FIELD OF THE INVENTION
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
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.
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).
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).
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.
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
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.
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.
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.
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.
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.
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
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.
Prior to the present invention, no myelostimulative or colony stimulating activity had been attributed to this glycosaminoglycan-degrading enzyme. The HYAL1 enzyme has high specificity and is present predominantly in human plasma at a concentration of 20-50 μg/ml (Frost et al, 1997). Because of its CSF activity, the HYAL1 gene product should be redefined as CSF5-hyaluronidase.
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.
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 μg/ml and 100 mg/ml, preferably between about 0.1 μg/ml and 10 mg/ml, and more preferably between about 1 μg/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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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..
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 μg/kg and 1,000 mg/kg, preferably between about 0.1 μg/kg and 100 mg/kg, and more preferably between about 1 μg/kg and 10 mg/kg. Dosage optimization can be performed using standard dose-response curves.