U.S. patent application number 13/068025 was filed with the patent office on 2011-09-01 for myeloid colony stimulating factor and uses thereof.
Invention is credited to Per Borgstrom, Gregory I. Frost.
Application Number | 20110212074 13/068025 |
Document ID | / |
Family ID | 29400881 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110212074 |
Kind Code |
A1 |
Frost; Gregory I. ; et
al. |
September 1, 2011 |
Myeloid colony stimulating factor and uses thereof
Abstract
The identification of the HYAL 1 hyaluronidase enzyme as a human
plasma-derived myeloid colony-stimulating factor (CSF), herein
designated CSF5-hyaluronidase, its recombinant production and
methods of use are described. This protein may be used for the
treatment of myelosuppression as may occur after irradiation,
chemotherapy or other diseases where an increase in leukocyte
levels may be beneficial. For example, CSF5-may be used to enhance
the immune response to viral infection or other diseases associated
with immune suppression.
Inventors: |
Frost; Gregory I.; (Solana
Beach, CA) ; Borgstrom; Per; (La Jolla, CA) |
Family ID: |
29400881 |
Appl. No.: |
13/068025 |
Filed: |
April 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12753046 |
Apr 1, 2010 |
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13068025 |
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11431181 |
May 9, 2006 |
7718428 |
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12753046 |
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10182088 |
Nov 26, 2002 |
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PCT/US01/02575 |
Jan 25, 2001 |
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11431181 |
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60177913 |
Jan 25, 2000 |
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Current U.S.
Class: |
424/94.62 |
Current CPC
Class: |
A61P 37/00 20180101;
A61K 38/193 20130101; C12N 9/2408 20130101; A61K 48/00 20130101;
A61K 38/47 20130101 |
Class at
Publication: |
424/94.62 |
International
Class: |
A61K 38/47 20060101
A61K038/47; A61P 37/00 20060101 A61P037/00 |
Claims
1. A method for treating a mammal with a myelosuppressed condition,
comprising administering a CSF-5 hyaluronidase to the mammal.
2. The method of claim 1, wherein the myelosuppressed condition is
a myeloid-cell insufficiency that results from radiation treatment,
chemotherapy, or viral infection.
3. The method of claim 1, wherein the myelosuppressed condition is
characterized by insufficient production of at least one cytokine
by a myeloid cell, and the CSF5-hyaluronidase facilitates
production of that cytokine.
4. The method of claim 3, wherein the cytokine is selected from
among an interferon, interleukin, tumor necrosis factor and myeloid
colony stimulating factor.
5. The method of claim 1, wherein the CSF5-hyaluronidase comprises
a sequence of amino acids that is produced upon expression in a
cell of a sequence of nucleotides set forth as nucleotides 617-1921
of SEQ ID NO:8.
6. The method of claim 1, wherein the CSF5-hyaluronidase comprises
the sequence of amino acids set forth in SEQ ID NO:9 without the
leader sequence.
7. The method of claim 1, wherein the myelosuppressed condition is
associated with monocytopenia.
8. The method of claim 1, wherein the mammal is a human.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of copending U.S.
application Ser. No. 12/753,046, filed Apr. 01, 2010; which is a
divisional application of U.S. application Ser. No. 11/431,181
filed May 9, 2006, now issued as U.S. Pat. No. 7,718,428; which is
a divisional application of U.S. application Ser. No. 10/182,088
filed Nov. 26, 2002, now abandoned; which is a 35 USC .sctn.371
National Stage application of International Application No.
PCT/US01/02575 filed Jan. 25, 2001; which claims the benefit under
35 USC .sctn.119(e) to U.S. application Ser. No. 60/177,913 filed
Jan. 25, 2000, now expired. The disclosure of each of the prior
applications is considered part of and is incorporated by reference
in the disclosure of this application.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the discovery that the
previously-reported human hyaluronidase, HYAL1, is actually a new
member of a class of molecules known collectively as myeloid colony
stimulating factors.
[0004] 2. Background Information
[0005] Colony stimulating factors are proteins capable of
influencing the growth and differentiation of cells responsible for
the cellular components of blood in the body. Colony Stimulating
factors have traditionally been defined by their ability to
stimulate growth of colonies of bone marrow cells in semi-solid
media. Macrophage colony stimulating factors are a subclass of
colony stimulating factors that play a role in the regulation of
immune responses by potentiating the proliferation and
differentiation of macrophages from immature hematopoietic
progenitor cells, and inducing effector functions of mature
macrophages including secretion of interferon-.gamma, tumor
necrosis factor and non-M-CSF colony stimulating activities.
[0006] The ability of certain factors produced in very low
concentration in a variety of tissues to stimulate the growth and
development of bone marrow progenitor cells into granulocytes
and/or macrophages has been known for many years. The presence of
such factors in sera, urine samples, and tissue extracts from a
number of species is demonstrable using assays which measure the
stimulation of colony formation by bone marrow cells plated in
semi-solid culture medium. There is no known in vivo assay. As
these factors induce the formation of such colonies, the factors
collectively have been called Colony Stimulating Factors (CSF).
[0007] Colony Stimulating Factors have been purified from a number
of tissue sources and species. Japanese Pat. No. 8,020,599 teaches
of a rat myoid cell derived colony-stimulating factor capable of
stimulating rat thymic macrophages and migroglia cells. Some colony
stimulating factors are species restricted in their activity, such
that CSF's derived from one species may lack colony forming
activity in distantly related species (Shanafelt et al. J Biol Chem
1991 Jul. 25;266(21): 13804-10).
[0008] It has been shown that there are at least three subclasses
of human CSF proteins defined according to the types of cells found
in the resultant colonies. One subclass, CSF-1 results in colonies
containing predominantly macrophages. Other subclasses produce
colonies of both neutrophilic granulocytes and macrophages; which
contain exclusively neutrophilic granulocytes; and which contain
neutrophilic and eosinophilic granulocytes and macrophages.
[0009] Treatment of patients suffering from AIDS with colony
stimulating factors, alone or together with erythropoietin and/or
an antiviral agent and/or IL-2, is reported in PCT W087/03204 and
U.S. Pat. No. 4,482,485. These references teach that CSF can be
used for a supporting role in the treatment of cancer. In addition,
EP 118,915 reports production of CSF for preventing and treating
granulocytopenia and macrophagocytopenia in patients receiving
cancer therapy, for preventing infections, and for treating
patients with implanted bone marrow. In addition, CSFs stimulate
nonspecific tumoricidal activity (Ralph et al., Immunobiol
172:194-204,1986). CSF has no immediate direct role in activation
of macrophages for tumoricidal and microbiocidal activities against
fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (Ralph
et al., 76:10-21, 1983). The combination of CSF-1 and lymphokine
has an added tumoricidal effect on murine sarcoma TU5 targets
(Ralph et al., Cell. Immunol. 105:270-279, 1987). Warren et al. (J
Immunol. 137:2281-2285, 1986) disclose that CSFs stimulate monocyte
production of interferon, TNF and colony stimulating activity. Lee
et al. (J. Immunol. 138:3019-3022, 1987) disclose CSF-induced
resistance to viral infection in murine macrophages.
SUMMARY OF THE INVENTION
[0010] The present invention is based on the discovery that the
human protein HYAL1, with previously reported hyaluronidase
activity, has potent colony-stimulating activity. For this reason,
this molecule is renamed herein as CSF5-hyaluronidase.
[0011] One embodiment of the invention is a process for purifying
human CSF5-hyaluronidase protein comprising subjecting a biological
sample of human or human tissue origin to the steps of phase
extraction, cation exchange chromatography and hydroxyapatite
chromatography, such that purified human CSF5-hyaluronidase is
recovered.
[0012] The invention also includes a method for increasing the
number of myeloid progenitors in a cell population, comprising the
step of contacting the cell population with an exogenously-derived
CSF5-hyaluronidase.
[0013] The invention further provides a method for treating a
mammal with a myelosuppressed condition, comprising the step of
administering to a mammal an effective amount of
exogenously-derived CSF5-hyaluronidase. In one embodiment,
CSF5-hyaluronidase is administered in conjunction with a treatment
selected from the group consisting of surgery, radiation therapy
and chemotherapy. In certain embodiments, the myelosuppression is
associated with radiation, chemotherapy or viral infection.
[0014] The invention further includes a method for treating a
mammal with a myelosuppressed condition, comprising the step of
administering to said mammal nucleic acid operatively encoding
CSF5-hyaluronidase such that SCF5-hyaluronidase is expressed in
said mammal. The nucleic acid may advantageously be in an
expression vector, preferably operatively linked to a promoter,
which may be, for example, an exogenous promoter, an inducible
promoter, a viral promoter, a constituitive promoter, or a
heterologous human promoter.
[0015] A further aspect of the present invention is a method for
enhancing the production of cytokines by myeloid cells, comprising
the step of contacting said myeloid cells with exogenously-derived
CSF5-hyaluronidase. Cytokines contemplated in the present invention
include, for example, interferon, interleukin, tumor necrosis
factor and myeloid colony stimulating factor.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention relates to the discovery of
colony-stimulating activity associated with a protein known in the
literature as HYAL1. This protein represents a new member of the
colony stimulating factor family of the monocytic subclass, and has
a unique dual function in that the biochemically purified and
recombinant protein also possesses glycosaminoglycan degrading
activity towards chondroitin sulfates and hyaluronan under acidic
conditions. This protein, previously known as HYAL1, has been
recently purified, cloned and sequenced by virtue of its
glycosaminoglycan degrading, or hyaluronidase activity (Frost et
al., Biochem. Biophys. Res. commun. 236:5-10, 1997). Six paralogous
sequences to HYAL1 have been identified in the human genome (Csoka
et al., Genomics 1999 Sep. 15;60(3):356-61). Hyaluronidase like
genes have been identified in other mammalian species, including
mouse and rat (Strobl et al., Genomics 1998 Oct 15;53 (2):214-9)
(Genbank Accession Number 4104235). The orthologous relationship
between such genes has not been established in some species.
[0017] Prior to the present invention, no myelostimulative or
colony stimulating activity had been attributed to this
glycosaminoglycandegrading enzyme. The HYAL 1 enzyme has high
specificity and is present predominantly in human plasma at a
concentration of 20-50.mu.g/ml (Frost et al., 1997). Because of its
CSF activity, the HYAL1 gene product should be redefined as
CSF5-hyaluronidase.
[0018] Human CSF5-hyaluronidase supports monocyte proliferation
and/or differentiation in vitro. This novel property of the gene
product was identified from the treatment of human peripheral blood
monocytes in vitro with recombinant CSF5-hyaluronidase produced as
described in the examples below. Based on this discovery,
CSF5-hyaluronidase and vectors encoding this protein are suitable
for use in supporting hematopoiesis in vivo, and in treating immune
deficiencies associated with chemotherapy or viral infection.
[0019] CSF5-hyaluronidase is also used in the present invention to
increase the number of monocytes in a cell population by contacting
the cell population with an effective amount of the protein. This
effective amount is, in general, between about 0.01 .mu.g/ml and
100 mg/ml, preferably between about 0.1 .mu.g/ml and 10 mg/ml, and
more preferably between about 1 ng/ml and 1 mg/ml. These amounts
can be optimized for any cell population using standard
dose-response curves. This is useful for producing large numbers of
cultured monocytes which can be used therapeutically or for
screening assays to discover compounds capable of stimulating
release of cytokines from monocytes. It can also be used in vivo to
treat myeloid-cell insufficiency.
[0020] Note that referred embodiments of the present invention
utilize exogenously-derived CSF5-hyaluronidase.
"Exogenously-derived," in the context of treatment of a cell
population or a mammal, is defined as CSF5-hyaluronidase that has
been introduced into a system, such as recombinantly-produced
CSF5-hyaluronidase, purified or isolated CSF5-hyaluronidase,
CSF5-hyaluronidase produced from another organism, or
CSF5-hyaluronidase previously purified from tissues or fluids of
the same organism, at a different point in time. CSF5-hyaluronidase
produced by exogenously-introduced polynucleotide encoding that
protein is also defined as "exogenously-derived" for purposes of
the present invention.
[0021] Although various methods of treatment of cell populations
and mammals (including human and non-human mammals) are described
herein, it will be appreciated that the present invention also
contemplates use of CSF5-hyaluronidase (or polynucleotide encoding
CSF5-hyaluronidase) in the preparation of a medicament for the
practice of each and every treatment method described herein. Such
medicaments are typically prepared by formulating the
CSF5-hyaluronidase with a pharmaceutically-acceptable carrier, of
well-known type. Such carriers are typically injectable carriers,
although inhalable formulations and other methods of protein
delivery are also contemplated.
[0022] In one aspect, the invention relates to methods of enhancing
production of cytokines by monocytes, particularly interferon,
tumor necrosis factor and myeloid CSF, by treating the monocytes
with an effective amount of CSF5-, either native or recombinant. In
another aspect, the invention relates to methods of enhancing the
killing of target cells by macrophages, of enhancing the production
of white blood cells from stem cells or enhancing the immune system
of a subject, of inducing resistance to viral infections in
macrophages, of promoting wound healing, and of treating tumor
cells by using an effective tumor-treating amount of
CSF5-hyaluronidase of the present invention. In addition, the
invention relates to pharmaceutical and therapeutic compositions
comprising CSF5-hyaluronidase, and to a mixture thereof with an
excipient or a cytokine or lymphokine.
[0023] In another embodiment of the present invention, there are
provided methods for the stimulation of cells of the monocytic
lineage by way of gene transfer of CSF5-hyaluronidase encoding
nucleic acids. As will be appreciated by those of skill in the art,
there are numerous methods available to express a gene, all of
which are contemplated for use in accordance with the present
invention. In a particular aspect of the present invention,
CSF5-hyaluronidase gene expression is accomplished by introduction
of the cDNA encoding the CSF5-hyaluronidase in a gene construct
(See, e.g., SEQ ID NO: 8 for the sequence of human CSF5
hyaluronidase mRIMA). Expression of CSF5 by way of virus-mediated
transfer (e.g. retroviruses, adenoviruses), naked nucleic acids and
other means known by those skilled in the art are available methods
to transfer the CSF5-hyaluronidase gene into a patient. Gene
delivery systems are described by Feigner et al. (Hum. Gene Ther.
8:511-512, 1997) and include cationic lipid-based delivery systems
(lipoplex), polycation-based delivery systems (polyplex) and a
combination thereof (lipopolyplex), all of which are contemplated
for use in the present invention.
[0024] Host-vector systems for the expression of CSF5-hyaluronidase
may be prokaryotic or eukaryotic, although eukaryotic expression
vectors are preferred. Many such expression vectors are known and
commercially available. Standard techniques for the construction of
these expression vectors are well known and can be found in
references such as Sambrook et al., or in any of the widely
available laboratory manuals on recombinant DNA technology.
Expression may be accomplished, for example, by transforming
prokaryotic or eukaryotic cells with a suitable vector encoding
CSF5-hyaluronidase. The DNA sequence can be expressed directly in
mammalian cells under the control of a suitable promoter.
Heterologous promoters well-known by those skilled in the art can
be used. Examples of such promoters include the human
cytomegalovirus (CMV) promoter, the SV40 promoter, the herpes
simplex virus (HSV) thymidine kinase (TK) gene promoter, the
adenovirus immediate early gene promoter and retroviral long
terminal repeats. The use of constitutive, inducible and
tissue-specific promoters are all within the scope of the present
invention. The expression vector also typically contains a
selectable marker, such as antibiotic resistance, to select for
cells which are expressing the protein. Other nucleotide sequence
elements can be incorporated into the expression vectors to
facilitate integration of DNA into chromosomes, expression of the
DNA and cloning of the vector. For example, the presence of
enhancers upstream of the promoter or terminators downstream of the
coding region can facilitate expression of the nucleic acid
contained within the expression vector.
[0025] In order to express CSF5-hyaluronidase in prokaryotic or in
yeast cells, the leader sequence (or secretory sequence) is
typically removed. This can be done using standard techniques known
by those skilled in the art. Once the desired CSF5- hyaluronidase
cDNA clone is obtained, known and appropriate means are utilized to
express the CSF protein, e.g., insertion into an appropriate
vector, and transfection of the vector into an appropriate host
cell, selection of transformed cells, and culture of these
transformants to express CSF activity. Such methods are described
in detail by Sambrook et al., Molecular Cloning: a Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY, latest edition and by Ausubel et al., Current Protocols in
Molecular Biology, latest edition. Suitable host cells include
bacteria, e.g. E. coli, yeast mammalian e.g. CHO, and insect cells,
e.g. Sf9 cells. The CSF5-hyaluronidase protein thus produced may
have a methionine group at the N-terminus of the protein (herein
called Met-CSF). The mature protein produced by prokaryotic and
eukaryotic cells will be otherwise identical in amino acid
sequence, but the eukaryotic product may be glycosylated to the
same or a different extent as in the natural product. Various
methods of obtaining CSF protein in accordance with the convention
are illustrated in the Examples described below. Various cell
transfection methods may be used, including electroporation,
calcium phosphate precipitation, microinjection and cell fusion.
Other methods or materials, e.g. vectors, will be readily apparent
to those skilled in the art on the basis of the Examples and the
foregoing description.
[0026] Pharmaceutically acceptable compositions of
CSF5-hyaluronidase may be used to treat mammals suffering from
monocytopenia, particularly those associated with radiation,
chemotherapy, and viral infections. Monocytopenia is defined as an
abnormal decrease in the proportion of monocytes in the blood. A
variety of mammalian hosts may be treated according to the subject
invention. Such hosts include rare or valuable mammals, pets and
livestock, humans, and the like.
[0027] As discussed above, the subject methods result in the
increase in cells of the monocytic lineage by administration of a
recombinant protein of CSF5-hyaluronidase or nucleic acid encoding
the same. CSF5-hyaluronidase may be used in combination with
additional treatment modalities, including surgery, radiation
therapy and chemotherapy. Methods of surgery for both biopsy and
reduction or elimination of tumor mass are known to those of skill
in the art. Radiation therapy is also known to those of skill in
the art and includes electromagnetic radiation, e.g., high
frequency x-rays, and subatomic particle radiation, e.g., alpha
particles, beta particles, neutrons, protons, mesons, and heavy
ions. Finally, a variety of chemotherapeutic agents and methods for
their use in cancer therapy are known and include: alkylating
agents, e.g., Mechlorethamine hydrochloride (Nitrogen Mustard,
Mustargen, HN2), Cyclophosphamide (Cytovan, Endoxana), Ifosfamide
(IFEX), Chlorambucil (Leukeran), Melphalan (Phenylalanine Mustard,
L-sarcolysin, Alkeran, LPAM), Busulfan (Myleran), Thiotepa
(Triethylenethiophosphoramide), Carmustine (BiCNU, BCNU), Lomustine
(CeeNU, CCNU), Streptozocin (Zanosar), and the like; plant
alkaloids, e.g., Vincristine (Oncovin), Vinblastine (Velban,
Velbe), Paclitaxel (Taxol), and the like; antimetabolites, e.g.,
Methotrexate (MTX), Mercaptopurine (Purinethol, 6-MP), Thioguanine
(6-TG), Fluorouracil (5-FU), Cytarabine (Cytosar-U, Ara-C),
Azacitidine (Mylosar, 5-AZA), and the like; antibiotics, e.g.,
Dactinomycin (Actinomycin D Cosmegen), Doxorubicin (Adriamycin),
Daunorubicin (duanomycin, Cerubidine), Idarubicin (Idamycin),
BJeomycin (Blenoxane), Picarnycin (Mithramycin, Mithracin),
Mitomycin (Mutarnycin), and the like, and other anticellular
proliferate agents, e.g., Hydroxyurea (Hydrea), Procarbazine
(Mutalane), Dacarbazine (DTIC-Dome), Cisplatin (Platinol)
Carboplatin (Paraplatin), Asparaginase (Elspar) Etoposide (VePesid,
VP-16213), Amsarcrine (AMSA, m-AMSA), Mitotane (Lysodren),
Mitoxantrone (Novatrone and the like.
[0028] In using the subject methods in combination with one or more
of the above reviewed conventional treatment modalities, the timing
of the different modalities may be controlled so as to obtain
optimum results with regard to beneficial effects upon the cells of
the monocytic lineage.
[0029] Pharmaceutically acceptable compositions contemplated for
use in the practice of the present invention can be used in the
form of a solid, a solution, an emulsion, a dispersion, a micelle,
a liposome, and the like, wherein the resulting composition
contains one or more of the active compounds contemplated for use
herein, as active ingredients thereof, in admixture with an organic
or inorganic carrier or excipient suitable for nasal, enteral or
parenteral applications. The active ingredients may be compounded,
for example, with the usual non-toxic, pharmaceutically or
physiologically acceptable carriers for tablets, pellets, capsules,
troches, lozenges, aqueous or oily suspensions, dispersible powders
or granules, suppositories, solutions, emulsions, suspensions, hard
or soft capsules, caplets or syrups or elixirs and any other form
suitable for use. The carriers that can be used include glucose,
lactose, gum acacia, gelatin, mannitol, starch paste, magnesium
trisilicate, talc, corn starch, keratin, colloidal silica, potato
starch, urea, medium chain length triglycerides, dextrans, and
other carriers suitable for use in manufacturing preparations, in
solid, semisolid, or liquid form. In addition auxiliary,
stabilizing, thickening and coloring agents may be used. The active
compounds contemplated for use herein are included in the
pharmaceutical composition in an amount sufficient to produce the
desired effect upon the target process, condition or disease.
[0030] In addition, such compositions may contain one or more
agents selected from flavoring agents (such as peppermint, oil of
wintergreen or cherry), coloring agents, preserving agents, and the
like, in order to provide pharmaceutically elegant and palatable
preparations. Tablets containing the active ingredients in
admixture with non-toxic pharmaceutically acceptable excipients may
also be manufactured by known methods. The excipients used may be,
for example, (1) inert diluents, such as calcium carbonate,
lactose, calcium phosphate, sodium phosphate, and the like; (2)
granulating and disintegrating agents, such as corn starch, potato
starch, alginic acid, and the like; (3) binding agents, such as gum
tragacanth, corn starch, gelatin, acacia, and the like; and (4)
lubricating agents, such as magnesium stearate, stearic acid, talc,
and the like. The tablets may be uncoated or they may be coated by
known techniques to delay disintegration and absorption in the
gastrointestinal tract, thereby providing sustained action over a
longer period. For example, a time delay material such as glyceryl
monostearate or glyceryl distearate may be employed. The tablets
may also be coated by the techniques described in the U.S. Pat.
Nos. 4,256,108; 4,160,452; and 4,265,874, to form osmotic
therapeutic tablets for controlled release.
[0031] When formulations for oral use are in the form of hard
gelatin capsules, the active ingredients may be mixed with an inert
solid diluent, for example, calcium carbonate, calcium phosphate,
kaolin, or the like. They may also be in the form of soft gelatin
capsules wherein the active ingredients are mixed with water or an
oil medium, for example, peanut oil, liquid paraffin, olive oil and
the like.
[0032] Formulations may also be in the form of a sterile injectable
suspension. Such a suspension may be formulated according to known
methods using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1,4-butanediol. Sterile, fixed oils are conventionally
employed as a solvent or suspending medium. For this purpose, any
bland fixed oil may be employed including synthetic mono- or
diglycerides, fatty acids (including oleic acid), naturally
occurring vegetable oils like sesame oil, coconut oil, peanut oil,
cottonseed oil, etc., or synthetic fatty vehicles like ethyl oleate
or the like. Buffers, preservatives, antioxidants, and the like can
be incorporated as required.
[0033] Formulations contemplated for use in the practice of the
present invention may also be administered in the form of
suppositories for rectal administration of the active ingredients.
These compositions may be prepared by mixing the active ingredients
with a suitable non-irritating excipient, such as cocoa butter,
synthetic glyceride esters of polyethylene glycols (which are solid
at ordinary temperatures, but liquify and/or dissolve in the rectal
cavity to release the active ingredients), and the like.
[0034] In addition, sustained release systems, including
semi-permeable polymer matrices in the form of shaped articles
(e.g., films or microcapsules) can also be used for the
administration of the active compound employed herein. The
CSF5-hyaluronidase can also be provided as a unit dosage such as a
septum-sealed vial, either lyophilized or in aqueous solution.
[0035] The amount of CSF5-hyaluronidase administered to a patient
will vary depending upon the condition to be treated, the severity
of the condition, and the response of the patient to the treatment.
In general, the amount of CSF5-hyaluronidase administered is
between about 0.01 .mu.g/kg and 1,000 mg/kg, preferably between
about 0.1 .mu.g/kg and 100 mg/kg, and more preferably between about
1 .mu.g/kg and 10 mg/kg. Dosage optimization can be performed using
standard dose-response curves.
EXAMPLE 1
PURIFICATION OF HUMAN HYALURONIDASE-CSF5
[0036] To two liters of human plasma (Irwin Memorial Blood Bank,
San Francisco, CA), 0.02% sodium azide, 50 mM NaCI, 5% sucrose and
7.5% Triton X-114 (Boehringer Mannheim, Indianapolis, IN) were
dissolved at 4.degree. C. with stirring for 90 min followed by
centrifugation at 10,000 x g for 30 min. The plasma was then
subjected to temperature-induced phase extraction at 37.degree. C.
The extract was centrifuged at 10,000 x g for 30 min at 37.degree.
C. to clarify the two phases. The detergent-rich phase was removed
and diluted to 2 L with ice cold 50 mM
(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (HEPES),
pH 7.5, 0.15M NaCI, followed by repartitioning at 37C.degree. with
centrifugation. This washing procedure was repeated three times.
The final detergent phase was diluted sixfold with 25 mM
(2-[N-Morpholino]ethanesulfonic acid) (MES), pH 6.0, and 20 mL of
equilibrated SPSepharose cation exchange resin was added
(Pharmacia, Piscataway NJ) and stirred overnight at 4.degree. C.
The beads were collected by centrifugation and washed with 25 mM
MES, pH 6.0, containing 46 mM octylglucoside (Boehringer Mannheim).
CSF5- Hyaluronidase was eluted from the beads by the addition of
0.3 M NaCI in MES buffer pH 6.0 with several washes. The
SP-Sepharose eluant was concentrated by ultrafiltration using a YM3
membrane (Amicon, Beverly, MA) and desalted into 10 mM PO4 pH 7.4
with 25 mM NaCI, 46 mM octylglucoside on a FPLC. Fast-Desalting
column (Pharmacia). The hyaluronidase preparation was then combined
with 10 mL of hydroxyapatite resin (Biorad, Richmond, CA)
equilibrated in the same buffer, and left on a rocker overnight at
4.degree. C. CSF5- hyaluronidase did not adsorb to the resin and
was recovered in the supernatant. The supernatant was then
concentrated to 0.5 mL on a Centriplus YM3 concentrator (Amicon,
Beverly, MA), and applied to a 12.5% polyacrylamide gel on a Phast
Gel System (Pharmacia), then silver-stained according to the
manufacturer's instructions to ensure purity. Protein
determinations were measured throughout the purification using the
Lowry (Pierce, Rockford, IL) or Bradford (Biorad) assays with BSA
as a standard.
[0037] CSF5-Hyaluronidase partitioned into the temperature-induced
Triton X-114 detergent phase and gave a 60-fold enrichment. The
activity was very stable at 37.degree. C. in the presence of
non-ionic detergents. Removal of Triton X-114 was performed by
batch absorption onto a SP-Sepharose cation exchanger resin. The
post SPSepharose preparation could be purified to homogeneity as
determined by silver staining. Batch adsorption using
hydroxyapatite resin, resulted in an overall purification of
1.5-million fold. The specific activity of the enzyme activity of
the CSF5-hyaluronidase (100,000 rTRU/mg) was approximately
equivalent that of the reported values for the sperm hyaluronidase,
PH-20 (Harrison, Biochem. J. 252:865-874, 1988), thereby ruling out
contamination of the enzyme factor with a minor colony stimulating
factor contaminant. The protein migrated on SDSPAGE with a relative
molecular mass of 57 kDa.
EXAMPLE 2
GENERATION OF ANTI-CSF5-HYALURONIDASE MONOCLONAL ANTIBODIES
[0038] Six week-old female BALB/c mice were immunized using
purified antigen from the post hydroxyapatite step described in
Example 1 using established procedures (Harlow, Antibodies: a
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, 1988). Hybridomas were obtained by fusion of spleen
cells and myeloma cells using standard Ed Harlow, D. L. Antibodies:
a laboratory manual Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY. 1988). Hybridomas secreting anti-CSF5-hyaluronidase
antibodies were screened by a modified enzyme capture assay. The
bHA (Frost et al. 1997a Anal Biochem. 251:263-9.) enzyme substrate
was coated onto Covalink plates (Placerville, NJ) under the same
conditions as those described for the microtiter based enzyme assay
(Frost et al. 1997a) except that 1.25/.mu.g/well of goat anti-mouse
IgG (Jackson Immunolabs, West Grove, PA) was included with the bHA
so that both bHA and goat anti-mouse IgG were covalently coupled to
the plates. Hybridoma supernatants were incubated with diluted
human plasma for 60 min at 37.degree. C. followed by incubation in
the bHA/antimouse-IgG plates for 60 min at 37.degree. C. Plates
were washed 5 times with PBS containing 1% Triton X-100, and 10
mg/ml BSA followed by the addition of formate assay buffer and
incubation at 37.degree. C. for 60 min. Digested bHA as a result of
immunoprecipitated CSF5-hyaluronidase was detected as in the
standard assay.
[0039] An enzyme capture assay was developed for screening
hybridomas that exploited the lack of activity of CSF5-
hyaluronidase at neutral pH and the fact that the protein had no
binding affinity for HA above pH 4.5, as determined by HA-Sepharose
affinity chromatography. The hybridoma supernatants were incubated
with crude plasma at neutral pH in the bHA/anti-mouse IgG
microtiter plates to immunoprecipitate the antibody-antigen
complex. Eight clones were identified from twenty hybridoma fusion
plates using this screening procedure. One clone of the IgG2a
class, 17E9, was used to generate ascites. Addition of serial
dilutions of the 17E9 antibody to human plasma followed by
immunoprecipitation with Protein-A resulted in precipitation of all
detectable acid-active hyaluronidase activity.
EXAMPLE 3
IMMUNOPRECIPITATION AND IMMUNOAFFINITV PURIFICATIONS
[0040] Purified IgG2a from the 17E9 anti-CSF5-hyaluronidase
hybridoma clone prepared as described in Example 2 was used for
routine immunoprecipitation and purifications. For the
immunoprecipitation of CSF5-hyaluronidase from plasma, serial
dilutions of purified 17E9 IgG or control mouse lgG2a were mixed
with plasma diluted in RIPA buffer (1% NP40,1% deoxycholate, 1%
Triton X-100, 5 mM EDTA in PBS), followed by immunoprecipitation
with protein-A beads. Residual CSF5-hyaluronidase activity in the
supernatant was then measured in the microtiter assay. For the
immunoaffinity purification of CSF5- hyaluronidase, 3 mg of
purified IgG from the 17E9 hybridoma clone was coupled to a 1 mL
HiTrap-NHS activated column (Pharmacia). Plasma or HEK-293 human
embryonic kidney cell recombinant CSF5-hyaluronidase conditioned
media was diluted 1:2 with RIPA buffer, and passed over the
anti-CSF5-hyaluronidase IgG column. The column was first washed
with PBS containing 2M NaCI, 100 mM octylglucoside followed by
washing with 100 mM citrate pH 4.0, 0.15M NaCI and octylglucoside,
and then eluted with the same buffer adjusted to pH 3.0.
[0041] Hyaluronidase could be purified to homogeneity in a single
step from human plasma by immunoaffinity chromatography using the
17E9 antibodies. After washing the column under stringent
conditions, the enzyme eluted at pH 4.0 and was purified to
homogeneity as determined by SDS-PAGE and amino acid sequencing.
Three sequences were obtained from CNBr digests of immunopurified
protein.
EXAMPLE 4
AMINO ACID SEQUENCING OF CSF5-HYALURONIDASE
[0042] For N-terminal amino acid sequencing, the immunoaffinity
purified protein was electroblotted from an SDS gel to a PVDF
membrane (ABI, Foster City, CA) and sequenced by Edman degradation.
Internal peptides of immunoaffinity purified CSF5- hyaluronidase
were obtained through digestion with cyanogen bromide (CNBr)
followed by fragment separation on an HPLC (Vydac C-18) column.
[0043] The nucleotide and amino acid sequences of CSF5-
hyaluronidase are shown in SEQ ID NOS: 8 and 9, respectively. The
N-terminal and internal amino acid sequences of CSF5-hyaluronidase
are 100% identical to the conceptual translation of the cDNA.
Alignment (Frohman et al., Proc. Natl. Acad. Sci. U.S.A.
85:8998-9002,1988)) of the predicted translation of colony
stimulating factor and human PH-20 indicated 40% sequence identity
and 60% homology at the amino acid level. PH-20 is a sperm specific
neutral-active hyaluronidase. The homology between a strictly
acid-active hyaluronidase and PH-20 suggests that all mammalian
f3,1-4 hyaluronidases may be members of a conserved family.
EXAMPLE 5
CSF5-HYALURONIDASE CDNA CLONING
[0044] A TBLASTN (Altschul et al., J Mol. Biol. 215:403-410, 1990)
homology search (compares a protein sequence against a nucleotide
sequence database translated in all reading frames) of the
Expressed Sequence Tag (EST) database (Lennon et al., Genomics
33:151-152, 1996) revealed an I.M.A.G.E. Consortium clone (Lennon
et al., supra.) (GenBank Accession No. AA223264) which was 100%
identical to the N-terminal amino acid sequence of determined in
accordance with Example 4. This EST is available from Genome
Systems (St. Louis, MO) and is 2 kb including the poly-A tail at
the 3' end. To obtain the 5' end of the cDNA, 5' RACE (Boguski et
al., Nature Genetics 4:332.333, 1993) was performed on a Marathon
Ready.TM. human heart cDNA library (Clontech Laboratories, Inc.,
Palo Alto, CA) according to the manufacturer's instructions, with
some modifications. Briefly, for the first PCR reaction, the
following primers were used: HPHRACE1
(5-ATCGAAGACACTGACATCCACGTCCACACC-3') (SEQ ID NO: 1) and the
Adapter Primer 2 (AP2) from Clontech (5'-
ACTCACTATAGGGCTCGAGCGGC-3') (SEQ ID NO: 2); annealing/extension was
at 73.degree. C. for 40 cycles. Advantage.sup.TM KlenTaq polymerase
mix (Clontech) was used to provide a "hot start". A diffuse band of
800 by was observed on agarose gel electrophoresis. The band was
excised using a QIAquick gel extraction kit (Qiagen Inc.
Chatsworth, CA) according to the manufacturer's instructions. The
excised DNA was used as a template for a second nested PCR using
primer HPHRACE2 (5'-TGCCTCTCCAGGCACCACTGGGTGTTTGC-3') (SEQ ID NO:
3) with the AP2 primer (SEQ ID NO: 2); annealing/extension was at
72.degree. C. for 15 cycles. A "hot start" was employed as
described above. A single sharp band of 800 by was observed on
agarose gel electrophoresis. 120 ng of the PCR product was ligated
into the TA cloning vector pCR2.1 (Invitrogen, San Diego, CA) and
used to transform One Shot TOP 1OF' competent cells according to
the manufacturer's instructions. Positive colonies were sequenced
as above. The 800bp product exhibited 100% overlap with the 5' end
of the EST by 300 bp.
[0045] For generation of the CSF5-hyaluronidase cDNA coding
sequence, a PCR reaction was performed using the EST as template
with the following primers: HPHF1 5'-GTGCCATGGCAGCCCACC-3' (SEQ ID
NO: 4) and HPHR1 5'-ATCACCACATGCTCTTCCGC-3' (SEQ ID NO: 5) with
annealing at 58.degree. C. for 35 cycles. 120 ng of the PCR product
was cloned into the TA expression vector pCR3.1-Uni (Invitrogen,
San Diego, CA) and used to transform One Shot TOPIOF' competent
cells according to the manufacturer's instructions. Colony
stimulating factor in the pCR3.1-Uni expression vector was purified
from positive colonies and verified by restriction mapping with Pst
I and Dra III. The insert was sequenced by standard methods and
found to contain a complete open reading frame which was 100%
identical to the HYAL1 gene (SEQ ID NO: 8) described in (Frost et
al. 1997) and in GenBank Accession No. UO3056 (Wei et
al.,1996).
EXAMPLE 6
EXPRESSION OF RECOMBINANT CSFS-HYALURONIDASE IN HUMAN EMBRYONIC
KIDNEY CELLS
[0046] To substantiate the identity of colony stimulating factor
with the cloned gene, the cDNA was stably transfected into human
embryonic kidney (HEK-293) cells. The cDNA was amplified from the
EST and then subcloned into a unidirectional expression vector.
This vector was used to generate HEK-293 clones overexpressing
hyaluronidase activity.
[0047] The CSF5-hyaluronidase containing vector was transfected
into 75% confluent T75 flasks of human embryonic kidney (HEK-293)
cells for five hours in the absence of serum using 9 .mu.g of
purified plasmid and 60 .mu.l of Lipofectin (Gibco BRL) in 20 mL of
DME/F12 50/50. The transfected cells were then grown for an
additional 48 h in DME/F12 50/50 mix containing 10% fetal bovine
serum (FBS). After 48 h, cells were plated by limited dilution into
24 well plates in the presence 500.mu.g/ml G418 to select for
neomycin resistance. After 14 days, the conditioned media of
resistant colonies was assayed for hyaluronidase activity using the
protocol described herein. Colonies with high level expression were
then expanded. For the analysis of the recombinant CSF5-
hyaluronidase and comparison with the biochemically purified
protein, a recombinant overexpressing hyaluronidase HEK 293 cell
line was grown for 48 h in serum free medium, and the conditioned
medium was passed over a 17E9 anti-CSF immunoaffinity column.
Recombinant enzyme eluted using the same protocol as for human
plasma. Purified recombinant hyaluronidase was then blotted to PVDF
and subjected to N-terminal amino acid sequencing to ensure
authenticity.
[0048] The parental HEK 293 cell line produced undetectable levels
of hyaluronidase in the conditioned media and cell layer whereas
the stably transfected clones secreted approximately 15 rTRU/ml, a
3,000 fold increase. To ensure that the hyaluronidase activity
found in the recombinant HEK-293 cell clones was the product of the
transfected cDNA, the hyaluronidase was immunoaffinity purified
from serum free conditioned medium of the HEK-293 overexpressing
clone and sequenced the eluent from the 17E9 column. This yielded
the same processed N-terminus (FR6PLLVP) found in human plasma and
a migrated as a single band on SDSPAGE. This band aligned with the
purified plasma using both silver stain and substrate gel
zymography. A commercial preparation of testicular hyaluronidase
(3,000 TRU/mg solid) was run for comparison of the specific
activity. The pH activity curve of recombinant colony stimulating
factor has the same profile as the immunoaffinity-purified plasma
enzyme, with no activity in vitro above pH 4.5, in contrast to
bovine testicular hyaluronidase, which has maximal activity above
pH 7.
EXAMPLE 7
ORGAN SURVEY OF CSF5-HYALURONIDASE TRANSCRIPTS
[0049] Nested PCR primers amplifying the 1.3 kb coding region of
the colony stimulating factor cDNA were used to analyze the tissue
distribution of transcripts in .lamda.gt10 cDNA libraries. For the
first round of PCR the following primers were used: I-IPHF2
(5'-AGGTTGTCCTCGACCAGTC-3') (SEQ ID NO: 6) and HPHR2
(5'ATGTGCAACTCAGTGTGTGGC-3') (SEQ ID NO: 7) at an annealing
temperature of 58''C. The second PCR reaction consisted of 15
cycles at an annealing temperature of 58.degree. C. with primers
HPHF1 and HPHR1 (see above). PCR products were found in heart,
kidney, liver, lung, placenta, and skeletal muscle, but were not
detected in brain.
EXAMPLE 8
STIMULATION OF MONOCYTE COLONY FORMATION BY CSF5-HYALURONIDASE
[0050] Colony stimulating activity of CSF5-hyaluronidase was
determined in serum free culture using recombinant
CSF5-hyaluronidase supernatant from HEK293 cells. Briefly, whole
blood from normal donors was collected in EDTA. Blood was diluted
1:2 in phosphate buffered saline (PBS) and overlayed in a 2:1 ratio
onto Lymphoprep. Samples were centrifuged at 1,500 x g for 20 min
and the lymphocyte band was removed. Cells were washed twice with
serum-free Dulbecco's Modified Eagle Medium (DMEM) and plated serum
free in 24 well dishes in DMEM for 1 hour at 37.degree. C. Plates
were then washed twice with serum free DMEM, and remaining adherent
peripheral blood mononuclear cells were used for colony forming
assays.
[0051] In order to determine the colony forming activity of
CSF5-hyaluronidase, HEK293 cells overexpressing CSF5-hyaluronidase
as described in Example 6 were grown serum free in HEK293SFM medium
(Gibco BRL) for six days with an innoculum of 1 x 10.sup.5
cells/ml. As a control, HEK293 cells not expressing CSF5- were
grown under identical conditions with the same innoculum for six
days. The amount of CSF5-hyaluronidase activity present in the
media after six days was determined by an enzyme based assay based
upon an approximate specific activity of 100,000 TRU/mg protein
(Frost et al. 1997a). The results are shown in Table 3. The half
maximal stimulation of monocyte colony formation occurred at about
5 ng/ml hyaluronidase.
TABLE-US-00001 Concentration of HYAL1 Monocyte Colony Control HEK
Media Monocyte Colony (ng/ml) via specific activity Formation % of
FBS Dilutions Formation 100 ng + + + + 1:1 0 (10 TRU/ml) (OTRU/ml)
50 ng/ml + + + + 1:2 0 (5 TRU/ml) (OTRU/ml) 25 + + + + 1:4 0 (2.5
TRU/ml) (OTRU/ml) 12.5 + + 1:8 0 (1.25 TRU/ml) (OTRU/ml) 6.26 +
1:16 0 (0.625 TRU/ml) (OTRU/ml) 1 ng/ml 0 1:32 0 (0.1 TRU/ml)
(OTRU/ml)
[0052] CSF5-hyaluronidase media or corresponding control media from
HEK control cells was applied in serial dilutions in HEK293SFM to
adherent peripheral blood mononuclear cells (PBMC). Cells were
cultured in diluted CSF5-hyaluronidase for ten days. Cellular
proliferation was observed at day 10 by fixation of cells in
methanol containing 1% crystal violet and observed under an
inverted Leitz microscope. Resultant colonies were determined to be
of monocytic morphology by nuclear staining with Giemsa.
[0053] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit and scope of that which is described and claimed.
Sequence CWU 1
1
10130DNAArtificial SequencePCR primer 1atcgaagaca ctgacatcca
cgtccacacc 30223DNAArtificial SequencePCR primer 2actcactata
gggctcgagc ggc 23329DNAArtificial SequencePCR primer 3tgcctctcca
ggcaccactg ggtgtttgc 29418DNAArtificial SequencePCR primer
4gtgccatggc agcccacc 18520DNAArtificial SequencePCR primer
5atcaccacat gctcttccgc 20619DNAArtificial SequencePCR primer
6aggttgtcct cgaccagtc 19721DNAArtificial SequencePCR primer
7atgtgcaact cagtgtgtgg c 2182517DNAHomo sapiensCDS(617)..(1921)
8ttcctccagg agtctctggt gcagctgggg tggaatctgg ccaggccctg cttaggcccc
60catcctgggg tcaggaaatt tggaggataa ggcccttcag ccccaaggtc agcagggacg
120agcgggcaga ctggcgggtg tacaggaggg ctgggttgac ctgtccttgg
tcactgaggc 180cattggatct tcctccagtg gctgccagga tttctggtgg
aagagacagg aaggcctccc 240ccccttggtc gggtcagcct gggggctgag
ggcctggctg tcagccactc ttcccagaac 300atatgtcatg gcctcagtgg
ctcatgggga agcaggggtg ggcgagctta ggctagagca 360agtcctgtgg
gagatggcag aggcctggtc tgagaggcaa ctcggatgtg ccctccagtg
420gccatgctcc cctccatgcg tctcccctgc cctcctggag ccctgcaggt
caatgtttaa 480cagaaaccag agcagcggtg gattaatgcg caagggctca
gccccccagc cctgagcagt 540gggggaatcg gagactttgc aacctgttct
cagctctgcc tcccctggcc aggttgtcct 600cgaccagtcc cgtgcc atg gca gcc
cac ctg ctt ccc atc tgc gcc ctc ttc 652Met Ala Ala His Leu Leu Pro
Ile Cys Ala Leu Phe1 5 10ctg acc tta ctc gat atg gcc caa ggc ttt
agg ggc ccc ttg cta ccc 700Leu Thr Leu Leu Asp Met Ala Gln Gly Phe
Arg Gly Pro Leu Leu Pro 15 20 25aac cgg ccc ttc acc acc gtc tgg aat
gca aac acc cag tgg tgc ctg 748Asn Arg Pro Phe Thr Thr Val Trp Asn
Ala Asn Thr Gln Trp Cys Leu 30 35 40gag agg cac ggt gtg gac gtg gat
gtc agt gtc ttc gat gtg gta gcc 796Glu Arg His Gly Val Asp Val Asp
Val Ser Val Phe Asp Val Val Ala45 50 55 60aac cca ggg cag acc ttc
cgc ggc cct gac atg aca att ttc tat agc 844Asn Pro Gly Gln Thr Phe
Arg Gly Pro Asp Met Thr Ile Phe Tyr Ser 65 70 75tcc cag ctg ggc acc
tac ccc tac tac acg ccc act ggg gag cct gtg 892Ser Gln Leu Gly Thr
Tyr Pro Tyr Tyr Thr Pro Thr Gly Glu Pro Val 80 85 90ttt ggt ggt ctg
ccc cag aat gcc agc ctg att gcc cac ctg gcc cgc 940Phe Gly Gly Leu
Pro Gln Asn Ala Ser Leu Ile Ala His Leu Ala Arg 95 100 105aca ttc
cag gac atc ctg gct gcc ata cct gct cct gac ttc tca ggg 988Thr Phe
Gln Asp Ile Leu Ala Ala Ile Pro Ala Pro Asp Phe Ser Gly 110 115
120ctg gca gtc atc gac tgg gag gca tgg cgc cca cgc tgg gcc ttc aac
1036Leu Ala Val Ile Asp Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe
Asn125 130 135 140tgg gac acc aag gac att tac cgg cag cgc tca cgg
gca ctg gta cag 1084Trp Asp Thr Lys Asp Ile Tyr Arg Gln Arg Ser Arg
Ala Leu Val Gln 145 150 155gca cag cac cct gat tgg cca gct cct cag
gtg gag gca gta gcc cag 1132Ala Gln His Pro Asp Trp Pro Ala Pro Gln
Val Glu Ala Val Ala Gln 160 165 170gac cag ttc cag gga gct gca cgg
gcc tgg atg gca ggc acc ctc cag 1180Asp Gln Phe Gln Gly Ala Ala Arg
Ala Trp Met Ala Gly Thr Leu Gln 175 180 185ctg ggg cgg gca ctg cgt
cct cgc ggc ctc tgg ggc ttc tat ggc ttc 1228Leu Gly Arg Ala Leu Arg
Pro Arg Gly Leu Trp Gly Phe Tyr Gly Phe 190 195 200cct gac tgc tac
aac tat gac ttt cta agc ccc aac tac acc ggc cag 1276Pro Asp Cys Tyr
Asn Tyr Asp Phe Leu Ser Pro Asn Tyr Thr Gly Gln205 210 215 220tgc
cca tca ggc atc cgt gcc caa aat gac cag cta ggg tgg ctg tgg 1324Cys
Pro Ser Gly Ile Arg Ala Gln Asn Asp Gln Leu Gly Trp Leu Trp 225 230
235ggc cag agc cgt gcc ctc tat ccc agc atc tac atg ccc gca gtg ctg
1372Gly Gln Ser Arg Ala Leu Tyr Pro Ser Ile Tyr Met Pro Ala Val Leu
240 245 250gag ggc aca ggg aag tca cag atg tat gtg caa cac cgt gtg
gcc gag 1420Glu Gly Thr Gly Lys Ser Gln Met Tyr Val Gln His Arg Val
Ala Glu 255 260 265gca ttc cgt gtg gct gtg gct gct ggt gac ccc aat
ctg ccg gtg ctg 1468Ala Phe Arg Val Ala Val Ala Ala Gly Asp Pro Asn
Leu Pro Val Leu 270 275 280ccc tat gtc cag atc ttc tat gac acg aca
aac cac ttt ctg ccc ctg 1516Pro Tyr Val Gln Ile Phe Tyr Asp Thr Thr
Asn His Phe Leu Pro Leu285 290 295 300gat gag ctg gag cac agc ctg
ggg gag agt gcg gcc cag ggg gca gct 1564Asp Glu Leu Glu His Ser Leu
Gly Glu Ser Ala Ala Gln Gly Ala Ala 305 310 315gga gtg gtg ctc tgg
gtg agc tgg gaa aat aca aga acc aag gaa tca 1612Gly Val Val Leu Trp
Val Ser Trp Glu Asn Thr Arg Thr Lys Glu Ser 320 325 330tgt cag gcc
atc aag gag tat atg gac act aca ctg ggg ccc ttc atc 1660Cys Gln Ala
Ile Lys Glu Tyr Met Asp Thr Thr Leu Gly Pro Phe Ile 335 340 345ctg
aac gtg acc agt ggg gcc ctt ctc tgc agt caa gcc ctg tgc tcc 1708Leu
Asn Val Thr Ser Gly Ala Leu Leu Cys Ser Gln Ala Leu Cys Ser 350 355
360ggc cat ggc cgc tgt gtc cgc cgc acc agc cac ccc aaa gcc ctc ctc
1756Gly His Gly Arg Cys Val Arg Arg Thr Ser His Pro Lys Ala Leu
Leu365 370 375 380ctc ctt aac cct gcc agt ttc tcc atc cag ctc acg
cct ggt ggt ggg 1804Leu Leu Asn Pro Ala Ser Phe Ser Ile Gln Leu Thr
Pro Gly Gly Gly 385 390 395ccc ctg agc ctg cgg ggt gcc ctc tca ctt
gaa gat cag gca cag atg 1852Pro Leu Ser Leu Arg Gly Ala Leu Ser Leu
Glu Asp Gln Ala Gln Met 400 405 410gct gtg gag ttc aaa tgt cga tgc
tac cct ggc tgg cag gca ccg tgg 1900Ala Val Glu Phe Lys Cys Arg Cys
Tyr Pro Gly Trp Gln Ala Pro Trp 415 420 425tgt gag cgg aag agc atg
tgg tgattggcca cacactgagt tgcacatatt 1951Cys Glu Arg Lys Ser Met
Trp 430 435gagaacctaa tgcactctgg gtctggccag ggcttcctca aatacatgca
cagtcataca 2011agtcatggtc acagtaaaga gtacactcag ccactgtcac
aggcatattc cctgcacaca 2071catgcatact tacagactgg aatagtggca
taaggagtta gaaccacagc agacaccatt 2131cattccatgt ccatatgcat
ctacttggca aggtcataga caattcctcc agagacactg 2191agccagtctt
tgaactgcag caatcacaaa ggctgacatt cactgagtgc ctactctttg
2251ccaatccccg tgctaagcgt tttatgtgga cttattcatt cctcacaatg
aggctatgag 2311gaaactgagt cactcacatt gagagtaagc acgttgccca
aggttgcaca gcaagaaaag 2371ggagaagttg agattcaaac ccaggctgtc
tagctccggg ggtacagccc ttgcactcct 2431actgagtttg tggtaaccag
ccctgcacga cccctgaatc tgctgagagg caccagtcca 2491gcaaataaag
cagtcatgat ttactt 25179435PRTHomo sapiens 9Met Ala Ala His Leu Leu
Pro Ile Cys Ala Leu Phe Leu Thr Leu Leu1 5 10 15Asp Met Ala Gln Gly
Phe Arg Gly Pro Leu Leu Pro Asn Arg Pro Phe 20 25 30Thr Thr Val Trp
Asn Ala Asn Thr Gln Trp Cys Leu Glu Arg His Gly 35 40 45Val Asp Val
Asp Val Ser Val Phe Asp Val Val Ala Asn Pro Gly Gln 50 55 60Thr Phe
Arg Gly Pro Asp Met Thr Ile Phe Tyr Ser Ser Gln Leu Gly65 70 75
80Thr Tyr Pro Tyr Tyr Thr Pro Thr Gly Glu Pro Val Phe Gly Gly Leu
85 90 95Pro Gln Asn Ala Ser Leu Ile Ala His Leu Ala Arg Thr Phe Gln
Asp 100 105 110Ile Leu Ala Ala Ile Pro Ala Pro Asp Phe Ser Gly Leu
Ala Val Ile 115 120 125Asp Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe
Asn Trp Asp Thr Lys 130 135 140Asp Ile Tyr Arg Gln Arg Ser Arg Ala
Leu Val Gln Ala Gln His Pro145 150 155 160Asp Trp Pro Ala Pro Gln
Val Glu Ala Val Ala Gln Asp Gln Phe Gln 165 170 175Gly Ala Ala Arg
Ala Trp Met Ala Gly Thr Leu Gln Leu Gly Arg Ala 180 185 190Leu Arg
Pro Arg Gly Leu Trp Gly Phe Tyr Gly Phe Pro Asp Cys Tyr 195 200
205Asn Tyr Asp Phe Leu Ser Pro Asn Tyr Thr Gly Gln Cys Pro Ser Gly
210 215 220Ile Arg Ala Gln Asn Asp Gln Leu Gly Trp Leu Trp Gly Gln
Ser Arg225 230 235 240Ala Leu Tyr Pro Ser Ile Tyr Met Pro Ala Val
Leu Glu Gly Thr Gly 245 250 255Lys Ser Gln Met Tyr Val Gln His Arg
Val Ala Glu Ala Phe Arg Val 260 265 270Ala Val Ala Ala Gly Asp Pro
Asn Leu Pro Val Leu Pro Tyr Val Gln 275 280 285Ile Phe Tyr Asp Thr
Thr Asn His Phe Leu Pro Leu Asp Glu Leu Glu 290 295 300His Ser Leu
Gly Glu Ser Ala Ala Gln Gly Ala Ala Gly Val Val Leu305 310 315
320Trp Val Ser Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile
325 330 335Lys Glu Tyr Met Asp Thr Thr Leu Gly Pro Phe Ile Leu Asn
Val Thr 340 345 350Ser Gly Ala Leu Leu Cys Ser Gln Ala Leu Cys Ser
Gly His Gly Arg 355 360 365Cys Val Arg Arg Thr Ser His Pro Lys Ala
Leu Leu Leu Leu Asn Pro 370 375 380Ala Ser Phe Ser Ile Gln Leu Thr
Pro Gly Gly Gly Pro Leu Ser Leu385 390 395 400Arg Gly Ala Leu Ser
Leu Glu Asp Gln Ala Gln Met Ala Val Glu Phe 405 410 415Lys Cys Arg
Cys Tyr Pro Gly Trp Gln Ala Pro Trp Cys Glu Arg Lys 420 425 430Ser
Met Trp 435108PRTHomo sapiens 10Phe Arg Gly Pro Leu Leu Val Pro1
5
* * * * *