U.S. patent application number 10/182088 was filed with the patent office on 2003-11-13 for myeloid colony stimulating factor and uses thereof.
Invention is credited to Borgstrom, Per, Frost, Gregory I..
Application Number | 20030212021 10/182088 |
Document ID | / |
Family ID | 29400881 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030212021 |
Kind Code |
A1 |
Frost, Gregory I. ; et
al. |
November 13, 2003 |
Myeloid colony stimulating factor and uses thereof
Abstract
The identification of the HYAL1 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) |
Correspondence
Address: |
Lisa A Haile J D
Gray Cary Ware & Freidenrich
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Family ID: |
29400881 |
Appl. No.: |
10/182088 |
Filed: |
November 26, 2002 |
PCT Filed: |
January 25, 2001 |
PCT NO: |
PCT/US01/02575 |
Current U.S.
Class: |
514/44R ;
424/94.61; 435/200 |
Current CPC
Class: |
C12N 9/2408 20130101;
A61K 48/00 20130101; A61K 38/193 20130101; A61K 38/47 20130101;
A61P 37/00 20180101 |
Class at
Publication: |
514/44 ;
424/94.61; 435/200 |
International
Class: |
A61K 048/00; A61K
038/47; C12N 009/24 |
Claims
What is claimed is:
1. 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.
2. A method for increasing the number of myeloid progenitors in a
cell population, comprising the step of contacting said cell
population with an exogenously-derived CSF5-hyaluronidase.
3. A method for treating a mammal with a myelosuppressed condition,
comprising the step of administering to said mammal an effective
amount of exogenously-derived CSF5-hyaluronidase.
4. The method of claim 3, wherein said CSF5-hyaluronidase is
administered in conjunction with a treatment selected from the
group consisting of surgery, radiation therapy and
chemotherapy.
5. The method of claim 3, wherein said myelosuppression is
associated with radiation, chemotherapy or viral infection.
6. 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.
7. The method of claim 6, wherein said nucleic acid is in an
expression vector.
8. The method of claim 7, wherein said nucleic acid is operably
linked to a promoter.
9. A method for enhancing the production of cytokines by myeloid
cells, comprising the step of contacting said myeloid cells with
exogenously-derived CSF5-hyaluronidase.
10. The method of claim 9, wherein said cytokine is selected from
the group consisting of interferon, interleukin, tumor necrosis
factor and myeloid colony stimulating factor.
11. Use of CSF5-hyaluronidase or DNA encoding CSF5-hyaluronidase in
the preparation of a medicament for treating myeloid-cell
insufficiency.
12. The use of claim 11, wherein said myeloid-cell insufficiency is
myelosuppression.
13. The use of claim 12, wherein said myeloid-cell insufficiency
results from radiation treatment, chemotherapy, or viral
infection.
14. The use of claim 11, wherein said myeloid-cell insufficiency is
characterized by insufficient production of at least one cytokine,
and the medicament facilitates production of that cytokine.
15. The use of claim 14, wherein said cytokine is selected from the
group consisting of interferon, interleukin, tumor necrosis factor
and myeloid colony stimulating factor.
Description
FIELD OF THE INVENTION
[0001] 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.
BACKGROUND THE INVENTION
[0002] 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.
[0003] 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).
[0004] 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
Jul. 25, 1991;266(21):13804-10).
[0005] 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.
[0006] 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 WO87/03204 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
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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
[0013] 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 Sep. 15, 1999 ;60(3):356-61). Hyaluronidase like
genes have been identified in other mammalian species, including
mouse and rat (Strobl et al, Genomics Oct. 15, 1998;53 (2):214-9)
(Genbank Accession Number 4104235). The orthologous relationship
between such genes has not been established in some species.
[0014] Prior to the present invention, no myelostimulative or
colony stimulating activity had been attributed to this
glycosaminoglycan-degrad- ing enzyme. The HYAL1 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.
[0015] 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.
[0016] 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 .mu.g/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.
[0017] 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.
[0018] 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.
[0019] 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
ot 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.
[0020] 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 mRNA). 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.
[0021] 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.
[0022] 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, N.Y., 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.
[0023] 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.
[0024] 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
proliferative 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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..
[0032] 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
[0033] To two liters of human plasma (Irwin Memorial Blood Bank,
San Francisco, Calif.), 0.02% sodium azide, 50 mM NaCl, 5% sucrose
and 7.5% Triton X-114 (Boehringer Mannheim, Indianapolis, Ind.)
were dissolved at 4.degree. C. with stirring for 90 min followed by
centrifugation at 10,000.times.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.times.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-hydroxyethyllpiperazine-N'-]2-ethanesulfonic acid]) (HEPES),
pH 7.5, 0.15M NaCl, followed by repartitioning at 37 C..degree.
with centrifugation. This washing procedure was repeated three
times. The final detergent phase was diluted six-fold with 25 mM
(2-[N-Morpholino]ethanesulfonic acid) (MES), pH 6.0, and 20 mL of
equilibrated SP-Sepharose cation exchange resin was added
(Pharmacia, Piscataway N.J.) 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 NaCl in MES buffer pH 6.0 with several washes. The SP-Sepharose
eluant was concentrated by ultrafiltration using a YM3 membrane
(Amicon, Beverly, Mass.) and desalted into 10 mM PO.sub.4 pH 7.4
with 25 mM NaCl, 46 mM octylglucoside on a FPLC. Fast-Desalting
column (Pharmacia). The hyaluronidase preparation was then combined
with 10 mL of hydroxyapatite resin (Biorad, Richmond, Calif.)
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,
Mass.), 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, Ill.) or Bradford (Biorad) assays with BSA
as a standard.
[0034] 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 SP-Sepharose 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 SDS-PAGE with a
relative molecular mass of 57 kDa.
EXAMPLE 2
Generation of Anti-CSF5-hyaluronidase Monoclonal Antibodies
[0035] 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, N.Y., 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, N.Y. 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, N.J.) 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/anti-mouse-IgG plates for 60 min at 37.degree. C. Plates
were washed 5 times with PBS containing 1% Triton X-100, and 10
mg/l 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.
[0036] 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 plasm followed by
immunoprecipitation with Protein-A resulted in precipitation of all
detectable acid-active hyaluronidase activity.
EXAMPLE 3
Immunoprecipitation and Immunoaffinity Purifications
[0037] 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 IgG2a 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 Hi-Trap-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 NaCl, 100 mM octylglucoside followed by
washing with 100 mM citrate pH 4.0, 0.15M NaCl and octylglucoside,
and then eluted with the same buffer adjusted to pH 3.0.
[0038] 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
[0039] For N-terminal amino acid sequencing, the immunoaffinity
purified protein was electroblotted from an SDS gel to a PVDF
membrane (ABI, Foster City, Calif.) 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.
[0040] The nucleotide and amino acid sequences of
CSF5-hyaluronidase are shown in SEC 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
.beta., 1-4 hyaluronidases may be members of a conserved family
EXAMPLE 5
CSF5-hyaluronidase cDNA Cloning
[0041] 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, Calif.) 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.TM. KlenTaq polymerase mix (Clontech) was used to provide
a "hot start". A diffuse band of 800 bp was observed on agarose gel
electrophoresis. The band was excised using a QIAquick gel
extraction kit (Qiagen Inc. Chatsworth, Calif.) according to the
manufacturer's instructions. The excised DNA was used as a template
for a second nested PCR using primer HPHRACE2
(5'-TGCCTCTCCAGGCACCACTGGGT- GTTTGC -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 bp was observed on agarose gel
electrophoresis. 120 ng of the PCR product was ligated into the TA
cloning vector pCR2.1 (Invitrogen, San Diego, Calif.) and used to
transform One Shot TOP10F' competent cells according to the
manufacturer's instructions. Positive colonies were sequenced as
above. The 800 bp product exhibited 100% overlap with the 5' end of
the EST by 300 bp.
[0042] For generation of the CSF5-hyaluranidase 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, Calif.) and used to transform One Shot TOP10F' 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. U03056 (Wei et al.,
1996).
EXAMPLE 6
Expression of Recombinant CSF5-hyaluronidase in Human Embryonic
Kidney Cells
[0043] 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.
[0044] 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.
[0045] 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 (FRGPLLVP) found in human plasma and
a migrated as a single band on SDS-PAGE. 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
[0046] 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 .lambda.gt10 cDNA libraries. For the
first round of PCR the following primers were used: HPHF2
(5'-AGGTTGTCCTCGACCAGTC-3') (SEQ ID NO: 6) and HPHR2
(5'-ATGTGCAACTCAGTGTGTGGC-3') (SEQ ID NO: 7) at an annealing
temperature of 58.degree. 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
[0047] 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.times.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.
[0048] 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.times.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.
1 Monocyte Concentration of Colony Control Monocyte HYAL1 (ng/ml)
via Formation HEK Media Colony specific activity % of FBS Dilutions
Formation 100 ng (10TRU/ml) + + + + 1:1 0 (0 TRU/ml) 50 ng/ml + + +
+ 1:2 0 (5 TRU/ml) (0 TRU/ml) 25 + + + + 1:4 0 (2.5 TRU/ml) (0
TRU/ml) 12.5 + + 1:8 0 (1.25 TRU/ml) (0 TRU/ml) 6.26 + 1:16 0
(0.625 TRU/ml) (0 TRU/ml) 1 ng/ml 0 1:32 0 (0.1 TRU/ml) (0
TRU/ml)
[0049] 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.
[0050] 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.
* * * * *