, the protein
of interest is desirably treated at low pH for a certain period of
time.
[0010] A protein, which is poor in stability at low pH, easily
forms aggregates. If the ratio of aggregates is high, a reduction
in purification efficiency or production amount occurs in the
production of protein pharmaceutical products. In addition, an
immune response is provoked by mixing aggregates into
pharmaceutical products, and as a result, serious side effects,
such as anaphylaxis, are likely to occur.
[0011] As such, the instability of a protein of interest at low pH
may be problematic in the production of protein pharmaceutical
products.
[0012] A polypeptide (immunoadheson) comprising an immunoglobulin
and an extracellular domain is disclosed in Patent Literature 1.
Patent Literature 1 discloses a high affinity IgE receptor as an
example of such immunoadheson. However, this publication does not
specifically describe a fused protein of a high affinity IgE
receptor and an immunoglobulin.
[0013] A fused protein (hereinafter referred to as "Fusion protein
A") of a high affinity IgE receptor .alpha.-chain (Fc.epsilon.RI
.alpha.-chain; hereinafter referred to as "FCER1A") and
immunoglobulin G1 (IgG1) is disclosed in Non Patent Literature 3.
However, the Fusion protein A disclosed in the aforementioned
publication is largely different from the protein of the invention
of the present application, in terms of the mode of binding FCER1A
to IgG1 (Fc). That is to say, the protein of the invention of the
present application has a characteristic amino acid sequence in a
linker fragment region between the Fc.epsilon.RI and the IgG1.
[0014] A fused protein (NPB301) formed by linking a water-soluble
fragment of the high affinity IgE receptor (Fc.epsilon.RI) to a
human Fc region via a peptide linker is disclosed in Patent
Literature 2. However, the protein of the invention of the present
application does not comprise the peptide linker disclosed in
Patent Literature 2. Moreover, Patent Literature 2 neither
discloses nor suggests the characteristic amino acid sequence of
the linker fragment region of the present invention.
[0015] A fused protein of FCER1A and immunoglobulin G2 (IgG2) is
disclosed in Patent Literature 3. However, this fused protein
comprising IgG2 is different from the protein of the invention of
the present application, in terms of the amino acid sequences of a
linker fragment region and an Fc region.
[0016] A fused protein of non-human primate FCER1A and IgG1 is
disclosed in Patent Literature 4. Moreover, a fused protein of
FCER1A and IgG1 is disclosed in Patent Literatures 5 to 7. However,
these publications neither disclose nor suggest the characteristic
amino acid sequence of the linker fragment region of the present
invention.
[0017] The aforementioned Non Patent Literature 3 and Patent
Literatures 1 to 7 neither disclose nor suggest the protein of the
present invention.
CITATION LIST
Patent Literature
[0018] Patent Literature 1: U.S. Pat. No. 5,565,335 [0019] Patent
Literature 2: International Publication WO 2012/169735 [0020]
Patent Literature 3: Chinese Patent Application Laid-Open No.
101633698 [0021] Patent Literature 4: International Publication WO
2008/028068 [0022] Patent Literature 5: International Publication
WO 2011/056606 [0023] Patent Literature 6: International
Publication WO 2008/099178 [0024] Patent Literature 7:
International Publication WO 2008/099188
Non Patent Literature
[0024] [0025] Non Patent Literature 1: Chisei Ra, "Allergy no
Bunshi Saibo Kiko (Molecular and Cellular Mechanisms of Allergy),"
BIO INDUSTRY, 2008, Vol. 25, No. 9, PP. 23-39 [0026] Non Patent
Literature 2: Chisei Ra et al., "International Immunology," 1993,
Vol. 5, No. 1, PP. 47-54 [0027] Non Patent Literature 3: M.
Haak-Frendscho et al., "Journal of Immunology," 1993, Vol. 151, No.
1, PP. 351-358
SUMMARY OF INVENTION
Technical Problem
[0028] It is an object of the present invention to provide an Fc
fusion high affinity IgE receptor .alpha.-chain having excellent
stability at low pH.
Solution to Problem
[0029] The present inventors have conducted intensive studies in
order to obtain an Fc fusion high affinity IgE receptor
.alpha.-chain having high stability against low pH or heat. As a
result, the inventors have found that an Fc fusion high affinity
IgE receptor .alpha.-chain having high stability can be obtained by
using a linker fragment comprising three Cys residues, in a fused
protein comprising a high affinity IgE receptor .alpha.-chain and
the Fc region of IgG1, thereby completing the present invention.
Specifically, the present invention is as follows.
[0030] The present invention relates to the following [1] to [5],
etc. [0031] [1] An Fc fusion protein comprising:
[0032] (i) a high affinity IgE receptor .alpha.-chain; and
[0033] (ii) an Fc region of IgG1, wherein
[0034] a linker fragment region between the (i) and the (ii) is the
amino acid sequence shown in SEQ ID NO: 2. [0035] [2] The Fc fusion
protein according to the above [1], comprising the amino acid
sequence shown in SEQ ID NO: 3, or an amino acid sequence
comprising a deletion of lysine (K) at the C-terminus of the amino
acid sequence shown in SEQ ID NO: 3. [0036] [3] The Fc fusion
protein according to the above [1] or [2], which is a dimer. [0037]
[4] The Fc fusion protein according to the above [3], wherein
cysteine residues in the linker fragment region form three
disulfide bonds. [0038] [5] A pharmaceutical composition
comprising, as an active ingredient, the Fc fusion protein
according to any one of the above [1] to [4].
[0039] The present description includes the contents as disclosed
in Japanese Patent Application No. 2015-032231 and 2015-252231,
which are priority documents of the present application.
Advantageous Effects of Invention
[0040] The protein of the present invention has excellent stability
at low pH. In addition, the protein of the present invention has
excellent neutralizing activity against IgE. Accordingly, the
protein of the present invention is useful as a protein
pharmaceutical product for preventing or treating type I allergic
diseases mediated by IgE.
BRIEF DESCRIPTION OF DRAWINGS
[0041] FIG. 1 shows the activity of inhibiting the binding of human
IgE. In the figure, the horizontal axis indicates the concentration
of each drug (mol/L); and the longitudinal axis indicates the value
of the amount of IgE bound to Protein 1 immobilized on a plate,
which is shown at a percentage (free IgE (percent with respect to a
control)), using, as a reference, the binding amount obtained when
a predetermined amount of IgE is added. In the figure, the circle
indicates the value of Protein 1, and the square indicates the
value of Omalizumab, respectively.
[0042] FIG. 2 shows a transition of a change in the content rate
(%) of aggregates at low pH. In the figure, the horizontal axis
indicates the number of days (Day), and the longitudinal axis
indicates a change in the content rate (%) of aggregates. In the
figure, the circle indicates the value of Protein 1, and the square
indicates the value of Fusion protein A, respectively.
[0043] FIG. 3 shows a transition of a change in the content rate
(%) of aggregates by a heat treatment. In the figure, the
horizontal axis indicates the number of days (Day), and the
longitudinal axis indicates a change in the content rate (%) of
aggregates. In the figure, the circle indicates the value of
Protein 1, and the square indicates the value of Fusion protein A,
respectively.
DESCRIPTION OF EMBODIMENTS
[0044] The embodiments of the present invention will be described
in detail below.
[0045] In the present invention, individual terms have the
following meanings, unless otherwise specified.
[0046] In the present invention, the "high affinity IgE receptor
.alpha.-chain (FCER1A)" means a protein comprising an .alpha.-chain
portion that is an extracellular domain of a high affinity IgE
receptor. The high affinity IgE receptor .alpha.-chain is, for
example, a protein shown in the following SEQ ID NO: 1.
TABLE-US-00001 SEQ ID NO: 1:
VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETN
SSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEG
QPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY
CTGKVWQLDYESEPLNITVIKAPREKYWL
[0047] The above-described high affinity IgE receptor .alpha.-chain
includes, for example, a protein having identity of 90% or more,
95% or more, 97% or more, or 99% or more to the amino acid sequence
shown in SEQ ID NO: 1, when the identity is calculated using BLAST
(Basic Local Alignment Search Tool at the National Center for
Biological Information) or the like (e.g., using default
parameters), and having binding ability to IgE. Moreover, the high
affinity IgE receptor .alpha.-chain also includes a protein
comprising an amino acid sequence having a substitution, deletion
and/or addition of one or more, or several amino acids (1 to 10,
preferably 1 to 5, and more preferably 1 or 2 amino acids) with
respect to the amino acid sequence shown in SEQ ID NO: 1, and
having binding ability to IgE.
[0048] In the present invention, the "Fc region of IgG1" means the
Fc fragment of immunoglobulin G1, namely, the CH2 and CH3 constant
domains of native immunoglobulin G1. The Fc region of IgG1 includes
all of a natural mutant, an artificial mutant, and a truncated
form.
[0049] In the present invention, the "linker fragment region
between the high affinity IgE receptor .alpha.-chain and the Fc
region of IgG1" means a region consisting of 14 amino acid residues
ranging from the junction point between the above-described high
affinity IgE receptor .alpha.-chain and the above described Fc
region of IgG1 towards the direction of the Fc region.
[0050] In the present invention, the "Fc fusion protein" means a
recombinant protein comprising the high affinity IgE receptor
.alpha.-chain and the Fc fragment of an immunoglobulin.
[0051] The protein of the present invention is characterized in
that the linker fragment region between the high affinity IgE
receptor .alpha.-chain and the Fc region of IgG1 is the amino acid
sequence shown in the following SEQ ID NO: 2.
TABLE-US-00002 SEQ ID NO: 2: EPKSCDKTHTCPPC
[0052] The protein of the present invention is preferably an Fc
fusion protein comprising the amino acid sequence shown in the
following SEQ ID NO: 3 (hereinafter referred to as "Protein
1").
TABLE-US-00003 SEQ ID NO: 3:
VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETN
SSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEG
QPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY
CTGKVWQLDYESEPLNITVIKAPREKYWLEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHY TQKSLSLSPGK
[0053] The amino acid sequence shown in SEQ ID NO: 3 has a sequence
formed by fusing the amino acid sequence shown in SEQ ID NO: 1
(from Val at position 1 to Leu at position 179 of the amino acid
sequence shown in SEQ ID NO: 3), the amino acid sequence shown in
SEQ ID NO: 2 (Glu at position 180 to Cys at position 193 of the
amino acid sequence shown in SEQ ID NO: 3), and the amino acid
sequence of the Fc fragment of an immunoglobulin (Pro at position
194 to Lys 411 of the amino acid sequence shown in SEQ ID NO: 3) in
this order. This Fc fusion protein includes a protein comprising an
amino acid sequence having identity of 90% or more, 95% or more,
97% or more, or 99% or more to the amino acid sequence shown in SEQ
ID NO: 3, which is other than the amino acid sequence portion from
Glu at position 180 to Cys at position 193 that corresponds to the
amino acid sequence shown in SEQ ID NO: 2, when the identity is
calculated using, for example, BLAST (Basic Local Alignment Search
Tool at the National Center for Biological Information) or the like
(e.g., using default parameters), and having binding ability to
IgE. Moreover, this Fc fusion protein includes a protein comprising
an amino acid sequence having a substitution, deletion and/or
addition of one or more, or several amino acids (1 to 10,
preferably 1 to 5, and more preferably 1 or 2 amino acids) with
respect to the amino acid sequence shown in SEQ ID NO: 3, which is
other than the amino acid sequence portion from Glu at position 180
to Cys at position 193 that corresponds to the amino acid sequence
shown in SEQ ID NO: 2, and having binding ability to IgE.
[0054] Upon the production of a recombinant antibody, there is a
case where the lysine at the C-terminus is deleted by
post-translational modification. Accordingly, the protein of the
present invention may be an Fc fusion protein comprising an amino
acid sequence having a deletion of the lysine (K) at the C-terminus
of the above-described Protein 1. For example, such an Fc fusion
protein comprising an amino acid sequence having a deletion of the
lysine (K) at the C-terminus of Protein 1 shown in SEQ ID NO: 3
consists of an amino acid sequence portion from the position 1 to
the position 410 of the amino acid sequence shown in SEQ ID NO:
3.
[0055] The protein of the present invention includes both a monomer
and a dimer of an Fc fusion protein formed by linking a high
affinity IgE receptor .alpha.-chain to the Fc region of IgG1 via a
linker fragment consisting of the amino acid sequence shown in SEQ
ID NO: 2. Three Cys residues are present in the above-described
linker fragment region (the Cys residues at positions 184, 190, and
193 of the amino acid sequence shown in SEQ ID NO: 3), and a dimer
can be formed by disulfide bonds. In general, two Fc fusion protein
monomers form a dimer as a result of the formation of three
disulfide bonds between Cys residues at the same positions as those
of the above-described three Cys residues, and the above-described
three Cys residues. Because of the above-described three disulfide
bonds, a dimer is stabilized, and it has high stability against low
pH and heat. The phrase "having high stability against low pH and
heat" means, for example, that only a few aggregates are formed
under conditions of low pH and under heating. Such high stability
against low pH or heat can be confirmed, for example, by performing
a low pH treatment or a heat treatment on a protein, and then
measuring the content of aggregates in the protein by gel
filtration chromatography. For example, even if the Fc fusion
protein of the present invention is preserved at a low temperature
of 2.degree. C. to 8.degree. C., and preferably 4.degree. C., at pH
1 to 5, and preferably at pH 2 to 4, for 1 day to 1 month,
preferably for 1 day to 14 days, and more preferably for 5 to 12
days, or even if the present Fc fusion protein is preserved at a
temperature of 25.degree. C. to 45.degree. C., and preferably
30.degree. C. to 40.degree. C., for 1 day to 1 month, preferably 1
day to 14 days, and more preferably 1 day to 7 days, a change in
the content rate of aggregates is small. For example, when a change
in the content rate of aggregates in the protein of the present
invention is calculated based on the peak area of gel filtration
chromatography, it is 10% or less, and preferably 8% or less.
Further, when compared with an Fc fusion protein having two or less
Cys residues as a linker fragment, a change in the content rate of
aggregates in the protein of the present invention is small.
[0056] The protein of the present invention can be produced, for
example, by the following method or a method equivalent thereto, or
by the methods described in publications or methods equivalent
thereto.
[0057] The protein of the present invention can be produced using a
genetic recombination technique that is well known to a person
skilled in the present technical field. For example, DNA encoding
the protein of the present invention is prepared, and an expression
vector comprising this DNA is then constructed. Subsequently,
prokaryotic or eukaryotic cells are transformed or transfected with
the above-described vector, and a protein of interest can be
isolated and purified from a culture supernatant of the obtained
cells.
[0058] The protein of the present invention can also be produced
using protein-expressing cells that are well known to a person
skilled in the present technical field. For example, cDNA encoding
the amino acid sequence shown in SEQ ID NO: 3 is incorporated into
a mammalian expression plasmid vector to prepare a protein
expression plasmid, and the prepared plasmid is then introduced
into animal cells such as Chinese hamster ovary cells (CHO), so as
to establish a stable expression cell line. The obtained cells are
cultured, and then, the protein of the present invention can be
obtained from a culture supernatant.
[0059] The protein of the present invention can be isolated and
purified, as necessary, by isolation and purification means that
are well known to a person skilled in the present technical field.
Examples of the isolation and purification method include affinity
chromatography, ion exchange chromatography, gel filtration
chromatography, hydrophobic chromatography, mixed mode
chromatography, dialysis, a fractional precipitation method, and
electrophoresis. The protein of the present invention can also be
isolated and purified by combining these methods with one another,
as appropriate.
[0060] The protein of the present invention may also undergo
chemical modification that is well known to a person skilled in the
present technical field. Examples of the chemical modification
include glycosylation, polyethylene glycolation (PEG), acetylation,
and amidation.
[0061] Since the protein of the present invention has neutralizing
activity against IgE, it can be used as a preventive or therapeutic
agent for various diseases mediated by IgE. For instance, the
protein of the invention of the present application is useful as a
preventive or therapeutic agent for diseases associated with type I
allergy, and the like, such as bronchial asthma, eosinophilic
otitis media, eosinophilic sinusitis, allergic conjunctivitis,
allergic rhinitis, pollinosis, food allergy, mite allergic disease,
hives, and anaphylactic shock.
[0062] The protein of the present invention has excellent affinity
for IgE. Hence, the protein of the present invention can also be
used as a "protein-drug conjugate" that utilizes such affinity,
such as an antibody-drug conjugate (ADC). Examples of such a
"protein-drug conjugate" include the modes of use, such as "Protein
1-drug" and "Protein 1-linker-drug." As a drug, an anti-allergic
agent or the like can be used. Such a conjugate can be produced by
a method well known to a person skilled in the present technical
field.
[0063] With regard to the pharmaceutical composition of the present
invention, various dosage forms are used depending on usage.
Examples of an oral agent include a tablet, a powder agent, a
granule, a fine granule, and a capsule. Examples of a parenteral
agent include an injection, inhalation powders, an inhalation
liquid, eye drops, a liquid agent, a lotion agent, a spray agent,
nasal drops, a drip, an ointment, a suppository, and a patch.
[0064] The pharmaceutical composition of the present invention is
administered by various administration methods, depending on usage.
Examples of the administration method include oral administration,
intravenous administration, intraperitoneal administration,
subcutaneous administration, topical administration, and
intramuscular administration.
[0065] The pharmaceutical composition of the present invention is
prepared using the protein of the present invention and at least
one pharmaceutical product additive. The pharmaceutical composition
of the present invention can be prepared by a known pharmaceutical
method, depending on its dosage form. Examples of such a
pharmaceutical product additive include an excipient, a
disintegrator, a binder, a lubricant, a diluent, a buffer, a
tonicity agent, a preservative, a stabilizer, and a solubilizer.
The above-described pharmaceutical product additive also includes a
physiological saline and water for injection. The pharmaceutical
composition of the present invention can be prepared by being mixed
with, being diluted with, or being dissolved in the above-described
pharmaceutical product additives.
[0066] When the pharmaceutical composition of the present invention
is used for prevention or treatment, the dose of the protein of the
present invention, which is comprised therein as an active
ingredient, is determined, as appropriate, depending on the age,
sex, and body weight of a patient, the degree of disease, a dosage
form, an administration route, etc. With regard to the dose applied
to an adult by oral administration, the applied dose can be
determined, for example, in the range of 0.1 .mu.g/kg to 1000
mg/kg/day. The daily dose in the case of oral administration is
preferably in the range of 0.1 mg/kg to 10 mg/kg/day, depending on
the dosage form. Such a daily dose may be administered at once, or
dividedly over two or three administrations. Moreover, the dose
applied to an adult by parenteral administration can be determined
in the range of 0.01 .mu.g/kg to 1000 mg/kg/day. The daily dose in
the case of parenteral administration is in the range of preferably
0.1 .mu.g/kg to 10 .mu.g/kg/day, 1 .mu.g/kg to 100 .mu.g/kg/day, or
10 .mu.g/kg to 1000 .mu.g/kg/day, depending on the dosage form.
EXAMPLES
[0067] The content of the present invention will be described in
more detail in the following examples and test examples. However,
these examples are not intended to limit the scope of the present
invention.
Example 1
Expression and Preparation of Protein 1
(1) Preparation of Protein 1 Expression Vector
[0068] cDNA encoding the amino acid sequence shown in SEQ ID NO: 3
was incorporated into a mammalian expression plasmid vector to
prepare a Protein 1 expression plasmid.
(2) Preparation of Protein 1-Expressing Cells
[0069] The Protein 1 expression plasmid was introduced into Chinese
hamster ovary cells (CHO) to establish a Protein 1 stable
expression cell line. Secretion of Protein 1 into a culture
supernatant was confirmed by SDS-PAGE.
Test Example 1
IgE Binding Inhibitory Activity (IgE Neutralizing Activity)
(1) Preparation of Assay Plate
[0070] Protein 1 was dissolved in a coating buffer, and an aliquot
of the obtained solution was added onto a microplate. The
microplate was left at 4.degree. C. for 18 or more hours, and was
then washed with a washing buffer (PBS-Tween 20). Thereafter, a
blocking solution (Assay Diluent) (BD Biosciences) was added
thereto. The microplate was left at room temperature for 1 hour,
and the blocking solution was then removed. The plate was washed
with a washing buffer, and was then used in the measurement of
binding inhibitory activity.
(2) Method of Measuring Binding Inhibitory Activity
[0071] Using omalizumab (anti-human IgE antibody) as a positive
control, the IgE binding inhibitory activity of Protein 1 was
measured by the following method.
[0072] A certain amount of human IgE (ANTIBODYSHOP) was mixed with
Protein 1 or omalizumab (Novartis) having any given concentration.
The obtained mixture was added to the plate prepared in the above
(1), and it was then left at room temperature for approximately 2
hours. After the mixed solution had been discarded, the plate was
washed with a washing buffer. A HRP-labeled anti-human IgE antibody
(BD Biosciences) was added to the resulting plate, and it was then
left at room temperature for approximately 1 hour. After the
antibody solution had been discarded, the plate was washed with a
washing buffer. A TMB (3,3',5,5'-tetramethylbenzidine) solution was
added to the plate. After a certain period of time had passed,
phosphoric acid was added to the plate to terminate a coloring
reaction. Thereafter, using a plate reader, absorbance (OD450) was
measured. Based on the amount of IgE bound to Protein 1 immobilized
on the plate, Protein 1 and omalizumab were evaluated in terms of
IgE binding inhibitory activity (IgE neutralizing activity) (FIG.
1).
(3) Results
[0073] The Protein 1 of the present invention inhibited the binding
of IgE to FCER1A in a concentration-dependent manner.
Test Example 2
Test Regarding Stability at Low pH
(1) Preparation of Sample
[0074] A purification operation was carried out using AKTA Explorer
10 S (GE Healthcare). Protein 1 was allowed to be expressed by the
same method as that described in Example 1, and then, a culture
supernatant thereof was 2 times diluted with D-PBS(-) (Dulbecco's
Phosphate Buffered Saline). The diluted solution was loaded on
HiTrap rProtein A FF (GE Healthcare, 17-5079-01). The
above-described column was washed with D-PBS(-), and was then
eluted with a 100 mM glycine-HCI buffer (pH 2.2). A protein A
adsorption fraction was fractionated at 1.0 mL/tube. The peak
fractions were mixed with one another to obtain a low pH-treated
sample (pH 2.9). The low pH-treated sample that had been preserved
at 4.degree. C. was used as an evaluation sample. 0.25 mL of an
aliquot was sampled from the evaluation sample at a predetermined
timing (5 to 12 days after the preservation at 4.degree. C.), and
0.05 mL of a 1 M Tris-HCI buffer (pH 9.0) was added to the aliquot
to neutralize it, thereby obtaining a neutralized sample.
[0075] As a control, Fusion protein A described in Non Patent
Literature 3 was used. Fusion protein A was allowed to be expressed
by the same method as that described in Example 1, and then, a
neutralized sample was obtained by the same method as the
aforementioned method. It is to be noted that the Fusion protein A
used in the present test is a protein consisting of the amino acid
sequence shown in the following SEQ ID NO: 4. The amino acid
sequence shown in SEQ ID NO: 4 comprises a linker fragment
consisting of Asp at position 180 to Cys at position 188, and the
number of Cys residues contained in this linker fragment is 2.
TABLE-US-00004 SEQ ID NO : 4:
VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETN
SSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEG
QPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY
CTGKVWQLDYESEPLNITVIKAPREKYWLDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK
(2) Method of Analyzing Content of Aggregates
[0076] With regard to the operation of analyzing aggregates, the
content of aggregates was confirmed by performing gel filtration
chromatography of using Superdex 200 10/300GL (GE Healthcare,
17-5175-01), employing AKTA Explorer 10 S (GE Healthcare), and also
using D-PBS(-) as a mobile phase. The areas of a peak whose eluted
position was a monomer and an aggregate peak eluted in a high
molecular region were calculated, and a transition of a change in
the content rate of aggregates (%) in Protein 1 and Fusion protein
A, which was caused by a low pH treatment, was evaluated (FIG.
2).
(3) Results
[0077] In the protein of the present invention, the increased level
of the formation of aggregates associated with a low pH treatment
time was significantly smaller than that in the case of Fusion
protein A, and thus, the protein of the present invention exhibited
high stability against exposure to low pH. Therefore, the protein
of the present invention is excellent in terms of stability at low
pH, and thus, the improvement of purification efficiency and
productivity is expected in the production process.
Test Example 3
Test Regarding Stability Against Heat
(1) Preparation of Sample
[0078] A purification operation was carried out using AKTA Explorer
10 S (GE Healthcare). Protein 1 was allowed to be expressed by the
same method as that described in Example 1, and a culture
supernatant thereof was then loaded on HiTrap Mab Select SuRe (GE
Healthcare, 17-0034-94). The above-described column was washed with
D-PBS(-) and a 100 mM citrate buffer (pH 4.0), and a protein A
adsorbate was then eluted with a 100 mM glycine-HCI buffer (pH
3.3). A 1 M Tris-HCI buffer (pH 9.0) was added at a volume of 1/10
to the recovered fraction to neutralize it, so as to obtain a
protein A-purified protein. The pH of this protein A-purified
protein was adjusted to pH 4.0 by addition of 1 N HCI, and it was
then loaded on a column filled with a mixed mode resin of
hydrophobic interaction and cation exchange. A non-adsorbed protein
was washed with a 50 mM acetate buffer (pH 4.0), and 100% linear
gradient elution was then carried out with a 50 mM Tris-Hcl buffer
(pH 9.0). A peak fraction was recovered to obtain a purified
protein. The obtained protein was subjected to gel filtration
fractionation, using D-PBS(-) as a mobile phase, and employing
HiLoad 16/60 Superdex 200 prep grade (GE Healthcare, 17-1069-01). A
peak fraction corresponding to a monomer was recovered to obtain a
gel filtration purified sample. This gel filtration purified sample
was adjusted again with D-PBS(-), and it was then poured into a
microtube, followed by incubation at 37.degree. C., so as to obtain
an evaluation sample. An aliquot was sampled from this evaluation
sample at a predetermined timing (1 day to 7 days after the
preservation at 37.degree. C.), so as to obtain a heat treatment
sample.
[0079] As a control, Fusion protein A described in Non Patent
Literature 3 was used. Fusion protein A was allowed to be expressed
by the same method as that in Example 1, and then, a heat treatment
sample was obtained by the same method as the aforementioned
method. It is to be noted that the Fusion protein A used in the
present test is a protein consisting of the same amino acid
sequence as that of the protein used in Test Example 2.
(2) Method of Analyzing Content of Aggregates
[0080] With regard to the operation of analyzing aggregates, the
content of aggregates was confirmed by performing gel filtration
chromatography of using Superdex 200 10/300GL (GE Healthcare,
17-5175-01), employing AKTA Explorer 10 S (GE Healthcare), and also
using D-PBS(-) as a mobile phase. The areas of a peak whose eluted
position was a monomer and an aggregate peak eluted in a high
molecular region were calculated, and a transition of a change in
the content rate of aggregates (%) in Protein 1 and Fusion protein
A, which was caused by a heat treatment, was evaluated (FIG.
3).
(3) Results
[0081] In the protein of the present invention, the increased level
of the content of aggregates after preservation at 37.degree. C.
was smaller than that in the case of Fusion protein A, and thus,
the protein of the present invention exhibited more stability
against exposure at 37.degree. C. Therefore, the protein of the
present invention is excellent in terms of stability against heat,
as well as stability at low pH, and thus, the improvement of
purification efficiency and productivity is expected in the
production process.
INDUSTRIAL APPLICABILITY
[0082] Since the protein of the present invention has excellent
neutralizing activity against IgE, it can be used as a protein
pharmaceutical product for preventing or treating various diseases
mediated by the IgE.
SEQUENCING FREE TEXT
[0083] SEQ ID NO: 2 Synthesized
[0084] All publications, patents and patent applications cited in
the present description are incorporated herein by reference in
their entirety.
Sequence CWU 1
1
41179PRTHomo sapiens 1Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro
Pro Trp Asn Arg Ile 1 5 10 15 Phe Lys Gly Glu Asn Val Thr Leu Thr
Cys Asn Gly Asn Asn Phe Phe 20 25 30 Glu Val Ser Ser Thr Lys Trp
Phe His Asn Gly Ser Leu Ser Glu Glu 35 40 45 Thr Asn Ser Ser Leu
Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly 50 55 60 Glu Tyr Lys
Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr 65 70 75 80 Leu
Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val 85 90
95 Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn
100 105 110 Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala
Leu Lys 115 120 125 Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn
Ala Thr Val Glu 130 135 140 Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys
Val Trp Gln Leu Asp Tyr 145 150 155 160 Glu Ser Glu Pro Leu Asn Ile
Thr Val Ile Lys Ala Pro Arg Glu Lys 165 170 175 Tyr Trp Leu
214PRTArtificialSynthetic 2Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 1 5 10 3411PRTHomo sapiens 3Val Pro Gln Lys Pro Lys
Val Ser Leu Asn Pro Pro Trp Asn Arg Ile 1 5 10 15 Phe Lys Gly Glu
Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe 20 25 30 Glu Val
Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu 35 40 45
Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly 50
55 60 Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val
Tyr 65 70 75 80 Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser
Ala Glu Val 85 90 95 Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys
His Gly Trp Arg Asn 100 105 110 Trp Asp Val Tyr Lys Val Ile Tyr Tyr
Lys Asp Gly Glu Ala Leu Lys 115 120 125 Tyr Trp Tyr Glu Asn His Asn
Ile Ser Ile Thr Asn Ala Thr Val Glu 130 135 140 Asp Ser Gly Thr Tyr
Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr 145 150 155 160 Glu Ser
Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys 165 170 175
Tyr Trp Leu Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 180
185 190 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro 195 200 205 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr 210 215 220 Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn 225 230 235 240 Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg 245 250 255 Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val 260 265 270 Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 275 280 285 Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 290 295 300
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 305
310 315 320 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe 325 330 335 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu 340 345 350 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe 355 360 365 Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly 370 375 380 Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr 385 390 395 400 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 405 410 4406PRTHomo sapiens 4Val
Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile 1 5 10
15 Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe
20 25 30 Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser
Glu Glu 35 40 45 Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe
Glu Asp Ser Gly 50 55 60 Glu Tyr Lys Cys Gln His Gln Gln Val Asn
Glu Ser Glu Pro Val Tyr 65 70 75 80 Leu Glu Val Phe Ser Asp Trp Leu
Leu Leu Gln Ala Ser Ala Glu Val 85 90 95 Val Met Glu Gly Gln Pro
Leu Phe Leu Arg Cys His Gly Trp Arg Asn 100 105 110 Trp Asp Val Tyr
Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys 115 120 125 Tyr Trp
Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu 130 135 140
Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr 145
150 155 160 Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg
Glu Lys 165 170 175 Tyr Trp Leu Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu 180 185 190 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp 195 200 205 Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp 210 215 220 Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 225 230 235 240 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 245 250 255 Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 260 265
270 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
275 280 285 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 290 295 300 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn 305 310 315 320 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 325 330 335 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 340 345 350 Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 355 360 365 Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 370 375 380 Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 385 390
395 400 Ser Leu Ser Pro Gly Lys 405
* * * * *