KR0140234B1 - Medical application of m-csf - Google Patents

Abstract

The present invention provides a medicinal use of M-CSF, in particular, a method for treating and treating inflammatory diseases and allergies using natural M-CSF or recombinant M-CSF or derivatives thereof.

Description

[Name of invention]

Medical use of M-CSF

Detailed description of the invention

(Technology)

The present invention relates to a method for treating and treating inflammatory diseases and allergies.

More curiously, the present invention is characterized by administering an effective amount of macrophage colony stimulator (hereinafter M-CSF) to inhibit interleukin 1 (hereinafter IL-1) biological activity and reduce the symptoms of inflammatory diseases. It relates to the treatment of patients with inflammatory diseases. The present invention is directed to a method for treating inflammatory disease patients characterized by administering an amount of M-CSF effective to produce an inhibitory factor of IL-1. The present invention also relates to a method of inhibiting IL-1 biological activity characterized by a pharmaceutically effective amount of M-CSF.

The present invention relates to a method of treating allergic diseases characterized by administering a pharmaceutically effective amount of M-CSF.

The invention also relates to novel anti-inflammatory and anti-allergic agents, more particularly anti-inflammatory and anti-allergic agents containing M-CSF as active ingredient.

(Background)

Inflammation is a complex chain change in body tissue that responds to injury caused by a number of agents, such as bacteria, trauma, chemicals, or fever. Inflammatory responses are a mechanism of resistance caused by the body in the presence of such agents, but inflammatory responses are severely painful for many patients suffering from inflammatory diseases such as rheumatoid arthritis and gout.

Inflammatory responses vary depending on the tissue involved, traits, etc., but there are some characteristics that characterize the response. These features include fluency in the microvascular system, leakage of blood elements into the interstitial space and migration of white blood cells to inflammatory tissues. This usually involves clinical signs of erythema, edema, sensitivity (ie hyperalgesia) and pain. This complex reaction also results in the release of chemical regulators such as histamine, 5-hydroxytryptamine (5-HT), an anaphylactic slow reactive substance (SRS-A), various chemotactic factors, bradykinin, and prostaglandins, and Action. In addition, phagocytes migrate to regions that cause disruption of the cell's lysosomal membrane and release of lysogenic enzymes. All of these may contribute to the inflammatory response.

Mechanisms of action on inflammatory responses have been the focus of attention since the earliest research periods for pharmaceuticals. Bark of willow and other plants has been used as a medicament in various civilizations for centuries. By the mid-18th century, it was reported that Leverend Edm unmd Stone explained the effectiveness of bark of willow in the treatment of intermittent fever (malaria), that is, fever.

In 1827, Laroux discovered that the active ingredient in the bark of the willow was alive, which eventually led to the production of silicic acid.

It is known that salicylic acid and aspirin-based drugs have been successful in alleviating various symptoms of the inflammatory response. However, this drug has serious side effects and disadvantages. For example, aspirin-based drugs inhibit or interfere with various other enzymes and cellular systems. In addition, in the treatment of inflammatory diseases associated with myasthenia disorders such as rheumatoid arthritis, osteoarthritis and ankylosing spondylitis, aspirin-based drugs only provide relief of symptoms from pain and inflammation associated with vaginal involvement, It does not prevent the progress of enemy injuries. Some doctors believe the drug may exacerbate the disease by allowing movement of arthritis joints, which would otherwise be impossible, which further promotes injury.

Aspirin-based drugs tend to cause gastric or intestinal ulceration and can sometimes be accompanied by secondary anemia resulting from resulting blood loss.

Other drugs previously used in the treatment of inflammatory diseases have the same serious side effects. For example, the pyrazolone derivative phenylbutazole was introduced in 1949 for the treatment of rheumatoid arthritis and related diseases. This is an effective anti-inflammatory agent, but there are serious toxicity problems with long term use. Phenylbutazone is poorly resistant in many patients with nausea, vomiting, dislocation discomfort and skin rashes. Diarrhea, dizziness, insomnia, gladness, neurosensitivity, hematuria and blurred vision symptoms have also been reported. More serious actions include peptic ulcer, hypersensitivity reaction, ulcerative stomatitis, hepatitis, nephritis, aplastic anemia and leukocytosis, and agranulocytosis accompanied by bleeding or perforation. In addition, many patients die due to aplastic anemia and agranulocytosis. Finally, treating the elderly with this drug is not suitable because the toxic effects of the drug are more severe for the elderly.

Indomethacin, the product of research findings to develop drugs with anti-inflammatory properties, was introduced in 1963 for the treatment of rheumatoid arthritis and related diseases such as acute gout. It is effective and widely used, but its use is often limited because of its toxicity. A very high percentage of patients (30-50%) experienced poor symptoms and about 20% reported that they had to stop using it. Gastrointestinal disorders include lack of appetite, nausea and abdominal pain. Central nervous system functioning includes severe frontal headache, dizziness, dizziness, mild mental disorders and delirium.

The aspirin-based medicines are generally used in accordance with stable and physical therapy for the treatment of inflammatory diseases such as rheumatoid arthritis. If the disease continues despite this treatment, the patient will generally receive the following treatment with gold or glucocorticoids.

The toxic effects of gold have severe toxicity within about 10% in the range of about 25-50% of patients, and are mainly associated with skin and mucous membranes. Mucosal lesions include stomatitis, gastritis, colitis and vaginitis. Serious blood diseases may arise from self-therapy. Thrombocytopenia sometimes occurs and causes many fatalities. Leukopenia, agranulocytosis and aplastic anemia may also occur. Synthetic homologs of glucocorticoids, cortisols and cordidazoles are also used in the treatment of inflammatory diseases such as rheumatoid arthritis because they have the activity of preventing or inhibiting the development of cognitive symptoms of inflammation, local fever, scar redness, swelling and sensitivity. Corticosteroids also inhibit the inflammatory response, whether the stimulant is radiation, mechanical causes, chemicals, infections or immune agents. However, corticosteroids merely inhibit the inflammatory response and inhibit the inflammatory response to the atrioventricular agent where the drug may simulate the progression of the disease. It is important that corticosteroid therapy may have to last for years or even once it begins.

Immunosuppressors developed in the study of therapies for neoplastic disease have been explored for use in inflammatory diseases such as body lupus erythema, hyperplastic vasculitis, scleroderma and rheumatoid arthritis. One of these agents, cyclophosphamide, has been the subject of many studies, but should only be used under warning. In addition to toxic effects such as nitrogen mustards, cyclophosphamide also has a high potential for causing infertility, germ cell development, mutations and cancer.

Despite the utility of these anti-inflammatory agents, scientists have continued to study the etiology of inflammatory diseases to find further better ways to treat inflammatory diseases. One of the most important recent findings is that the host response to injury and infection is associated with the production of interleukin 1 (IL-1). IL-1 is a polypeptide immunomodulatory cytokine produced mainly by mononuclear phenoxies having a sufficient effect in the body. IL-1 mediates tissue remodeling, repair and inflammation by helping to modulate the activity of cells such as endothelial cells, granulocytes, keel cells, chondrocytes, fibroblasts, hematopoietic cells, neurons and lymphocytes. Reference. J. W. Larrick Native interleukin 1 inhibitors, Reviews in Immunology Today 10; 61-66 (1989). Biological activities of IL-1 include activation of T helper cells, induction of fever, promotion of prostaglandin or collagenase production, neutrophil chemotaxis, induction of acute phase proteins and inhibition of iron levels in plasma. See U.S. Patent No. 4,794,114, Bender, RE et al., Inhibition of interleukin-1 production by monocytes and / or macrophages, December 27, 1988 publish.

Recent studies have exacerbated and / or aggravated diseases such as rheumatoid arthritis, osteoarthritis, toxic short symptoms, Reiter syndrome, gout, acute synovitis, and other acute or chronic inflammatory diseases such as inflammatory reactions caused by endotoxins. It is associated with excessive or unregulated IL-1 production in causing disease. See, U.S. Patent No. 4,794,114, Bender, et al., Inhibition of interleukin-1 production by monocytes and / or macrophages, issued December 27, 1988.

Therefore, researchers in this field have considered how to treat inflammatory diseases with inhibitors of IL-1. IL-1 inhibition mechanisms vary and include restriction of IL-1 transcription, interference with IL-1 binding by direct binding to the IL-1 receptor, and acting at other sites to limit the activity of the IL-1 receptor do.

Recent investigations by JW Larrick (Native interleukin linhibitors, in Immunology Today 10: 61-66 (1989)) include we-derived inhibitors (cf. PCT patent application WO 89/01946, PCT / US 88 / PCT). 02819, published by Dayer et al. March 9, 1989), uromodulin, monocyte-macrophage-derived inhibitors (see Hannum, CH, et al., Interleukin-l receptor antagonist activity of a human interleukin-1 Inhibitor, Nature 343: 336-3340 (January 25, 1990); Eisenberg, SP et al., Primary stucture and functional expression from complementary DNA of a human interleukin-1 receptor antagonist Nature 343: 341-346 (January 25, 1990) ), And IL-1 inhibitors, including virus-infected macrophages. More recent papers suggest that none of these inhibitors are purified and characterized. See, Hannum, C. H., et al., Interleukin-1 receptor antagonist activity of a humann interleukin-1 inhibitor, Nature 343: 336-3340 (January 25, 1990).

Non-oil inhibitors of IL-1 are also discussed. See, eg, US Patent No. 4,870,101, issued by G Ku, Method of inhiviting interleukin-1 release, 1989, 12. 26 (providing compounds having antioxidant properties), and US Patent No. 4,780,470 Bender et al., Inhibition of interleukin-1 by monocytes and / or macrophages published October 25, 1988 (about the use of 4,5-dinyl-2- (substituted imidazole)), 4,778,806 Bender et al., Inhibition of interleukin- 1 production by monocytes and / or macrophages published 1988, 10. 18 (for 2-2 '[1,3-propane-2-ondiyl-bis (thio)] bis-1H-imidazole). No. 4,794,114 Bender at al. Inhibition of interleukin-1 production by monocytes and / or macrophages published December 27, 1988 (pertaining to the use of diaryl-substituted imidazoles fused to thiazole pyrrolidine, thiazide or piperidine rings).

In spite of the many IL-1 inhibitors that are useful, it is also believed that only glycocorticoids inhibit IL-1 at the transcriptional and post-transcriptional levels. As mentioned above, glycocorticoids have serious side effects, especially when used in long-term liver therapy.

Thus, there is a need in the art for IL-1 inhibitors that can be used in the treatment of inflammatory diseases. This need can be best met by inhibitors of IL-1 that have been studied for beneficial effects in the treatment of other diseases. Side effects and any possible toxicology as well as resistance are known for such inhibitors. One of the agents of recent interest is a macrophage colony-stimulating factor, one of which acts as a hematopoietic growth factor. This agent is promising because it is known to be useful in the treatment of various diseases. For example, M-CSF can promote the function of leukocytes and therefore is effective as a medicament for the prevention and treatment of various infectious diseases. (Lopez, AF et al. J. Immunol., 131: 2983 (1983); Handam, E. et al., J. Immunol., 122: 1134 (1979); and Vadas. MA et al., J. Immunol , 130: 795 (1983). In addition, M-CSF is known to be effective for the treatment of myeloid leukemia because of its differentiation-inducing activity. (Metcalf. D. et al., Int. J. Cancer, 30: 773 (1982)). M-CSF is also thought to quench the white blood cell depletion resulting from cancer chemotherapy and radiation therapy. (European Patent Publication 261592).

However, the use of M-CSF in the treatment of inflammatory diseases is surprising because growth factor M-CSF was initially reported to promote rather than inhibit IL-1 production. For example, Moore, RN et al. Procedure of Lymphocyte Activating Factor [Ibterleukin 1] by Macrophages activated with colony stimulating factors, Journal fo Immunology, 125: 1302 (1980) and kurland, JI, et al. Induction of prostglandin E Synthesis in Normal and Neoplastic Macrophages: Role of Colony Stimulating Factors Distinct from Effects on Myeloid Progenitor Cell Proliferation, Proceedings of the National Academy of Sciences 76: 2326 (1979). Production of togglandin E ₂ (hereinafter PGE ₂ ) is discussed, and M-CSF is implicated in the progressive development of the inflammatory response because M-CSF is involved in the production of inflammatory mediators IL-1 and PGE ₂ .

(Initiation of invention)

The present invention can overcome the problems and disadvantages of the art, and administering an effective amount of macrophage-colony stimulator (hereinafter M-CSF) to inhibit interleukin 1 (hereinafter IL-1) biological activity In view of the prior art there is a surprising effect by providing a method for the treatment of patients with inflammatory diseases characterized. The present invention also relates to a method of treating an inflammatory disease patient characterized by administering an amount of M-CSF effective to cause the production of an inhibitor of IL-1. The present invention also relates to a method for inhibiting the biological activity of IL-1, characterized by administering a pharmaceutically effective amount of M-CSF.

The present invention also relates to a method for treating an allergic disease patient characterized by administering a pharmaceutically effective amount of M-CSF.

Based on the good anti-inflammatory and anti-allergic properties of M-CSF, the present invention also provides anti-inflammatory and anti-allergic agents which retain the M-CSF as an active ingredient.

Hereinafter, the therapeutic use of the above-mentioned inflammatory diseases and the induction of production of inhibitors of IL-1 will be described.

The accompanying drawings, which form a part of this specification, illustrate several embodiments of the present invention and, together with the description, support a description of the principles of the invention.

1 shows Northern blotting analysis of macrophage mRNAs illustrating the expression of IL-1, IL-6 and TGF-β.

2 is a radioautograph showing the expression of IL-1α and IL-6 mRNAs obtained from macrophages treated with LPS and M-CSF and a combination of LPS and M-CSF.

3 is a graph showing the bioavailability of IL-1 produced by macrophages in the presence of M-CSF, LPS, and a combination of LPS and M-CSF.

4 is a graph showing the production of IL-1 inhibitors in the presence of LPS, M-CSF, or a combination of LPS and M-CSF.

5 is a graph showing the lack of production of IL-6 inhibitors in the presence of LPS, M-CSF, or a combination of LPS and M-CSF.

FIG. 6 is a graph showing the separation of IL-1 inhibitors from IL-1 in QAE-52 column.

7 is a graph showing the binding capacity of IL-1 and IL-1 inhibitor.

8 is a graph showing the effect of IL-1 inhibitors on the action of TNF.

9 is a graph showing the effects of IL-1 inhibitors and IL-1 on T-cell responses to IL-2.

10 shows purification of IL-1 inhibitors in reversed phase HPLC C-4 columns, and the activity of IL-1 inhibitors bound to IL-1 receptors in terms of IL-1 bioassay, and inhibition of biologically active IL-1. It is a graph.

Reference is now made in more detail to preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings.

In the present invention, expressive inflammatory diseases include gout, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, scleroderma, shocken syndrome, mixed connective tissue vaginal swelling (MCTD), lighter syndrome, systemic hypertrophic vasculitis, sensitive vasculitis , Temporal arteritis, Wegener's granulomatosis, sarcoidosis, Kawasaki disease, Burger's disease, thickening infantoma, psoriatic arteritis, inflammatory diseases of the joints, insulin-resistant diabetes, histomoto thyroiditis, juvenile autoimmune diabetes, gravis gravis, Ulcerative colitis, cirrhosis and autoimmune uveitis.

Thus, the inflammatory conditions treated by the anti-inflammatory agents of the present invention include, but are not limited to, the foregoing allergic diseases, including inflammatory conditions resulting from the various diseases indicated by the inflammatory diseases exemplified above.

As used herein, the term M-CSF is a dense macrophage colony stimulating factor. In the present invention, M-CSF which can be used in the treatment of inflammatory diseases is one of the colony stimulating factor system which is a hematopoietic growth factor. Clony stimulating factor (hereafter CSF) promotes colony growth of hematopoietic cells derived from progenitor cells of the bone marrow, fetal liver and other hematopoietic organs. As indicated above, the CSF of particular interest is a macrophage colony stimulating factor (M-CSF, also known as CSF-1). M-CSF also promotes the proliferation and differentiation of the mononuclear phagocyte lineage of cells known as macrophages. Unlike other CSFs, such as granulocyte macrophage-CSF, which only exist only after inflammation, M-CSF is present in body fluids even in non-inflammatory states as a result of fibroblast production.

M-CSF refers to both resolving M-CSFs, including natural M-CSFs or derivatives, for example European Patent Publication Nos. 261592 and 328061; US Pat. Nos. 4,868,119 and 4,879,227. The M-CSF may be of human origin or of any other mammal, but is of human origin. A particularly desirable source of natural M-CSF is L929 fibroblasts obtained from the American type collection (hereinafter ATCC).

Recombinant M-CSF and its derivatives are described in detail below. For convenience, the M-CSF derivative is clearly indicated with reference to the amino acid primary sequence represented by the following formula (I).

Equation (1):

Wherein X represents Tyr or Asp.

Thus, based on the amino acid sequence from Glu at position 1 of Formula (1) to Val at position 522, various derivatives are shown which are obtained by modifying the amino acid sequence, for example by substitution, deletion and addition. . If desired, these derivatives are reduced in a conventional manner. For example, M-CSF- (3-153) or M-CSF- (4-153) has the amino acid sequence from Val at position 3 or Ser at position 4 to Thr at position 153 of formula (1) An active derivative consisting of a polypeptide is shown.

Recombinant M-CSF and its derivatives are suitably used in the present invention as long as it possesses the physiological activity of its own M-CSF. The M-CSF, or N-terminus, having the total amino acid primary sequence of Formula (1) is in the region from Glu at position 1 to Glu at position 5 of Formula (1) and the C-terminus is at position 153 M-CSF is present in the region from Thr to Val at position 522 and has an amino acid primary sequence to which Met may be added at the N-terminus. Particularly preferred examples are biologically active M-CSF derivatives and recombinant M-CSFs having an amino acid primary sequence extending from Val at position 3 of Formula (1) or Ser at position 4 to Thr at position 153 or Pro at position 214 Which may have Met added to the N-terminus.

In contrast to the techniques mentioned above, as described in an in vitro assay for measuring the expression of IL-1 and IL-6 in LPS treated macrophages, the M-CSF of the present invention is IL-1 or IL Does not promote gene expression of -6. In addition, as measured by in vitro analysis, the M-CSF of the present invention does not promote the production of IL-1, IL-6, or PGE ₂ from macrophages treated with LPS.

Instead, the M-CSF of the present invention can be used to treat patients diagnosed with inflammatory disease by administering an amount effective to inhibit IL-1 bioactivity. For example, as described in the Examples below, both pretreatment and adjuvant therapy with M-CSF are directed to lipopolysaccharide (LPS), a bacterial endotoxin known as an effective stimulator of in vitro macrophage function. It is possible to suppress the production of bioactive IL-1 produced in the reaction. Similarly, as described in the Examples below, pretreatment with M-CSF can inhibit the production of bioactive IL-1 produced in response to LPS in vivo.

The M-CSF of the present invention can also be used to treat a patient diagnosed with an inflammatory disease by administering an amount of M-CSF effective to cause the production of inhibitors of IL-1. As described in the Examples below, administration of M-CSF promotes the production of inhibitors of IL-1 that reduce the bioactivity of IL-1 in response to treatment with LPS in vitro. Interleukin-1 (hereinafter IL-1), as used herein, is a factor produced by macrophages, in particular after interaction with immune complexes, bacterial products or T cells. IL-1 promotes pleural cell proliferation and promotes the release of mature T cells with intrinsic growth promoting molecules. IL-1 also causes fever. Fever is considered to be an acute response to an antigen that makes T cell activity even higher.

As discussed above, IL-1 is an inflammatory short-term regulator. IL-1, as described in Larrick, JW et al., The role of tumer necrosis factor and interleukin-1 in the inflammation response, a review in Pharmaceutical Research 5 (3): 129-139 (1988). May modulate the inflammatory function of endothelial cells, leukocytes and fibroblasts, and may promote the accumulation of granulocytes at the site of inflammation. See, C. P. J. Maury, Interleukin and Pathogenesis of Inflammatory Diseases, Acta Medc Scand 220: 291-4 (1986).

IL-1 refers to native IL-1α, native IL-1β, recombinant IL-1α, recombinant IL-1β and derivatives of IL-1α or IL-1β, which are described in European Patent Publication No. 237073, European Patent Publication No. 237967 PCT Application Publication No. 89/01946 (Oppenheim, JJ et al., Immunol. Today 7: 45 (1986)). IL-1 may be of human origin or of any other mammal, and is preferably of human origin.

The term IL-1 inhibitor, as used herein, is a medicament capable of inhibiting the biological activity of IL-1, for example in the modulation or progression of inflammation. Biological activity is inhibited by Togawa, A. et al., Characterizaion of Lymphocyte Activating Factor Produced by Human Mononuclear Cells; Biochemical Relationship of High and Low Molecular weight Forms of LAF, J. Immunol. 122: 2112 (1979)] can be assessed using a number of IL-1 activity assays well known to those of skill in the art, such as the IL-1 bioassay described.

In addition, the IL-1 inhibitor of the present invention refers to a drug capable of binding to an IL-1 receptor. Such features are described, for example, in Kilian. T. L., et al., Interleukin l Alpha and Interleukin l Beta Bind to the Same Receptor on T Cells J. Immunol. 136: 4509 (1986), which can be readily determined by one skilled in the art using techniques as described.

The production of IL-1 inhibitors of the invention is influenced by M-CSF. In a preferred embodiment, the IL-1 inhibitor is regulated by M-CSF, so further administration of increased amounts of M-CSF results in greater inhibition of IL-1 bioactivity.

In a further preferred embodiment, the inhibitor is specific for IL-1 and does not modulate the activity of IL-2, IL-1-6 or TNF (Tumor Necrosis Factor).

The present invention relates to the inhibition of IL-1 biological activity by administration of a pharmaceutically effective amount of M-CSF. A pharmaceutically effective amount as used herein refers to an amount of a medicament capable of reducing the symptoms of the inflammatory disease described above. As is known to those skilled in the art, symptoms of inflammatory disease include erythema, edema, sensitivity (pain sensitivity) and pain. The dose of M-CSF that can reduce this symptom is in the range of 1 μ / kg to 100 mg / kg, and particularly preferably 1 mg / kg to 35 mg / kg.

Anyone skilled in the art can optimize the dosage of the M-CSF or IL-1 inhibitor of the invention using techniques known in the art. This technique is described, for example, in Goodman and Gilmanm, The Pharmacological Basis of Therapeutics, 5th Ed (1975) pages 19-28. Dose assays and routine dose-response studies can be used to confirm and optimize dose determination. The calculations needed to determine the appropriate dosage for treatment are usually further refined by those skilled in the art and belong to the shopkeeper of tasks routinely performed by those skilled in the art without undergoing inappropriate experimentation. In addition, the dosage of the M-CSF or IL-1 inhibitor administered in the methods of the invention will vary depending, for example, on the particular inflammatory disease to be treated, the mode of administration, the age, weight, and sex of the subject to be administered.

The present invention represents any manner of administering an M-CSF or IL-1 inhibitor. Examples of such modes of administration include intravenous administration, intramuscular administration, topical administration, transdermal administration, injection, oral administration and parenteral administration. Preferred administration is topical administration to the site. The M-CSF and IL-1 inhibitors of the invention may be administered alone or in combination with any of the conventional anti-inflammatory agents discussed above.

Suitable pharmaceutical acceptable carriers, diluents and adjuvants may be used with the compounds described herein to prepare the desired compositions for treatment of the patient. The pharmaceutical compositions of the present invention may contain M-CSF or IL-1 inhibitor compounds with pharmaceutically acceptable non-toxic carriers in solid or liquid. Such pharmaceutical carriers can be sterile liquids such as petroleum, animal, vegetable or synthetic oils such as oils and water, including peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, in particular for solutions for injection. In addition, the compounds of the present application can be combined with liposomes. [Described in U.S. Application No. 07 / 505,584, issued by Gideon Stassmann, issued April 6, 1990, specifically incorporated herein by reference] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin Malt, rice, wheat flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release preparations and the like. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions may contain a therapeutically effective amount of the active compound with an appropriate amount of carrier to provide the patient with an appropriate dosage form. The compounds of the present invention can be used in the treatment of patients. The term patient should be used in a broad sense to mean not only humans but also mammals including animals such as, for example, dogs, cats, guinea pigs, mice and mice.

As already mentioned, the present invention also relates to the N-terminus having the amino acid sequence from the 3-position Val or 4-position Ser of Formula (1) to the Thr 153-position or Pro of 214-position It relates to the use of an anti-allergic composition containing M-CSF as specified herein as an active ingredient, including M-CSF derivatives such as biologically active recombinant human M-CSF derivatives to which Met can be added.

Hereinafter, the preparation method of the M-CSF derivative used as the active ingredient in the present invention will be described in detail.

The derivative uses a gene encoding the same, for example, to prepare a recombinant DNA for the expression of the gene in the host cell, introduce the DNA into the host cell for transformation thereof, and culture the obtained transformant. It is manufactured by.

Genes for the preparation of human M-CSF derivatives of the present invention are described in various human cells with M-CSF production capacity, more specifically and advantageously described in AGR-ON [JP-A-SHO 59-169489]. Cultured cell lines derived from human leukemia T cells with characteristics: deposited as ATCC Accession No. CRT-8199 to the Collector (ATCC) over the American type]. Extraction process for the isolation of mRNA from the AGR-ON is for example guanidinium / high temperature phenol method (T. Maniatis, EF Fritsch and J. Sambrock, Molecular Coning, pp. 194-195 (Cold Spring Harbor Laboratory), 1982], guanidinium / cesium chloride method [T. Maniatis. E. F Fritsch and J. Sambrook, Molecular Cloning. P 196 (Cold Spring Harbor Laboratory), 1982], etc. Identity to cDNA Conversion of mRNA, ie synthesis of the gene of interest, is described, for example, in the Okayama-Berg method [H. Okayama and P. Berg, Molecular and Cellular Biology, Vol. 3, p. 280 (1983)], the Gulber-Hoffman method [V. Gubler and BJ Hoffama, Gene.Vol. 25, pp. 263-269 (1983), etc. Retain M-CSF cDNA from transformants and transformants by introducing the obtained DNA into host cells. The selection of the desired strain can be carried out by conventional methods: Japanese Patent Publication No. HEI 1-104176 describes this procedure or method. Is described in detail. An M-CSF gene described in said patent specification may also be used advantageously as a M-CSF gene for use in the practice of the invention.

The above-mentioned genes can also be produced by the method of compound synthesis of nucleotides using conventional methods such as the phosphite triester method [Nature, 31, 105 (1984)]. In any method, various procedures or steps may be performed in a conventional manner such as partial DNA synthesis by chemical means, DNA chain cleavage, deletion, addition, enzymatic treatment for ligation and ligation, DNA isolation, purification and replication, selection, and the like. . For example, the above-mentioned DNA isolation and purification can be performed by agarose gel electrophoresis, and partial variation of the cord in the nucleic acid sequence can be site-specific mutagenesis techniques [Proc. Natl. Acad. Sci., 81, 5662-5666 (1984) and the like. The codon selected for the desired amino acid is not particularly limited, but may be determined in a conventional manner in view of the use of codons for the host cell to be used and the like. The DNA sequence of the gene of interest to be used in the method can be found, for example, by the method of Enzymology, modified by Ginseng-Gilbert (Methods in Enzymology, 65, 499-560 (1980)) or by using a dedeoxynucleotide chain termination method using M13 phage. J. and Vieira, J., Gene, 19, 269-279 (1982).

M-CSF derivatives of the present invention can be easily produced in large quantities by recombinant DNA techniques using genes obtained in this manner. While the use of the above-mentioned specific genes is essential, the present invention can be basically carried out by general genetic engineering radix (see Molecular Cloing, T. Maniatis et al., Cold Spring Harbor Laboratory (1982), etc.).

More specifically, recombinant DNAs capable of transforming M-CSF genes in host cells are prepared, the DNAs are introduced into host cells for transformation, and the transformants are evacuated.

Useful host cells can be advanced or prokaryotic. Commonly used as prokaryotic hosts are E. coli and Bacillus subtilis. In the practice of the present invention, for example, a plasmid vector is used which allows replication in such a host and promoters and SD (Shine-Dalzano) are used to enable the expression of the M-CSF gene in the plasmid vector. -Dalgarno)) and additionally a expression expression vector containing an initiation codon (eg ATG) required for initiation of upstream protein synthesis from the gene can be used. Widely used as host E. coli is strain K12. pBR 322 is generally used as a vector. However, these are not limited, and various known strains and vectors can be used. Useful as promoters are, for example, tryptophan (trp) promoters, lpp promoters, lac promoters, P _L promoters, and the like, and in any case, the gene of interest may be expressed.

As a preferred method for preparing the human M-CSF derivative of the present invention using the above-mentioned genes, for example, a method of using a prokaryotic host such as E. coli as a host may be mentioned, in which the target protein is Transduced by 2-cistron technique. This is a gene expression system containing two cistrons in a sequence, and this method stably obtains and accumulates a large amount of a desired human M-CSF derivative in a host cell.

The production of M-CSF derivatives of the present invention by the 2-cistron method will now be described in detail. First, a transgenic plasmid containing two cystrons, the first cistron, which coats the appropriate polypeptide, and the second, the cystron M-CSF gene, are prepared. The plasmid contains a promoter and SD sequence for the expression of the gene upstream from the first cistron, and also an SD sequence for expressing the trait of the second sistron upstream from the first cistron and upstream from the second cistron. It is important to include in this order the synthetic linker, the termination codon for the first cistron and the starting codon for the second cistron.

The gene used as the first cistron may be a synthetic or natural gene in which the gene can be expressed in the host. The expression of the gene causes the polypeptide to be different from the M-CSF derivative to be produced in the same system, and since it is necessary to separate the polypeptide, the polypeptide must be hydrophobic and have a molecular weight that is significantly different from that of the M-CSF derivative. It is preferable. It is desirable that the above-mentioned first cistron should encode such a polypeptide. Preferred examples of the first cistron may refer to genes encoding IL-2, IFN-α, -β and -γ and the like, and fragments encoding about 50-100 amino acid residues and derived from such genes.

The promoter and SD sequences arranged upstream of the first cistron may be known per se. Examples of such promoters include trp promoter, tac promoter, P _L promoter, P _R promoter, lpp promoter, OmpA promoter, lac promoter and the like, among which trp promoter, P _L promoter, P _R promoter and the like are particularly preferred. Examples of SD sequences include 3-9 base pair sequences, such as GGAG and AGGA, which can form hydrogen bonds with the 3 'end of the 16S rRNA of prokaryotic cells.

The start codon and the end codon present in the synthetic linker between the first and second cistrons can be of any naturally occurring occurrence. These codons do not need to be used in their respective complete forms, eg TGA and ATG, but may be used in partially overlapped forms, eg TGATG, TAATG and the like. Transgenic plasmids for use in the 2-cistron method can be constructed by conventional methods.

Particularly preferred examples can be addressed by first cleaving the M-CSF gene-containing plasmid with an appropriate restriction enzyme, isolating and purifying the obtained M-CSF gene-containing fragment in a conventional manner, and separately in a conventional manner. By synthesizing the above-mentioned synthetic linker using, for example, a DNA synthesizer, and linking the linker to the fragment obtained in this manner upstream of the M-CSF gene using T4 DNA ligase and the like, It consists of mixing the DNA fragments obtained into plasmids containing the first cystron and capable of expressing it to the appropriate positions. As an alternative, a suitable expression expression plasmid for the desired 2-citron system is obtained by the above method and the first cistron is linked to the upstream end of the fragment, and then the resulting DNA fragment is plasmid with the appropriate protein expression system. It can be prepared from a DNA fragment containing a second cistron linked to a synthetic linker by incorporation into it.

The plasmid thus obtained is introduced into a host cell suitable for transformation of the cells, whereby the desired transformant can be obtained through the 2-cystron method. When using this transformant, the protein related to the first cistron and the target M-CSF derivative related to the second cistron are respectively expressed. This product can be analyzed or identified by conventional methods such as SDS-PAGE, western blotting and the like, and can be isolated and purified from each by various methods described below.

The desired transformant thus obtained can be cultured by conventional methods, whereby human M-CSF derivatives are produced and accumulated. The medium used for the culturing may be one appropriately selected from among those usually used for the host cells used. In order to culture transformants using E. coli or the like as host cells, for example, LB medium, E medium, M9 medium, M63 medium and the like can be used. Various known carbon sources, nitrogen sources, inorganic salts, vitamins, cell extracts, physiologically active substances and the like can be added to the medium if necessary.

In culturing the transformant, appropriate culture conditions may be used for growth of host cells. For example, in the case of E. coli, a pH of about 5-8, preferably 7 or about 7 and a temperature of about 20-43 ° C, preferably 37 ° C or about 37 ° C can be used.

In this manner, M-CSF derivatives of the present invention are produced and accumulated in transformant cells. This derivative can be isolated and purified by various separation procedures using its physical, chemical or other properties. Typical examples of such procedures include conventional refolding treatments, treatment with protein precipitants, centrifugation, osmotic shock, ultrasonication, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography. Graphics, affinity chromatography, high performance liquid chromatography (HPLC), various other liquid chromatography, high performance liquid chromatography (HPLC), various other liquid chromatography techniques, dialysis, and combinations thereof may be mentioned. By using this separation procedure, the desired human M-CSF derivative can be easily produced in high yield, with high purity, on a conventional scale.

The M-CSF derivative of the present invention thus obtained has CSF activity although there are slight differences in N-terminal amino acid sequence, molecular weight, etc., depending on the gene used for production and the expression system for the expression of the gene. It is characterized in that it has a Met residue corresponding to the start codon added to the N-terminus in the derivative of the present invention and the expression method.

As an active ingredient of the anti-allergic composition according to the present invention, it is preferred that the N-terminus is present at any position in the region from Glu at 1-position to Glu at 5-position of Formula (1) (the N-terminus Met may be added), wherein the C-terminus is an M-CSF derivative present at any position in the region from Thr in position 153- of the formula (1) to Val in position 522-.

Of these derivatives as mentioned above, particularly preferred are those having the amino acid primary sequence from 3-position Val or 4-position Ser of Formula (1) to Thr 153-position or Pro 214-position Met is a M-CSF derivative that may be added at the N-terminus.

Also useful are M-CSF derivatives that have a total amino acid sequence from Met at position-32 in Formula (1) to Val at position 52.

Anti-allergic agents of the present invention are usually formulated into pharmaceutical compositions containing an effective amount of M-CSF, including the M-CSF derivatives obtained above, in combination with conventional pharmacologically acceptable non-toxic carriers, which are in the form of a composition. Depending on the route of administration.

Such compositions are, for example, in the form of liquid preparations including solutions, suspensions, emulsions, and the like, which are usually administered orally, intravenously, subcutaneously, intradermal or intramuscularly. Such dosage forms or methods of administration are not specifically limited. Derivatives are also commonly used and can be prepared in a variety of other formulation forms suitable for, for example, oral or parenteral administration. The composition can also be prepared in form by the addition of a suitable carrier. The composition can also be provided as a dry formulation that can be recursed into a liquid by the addition of a suitable carrier. The dosage of this preparation is not specifically limited, but may be appropriately determined depending on the desired pharmacological effect, the type of disease, the age and sex of the patient, the severity of the disease, etc., and the preparation is generally active in terms of the amount of protein The dosage is such that the component is administered in an amount of about 0.001 to about 1 mg / kg / day. The composition may be provided in one daily or divided doses.

Biologically active M-CSF derivatives having the amino acid primary sequence from 3-position Val or 4-position Ser of Formula (1) to Thr 153-position and having Met added to the N-terminus of the present invention When used as the active ingredient M-CSF in, the obtained composition is characterized by not only inherent anti-inflammatory and anti-allergic properties, but also higher bioavailability compared to other M-CSFs. Thus, such anti-inflammatory and anti-allergic compositions are very useful in this respect.

The invention will be further clarified by the following examples, which are merely illustrative of the invention.

In the following Examples and Reference Examples, each of the M-CSF derivatives represented by reference to the aminosa sequence of Formula (1) represents X in the formula Asp.

Example 1

M-CSF does not stimulate the production of inflammatory regulators IL-1, IL-6, and PGE ₂ .

As set forth in FIGS. 1 and 2 and Table 1, M-CSF does not promote the production of macrophages of the inflammatory regulators IL-α, PGE ₂ and IL-6.

A. M-CSF does not promote the expression of IL-1 or IL-6.

Experimental Model

The active ingredient of endotoxins derived from bacterial serotypes, lipofuli saccharide (hereinafter LPS), is used as a model system to demonstrate the effect of M-CSF on the generation of inflammatory regulators in inflammatory macrophages.

To obtain inflammatory macrophages, nonpathogenic C57B1 / 6 mice from co-breeding females 3 days after injection of 1.0 ml of sterile Brewer's thioglycolate (TG) into mice (Charles River Breeding Laboratories, Wilmington, Mass.) Macrophages are harvested from. Cells are obtained by supplementing 10 U / ml of heparin and washing the peritoneum with 10 ml of ice-cold HBSS (Hanks Balanced Salt Solution) centrifuged at 250 X g for 5 minutes at 4 ° C.

10% (V / v) heat-inactivated low endotoxin (0.008 ng / ml), fetal calf serum (Hyclone, Logan, VT), 2.0 mM gulutamine, 100 U / ml penicillin and 100 μg / ml Cell pellets obtained are resuspended in PRMI 1640 (GIBCO, Grand Island, NY) (hereinafter complete media) supplemented with streptomycin.

Plate 1.0 ml aliquots of 0.5 × 10 ⁶ macrophages per 16 mm dwell (2.5 × 10 ⁵ macrophages / cm 2) in a tissue culture dish (Costar, Cambridge, NA). The dish is incubated 2.0 hours at 37 ° C. in 6.0% CO ₂ and washed three times with warm (37 ° C.) complete medium to remove non-adherent cells. As measured by non-specific esterase staining, the cell monolayer contains about 95% macrophages.

As described in Sections 2 and 3 below, cells are exposed to LPS and M-CSP at 37 ° C. The resulting control medium is collected by centrifugation at 570 × g for 15 minutes at 4 ° C., dialyzed against 100 volumes of PRMI 1640 medium, filtered sterilized and stored at −35 ° C. for further analysis.

2. M-CSF does not promote the expression of IL-1 or IL-6.

Each of the inflammatory macrophages was exposed to a control with varying time as follows; Exposure to LPS (0.5 μg / ml, E. coli 0.55: obtained from B5 (Difco, Detroit, MI)); 2.7 prepared as described in Takahashi, M. et al., Biochemical and Biophysical Research Communications 161 (2): 892-901 (June 15, 1989) and purified as in Reference Example 4 described below. Exposure to M-CSF (0.5 μg / ml recombinant M-CSF- (3-153)) with specific activity of 10 ⁷ U / mg (batch 058-2). M-CSF 1.0 units are defined as the maximal half stimulus of growth of M-CSF dependent human cell line NFS-60 [Nakoinz, I. et al., Exp. Hemotol. 17: 669 (1989)]

Tube content period (hours)

1 control group 6.0

2LPS (5 μg / ml) 0.75

3LPS (5 μg / ml) 1.5

4LPS (5 μg / ml) 3.0

5LPS (5 μg / ml) 6.0

6 M-CSF (0.58-2 0.5 μg / ml) 0.75

7 M-CSF (0.58-2 0.5 μg / ml) 1.5

8M-CSF (0.58-2 0.5 μg / ml) 3.0

9M-CSF (0.58-2 0.5 μg / ml) 6.0

Northern tubes of macrophage mRNA are used to analyze each tube for the expression of IL-1, IL-6 and TGF-β. Such assays are one of ordinary skill in the art, for example Nucleic Acid Hybridization: Determination of Genetic Homology in Recombinent DNA Methodology, Dillon JR, A. Nasim, ER Nestmann (eds) John Wiley Sons (1985), Section XI It is described in

Portions labeled IL-1α and IL-6 in FIG. 1 indicate that LPS can promote the expression of IL-1α and IL-6. Compare the LPS-only sample (lanes 2-5) with the control (lane 1). Expression of both IL-1α and IL-6 increases over time but peaks at 3 hours.

In contrast, the presence of M-CSF (lanes 7-10) does not promote the expression of IL-1α or IL-6. These two lanes are empty.

As can be seen in the portion labeled TGF-β, TGF-β is expressed by LPS as well as M-CSF, indicating that all proteins have activity.

This experiment was repeated twice to obtain the same results. Thus, unlike LPS, which promotes the production of inflammatory regulators, M-CSF does not promote the production of IL-1 or IL-6. The findings are surprising in light of early reports that M-CSF is known to promote the production of IL-1.

3. M-CSF does not stimulate IL-1α gene expression.

This experiment, performed with different preparations of M-CSF, confirms that M-CSF is incapable of inducing gene expression of IL-1. In FIG. 2, different preparations of M-CSF are tested to find that they do not stimulate IL-1α expression.

Inflammatory macrophages were treated with control (lane 1), 1 μg / ml LPS (obtained as above) (lane 2), μg / ml M-CSF- (3-153) (3.0 × 10 ⁷ U / mg Except for different batches with specific activity, as obtained above (lane 3), and the combination of LPS and M-CSF (lane 4). Cells are analyzed for expression of IL-1α as described in Molecular Colning: a laboratory manual, T. Maniatis, EF Fritsch, J. Sambrook (eds) Cold spring Harbor Press (1982).

Compared to the control lane (1), LPS (lane 2) stimulates the expression of IL-1α gene, and the addition of M-CSF (lane 4) does not affect the hemolytic expression of IL-1α. Thus, as described in section 1 above, M-CSF neither induces nor decreases the expression of IL-1α.

B. M-CSF does not promote the activity of IL-1, IL0-6, or PGE ₂ .

Experimental Model

a. Adjustable badge

Inflammatory macrophages obtained as described above were treated with M-CSF derived from two sources to obtain the following regulatory media:

Recombinant M-CSF prepared as above and having a specific activity of 3.0 × 10 ⁷ U / mg

(3-153) (batch EC-80-I); And

Natural M-CSF (obtained from ERStanley (Albert Einstein College of Medicine, NY)) with specific activity of 1.5 × 10 ⁷ U / mg derived from L929 fibroblasts.

2. Expose the cells to an increased dose of M-CSF from the species source up to a maximum of 1 μg / ml.

After a 24 hour warming period, the control medium is assayed for the activity of IL-1, IL-6 and PGE ₂ as described below.

b. IL-1 Thymic Cell Analysis

To assess the effect of regulatory batches on the production of biologically active IL-1, Togawa, a., Et al., Characterization of Lymphocyte Activqting Factor Produced by Human Mononuclear Cells; Biochemical Relationship of High and Low Molecular Weight Forms of LAF, J. Immunol. 122: 2112 (1979)], the cells are analyzed thymic cells. In this assay, single cell suspensions of pleural cells obtained from 6 to 10 weeks old female C3H / He mice (Bantin-Kingman (CA)) are prepared by gentle subdivision. Large aggregates are removed by warming with ice cooling for 10 minutes under unit gravity. The cells are washed twice with complete medium and resuspended to a density of 1.0 × 10 ² cells / ml in complete medium. The cells are then seeded in plate bottom microtiter plates at a density of 1.0 × 10 ⁶ cells / well in the absence or presence of 1 μg / ml Concanavalin A (hereafter Con A) (Sigma Chemical Co.).

Control medium or recombinant human IL-1β is added in serial dilutions to a final volume of 0.2 ml. The pleural cells are incubated for 72 hours and pulsed with 1.0 μCi of [methyl] ^-3 H-TdR (6.7 Ci / M moles, New England Nuclear, Boston, Mass.). Cells are harvested on glass fiber paper using a semi-automatic cell harvester (Skatrom, Sterling, VA) and the ³ H-TdR incorporation is measured by standard liquid scintillation counting procedures.

As shown in Table 1 below, native M-CSF (named M-CSF type L929) and recombinant M-CSF- (3-153) (E. coli [3-153] M-CSF or rh-M-CSF) (Labeled (3-153)) do not show a positive result in the analysis, indicating that no M-CSF from any source can produce IL-1 in inflammatory macrophages.

C. IL-1 Radioreceptor Analysis

The observation that both human IL-1α and IL-1β bind to the same receptor as murine IL-1 on murine EL-4.6.1 erythrocytes is described by kilian, TL, et al., Interleukin l Alpha and Interleukin l Beta Bind to the Same Receptor on T cells, J. Immunol. 136: 4509 (1986)], an IL-1 receptor assay described in the above was developed. In this assay, binding buffer containing murine EL-4.6.1 cells from ATCC, maintained in complete medium, by centrifugation and RPMI 1640 medium supplemented with bovine serum albumin 0.1 mg / ml and 25 nM HEPES, pH 7.2. Resuspend in. Cell density is adjusted to 8.0 × 10 ⁶ cells / ml and the 0.05 ml aliquots are dispensed into tubes under ice cooling.

275 p g of ¹²⁵ I-IL-1α (75000 CMP) (specific activity, 2000 Ci / mmol; Amersham, Arlington Heights, IL) are added to each tube. Tubes are kept warm at 4 ° C. for 3.0 hours with dilution control medium from macrophages treated with two source-derived M-CSFs of 1.0 μg / ml or less.

Non-specific binding is determined by the addition of 5.8 pM recombinant human IL-1β (specific activity, 4.0 × 10 ⁷ U / mg; stake from Hirai (Otsuka Seiyaku, Tokushima, Japan)). To isolate ¹²⁵ I-IL-1α bound to cells from free ¹²⁵ I-IL-1α, 0.2 ml of an oily mixture of dibutyl phthalate and dioxyl phthalate (10: 1 (VV)) was added to each assay tube. Inject and centrifuge each tube for 2.0 min at 4 ° C. in a macrorest. The fluid is then aspirated, the end of the tube is cut off, and cell binding radioactivity is measured with a γ counter.

As described in Table 1 below, no type of M-CSF can displace IL-1, indicating that no type of M-CSF can stimulate the production of IL-1 in macrophages.

d. IL-1 mRNA Analysis

The expression of IL-1 mRNA is measured as above. As in Figure 1, Table 1 shows that neither recombinant M-CSF nor native M-CSF can stimulate the expression of IL-1 from macrophages, whereby no type of IL- It is confirmed that 1 is not promoted.

e. IL-6 B-9 Assay

The IL-6 assay used was as described by Aarden et al., Eur. J. Immunol., 17: 1411-1416 (1987). IL-6-dependent hybridoma cell line B 13.29 clone B9 in Escorb's denatured Dulbecco's medium supplemented with 2.0% (V / V) MG 63 osteocosarcoma from ATCC regulatory medium, the source of IL-6 Keep it. 5.0% (V / V) fetal calf serum, 50 μM β-mercaptoethanol, 100 U / ml penicillin and 100 mg / ml streptomycin are added to these cells.

Clone B9 cells 5.0 × 10 ⁴ / ml are incubated in 0.2 ml of the medium in 96 well plates (Falcon) at the bottom of the plate. Control medium is added to the cells and the plate is warmed for 72 hours at 37 ° C. in air under an atmosphere of 5.0% CO ₂ . Plates are kept warm with 1.0 μCi [ ³ H] -titadine for the last 4 hours of incubation. Cells are harvested using Seppotger (Skatron, Sterling, VA) and [ ³ H] -thymidine incorporated in DNA is measured by liquid scintillation counter (LKB RockBeta). Samples are tested in duplicate and titrated in 3-fold dilutions. All analyzes are compared to a standard curve using recombinant human IL-6 (Amgen, Thousand Oaks, CA) 1.0 × 10 ⁷ U / mg.

As described in Table 1, both recombinant M-CSF and native M-CSF cannot stimulate the production of IL-6, indicating that no form can stimulate the production of IL-1 in macrophages. .

f. IL-6 mRNA Analysis

The expression of IL-6 mRNA is measured as above and the result obtained by B-9 analysis is confirmed. As described in Table 1, no form of M-CSF can stimulate the expression of IL-6.

g. PGE ₂ analysis

In order to measure the ability of M-CSF to stimulate PGE ₂ production, the presence of PGE ₂ is measured as follows. Control media from macrophages treated with recombinant and native M-CSF up to 1.0 μg / ml were diluted with phosphate buffered saline (PBS), acidified to about pH 2, and the same amount of chloroform: methanol (1: 1) (V / V)). The organic phase is dried under nitrogen and the amount of PGE ₂ is measured by standard radioimmunoassay equipment purchased from New England Nuclear, Boston, MA.

As described in Table 1 below, neither form of M-CSF can stimulate the production of PGE ₂ as in IL-1 and IL-6.

TABLE 1

Production of inflammatory regulators by macrophages in response to M-CSF

As can be seen in the experiment, M-CSF does not stimulate the production of inflammatory regulators such as IL-1, IL-6 or PGE ₂ .

Example 2

M-CSF regulates the production of inflammatory regulators that are produced in response to LPS treatment.

As shown in FIGS. 2-10 and Table 2-3 and as detailed below, M-CSF can reduce the activity of the inflammatory regulators IL-1 and PGE ₂ produced in response to LPS. .

A. M-CSF regulates the production of PGE ₂ .

1, pretreatment with M-CSF reduces PGE ₂ production.

The macrophages obtained as in Example 1 are divided into four sets. Two sets of macrophages are pretreated with culture medium and the other two sets are pretreated with recombinant M-CSF obtained as in Example 1. In the treatment step, LPS is injected at 1 μg / ml into one set of cells pretreated with medium and one set of cells pretreated with M-CSF as shown in Table 2 below. The remaining set is infused with medium. As mentioned in Example 1, the presence of PGE ₂ is determined and expressed in ng / mg (cell protein) / 24 hours or ng / ml / 24 hours.

As shown in Table 2a, the control value of PGE ₂ when pretreated with medium or M-CSF is 0.11-0.12. LPS treatment of cells pretreated with media resulted in PGE ₂ levels of about 10.00, almost 100-fold higher than control values. However, when LPS was added to cells pretreated with M-CSF, the increase in PGE ₂ levels was suppressed by 65%, resulting in about 3.6.

Thus, it can be seen that pretreatment with M-CSF inhibits the ability of LPS to stimulate PGE ₂ levels.

TABLE 2a

Pretreatment or adjuvant of M-CSF reduces LPS induced PGE ₂ formation by macrophages.

** This. Collie [3-153] M-CSF (M-CSF- (3-153))

2. Pretreatment or cotreatment with M-CSF reduces PGE ₂ production.

Macrophages obtained as in Example 1 are pretreated with culture medium or M-CSF as shown in Table 2 below. Recombinant M-CSF- (3-153) (commanded by rh) obtained as in Example 1 and native M-CSF (commanded by L929) obtained as in Example 1 were used to pretreat the cells.

Cells were then treated with media (to obtain a control value of PGE ₂₎ as shown in Table 2 below, treated with two M-CSF sources (sealing treatment effect with different M-CSF samples), 1 μg / Treated with LPS at ml concentration alone and with LPS combination with two M-CSF sources. Cells are analyzed to show the presence of PGE ₂ produced in ng / ml / 24 hours. Repeat this experiment 5 times.

As shown in Table 2, the resulting PGE ₂ values were 0.07 for control cells, 0.19 and 0.07 for cells pretreated with M-CSF only and 0.06 and 0.1 for cells treated with terminal M-CSF only. For cells pretreated only with medium, treatment with LPS increases the values to 1.27 and 1.10. In contrast, cells treated with LPS and cells pretreated or co-treated with M-CSF inhibited the production of PGE ₂ by 48% 87-89% or 100%. Thus, pretreatment or adjuvant of M-CSF reduces the production of PGE ₂ stimulated by LPS.

TABLE 2

Pretreatment or adjuvant of M-CSF reduces LPS induced PGE ₂ formation by macrophages.

* L929: Natural M-CSF purified from L929 fibroblasts

** rh: Recombinant M-CSF- (3-153) (E. coli [3-153] M-CSF)

B. M-CSF regulates production of IL-1 inhibitors.

Macrophages obtained as in Example 1 were cultured or rh-M-CSF- (3-153), ie recombinant M-CSF {. Coli [3-153] M-CSF} is pretreated for 24 hours at concentrations of 0.01, 0.03, 0.1, 0.3 and 1.0 μg / ml. Cells pretreated with medium were obtained in medium (control), LPS 1.0 μg / ml alone, rh-M-CSF- (3-153) alone at 1.0, 0.3, 0.01, 0.03 μg / ml concentrations, Or 1.0 μg / ml LPS with four concentrations of rh-M-CSF- (3-153). Cells pretreated with rh-M-CSF- (3-153) are treated with 1.0 μg / ml of medium or LPS.

In order to evaluate the effect on the production of bioactive IL-1, thymic cell analysis was performed as in Example 1 with the culture supernatant, with the result being the stimulation index. Stimulation index refers to cpm in the presence of macrophage regulatory medium in the presence of Con A and only Con A at a given dilution / cpm.

As shown in Table 3, the stimulation index values of the control samples were 2.5 (pretreatment and treatment with medium) and 1.19-1.67 (pretreatment and treatment with various concentrations of M-CSF). LPS-treated stimulus index values were 20.3, almost 10-fold higher. In contrast, the stimulation index values in the samples pretreated with M-CSF and stimulated with LPS ranged only from 6.02 to 10.09, with 51 to 70% inhibition of IL-1 bioactivity.

In a similar manner, the stimulation index values in the range of 5.65 to 11.8 for the samples supplemented with various concentrations of M-CSF and 72 to 42% for the IL-1 bioactivity inhibition rate. The inhibition rate is the highest at 1.0 μg / ml of M-CSF and the lowest at 0.03 μg / ml of M-CSF, indicating that it depends on the concentration. The EC ₅₀ (effective concentration capable of inhibiting 50%) for rh-M-CSF (3-153) is determined to be about 1.1 nM.

TABLE 3

Effect of Recombinant M-CSF on Bioactive IL-1 Production in Response to LPS

Similar experiments are performed with varying concentrations of native M-CSF purified from L929 cells. As shown in Table 3a, pretreatment of M-CSF inhibits the bioactivity of IL-1 produced from macrophages from 39 to 93% in response to LPS. As above, the maximum inhibition is observed at the highest concentration of M-CSF. EC ₅₀ for L929 M-CSF is found at about 0.6 nM.

TABLE 3a

Effect of Stagnant L929 M-CSF on Bioactive IL-1 Production

These two experiments are repeated at least 12 times with various M-CSF formulations and similar results are obtained in each experiment. Thus, these experiments suggest that pretreatment or cotreatment with M-CSF reduces the bioactivity of IL-1 in response to LPS. This effect also appears to be concentration dependent.

The results are shown graphically in FIG. When the bioactivity of IL-1 (CPM / 1000) is plotted against pretreatment and treatment, compared to pretreatment and treatment with medium (CC), pretreatment with medium and treatment with LPS (CL) Shows a maximum increase in bioactive IL-1. More increases in bioactive IL-1 are seen in pretreatment with medium and treatment with a combination of LPS and M-CSF (C-LM). Reduction of bioactive IL-1 is seen in samples pretreated with M-CSF and treated with LPS (M-L). Thus, it can be seen that M-CSF regulates the production of IL-1 in LPS-stimulated macrophages.

C. Potential Mechanism of M-CSF Action in IL-1 Bioactivity

As in FIGS. 1 and 2, LPS induces the expression of IL-1 (primarily IL-1α in macrophages), and LPS is also bioactive IL as in FIG. 3 and Tables 3 and 3a above. Increase the production of -1. When cells are treated with LPS and pretreated or co-treated with M-CSF, the level of bioactive IL-1 decreases. However, expression of IL-1 is not affected by the co-treatment of LPS and M-CSF. As in Figure 2, the IL-1 trait expression autoradiograph pattern of the combination of M-CSF and LPS is very similar to the pattern by LPS alone. This observation can be explained by the M-CSF-dependent stimulation of IL-1 inhibitors which in turn reduces the bioactivity of IL-1. The production of IL-1 inhibitors is tested by performing IL-1 pleural cell assays supplemented with both ConA and IL-1. The addition of these two components makes it possible to assess the action of IL-1 inhibitors by providing a detectable level of bioactive IL-1, thereby measuring any decrease in IL-1 levels.

As in FIG. 4, IL-1 thymic cell assay is performed on 6 sets of regulated media. The levels of bioactive IL-1 remain the same in the set pretreated and treated with media (C-C). In comparison, a set of pretreatment with medium or M-CSF and treatment with M-CSF or LPS or with a combination of M-CSF and LPS showed a slight reduction in bioactive IL-1, but not with IL-1 organisms. The true decrease in activity is only seen in the set pretreated with M-CSF and treated with LPS (ML). Thus, these studies indicate that M-CSF is involved in the production of inhibitors for IL-1.

To determine whether the inhibitor is specific for IL-1, a similar experiment is performed with IL-6. Six sets of macrophages are pretreated with medium (C) or M-CSF (M) as in FIG. 5 and treated with medium, M-CSF, LPS or a combination of M-CSF and LPS. The homogeneity of IL-6 is assessed using the assay of specific IL-6 B-9 above.

As shown in FIG. 5, similar patterns of IL-6 bioactivity were pretreated with medium, cells treated with LPS, cells treated with a combination of LPS and M-CSF, and with M-CSF. Observed in cells pretreated and treated with LPS. It was shown that inhibitors stimulated by M-CSF did not affect the IL-6 active phase. This pattern is very different from the pattern observed for IL-1 production in FIG. 4, which shows a sharp decrease in IL-1 in culture supernatants from cells pretreated with M-CSF and treated with LPS. Thus, this study is very different from the pattern observed for IL-1 production. Thus, these studies show that IL-1 inhibitors are specific for IL-1 and under the same conditions that M-CSF does not regulate the production of inhibitors specific for IL-6.

Example 3

Infusion of M-CSF in in vivo experiments reduces the IL-1 bioactivity produced in response to LPS.

Eight siblings of female non-pathogenic C57B1 / 6 mice (Charles River Breeding Laboratories, Wilmington, Mass.) Were injected intraperitoneally with 1.0 ml of sterile Brewer thioglycolate (TG) at 0 hours and divided into four groups. As shown in Table 4 below, mice at Group 1, Control and Group 2 were pretreated with 0.2 ml phosphate buffered saline (PBS) at 72 hours and mice in Groups 3 and 4 were treated with M-CSF- (3 PBS containing -153) is pretreated at 4 μg / mouse. At 90 hours, control group 1 was treated with PBS, volume 2 was treated with PBS containing LPS 50 μg / mouse (showing the effect of LPS treatment), and group 3 was treated with PBS (pretreatment with M-CSF alone). Group 4 is treated with PBS containing LPS 50 μg / mouse (showing the effect of M-CSF pretreatment in LPS treatment).

After 8 hours the mice are killed and their peritoneal cells are collected as in Example 1. 1 × 10 ⁶ cells / well of cells are plated on a costar plate and treated with conditioned medium.

After 20 hours, the control medium is collected and titrated and subjected to the IL-1 biopsy as above (thymus cells pulsed with ComA). Results are shown as mean cpm of three wells.

The percent inhibition of IL-1 bioactivity is determined as follows.

As shown in Table 4 below, at a quarter dilution of the control medium, treatment with LPS (group 2) yielded 13103 CPM of bioactive IL-1 and pretreatment with M-CSF (group 4) SMS 5050 CPM. Get Treatment with LPS at 1/12 dilution (Group 2) yields 5385 CPM compared to 2592 CPM obtained in pretreatment with M-CSF (Group 4). Thus, pretreatment with M-CSF at both 1/12 and 1/4 dilutions of the control medium inhibited 69.9% of the bioactivity of IL-1 produced in response to LPS, with M-CSF in vitro and in vivo. Shows that IL-1 bioactivity can be inhibited.

TABLE 4

Infusion of M-CSF in vivo reduces the IL-1 bioactivity produced in response to LPS.

Example 4

Isolation and Evaluation of IL-1 Inhibitors

A. Isolation and Characterization of IL-1 Inhibitors

To further evaluate the IL-1 inhibitor, the control medium treated with M-CSF and LPS is separated from the IL-1 inhibitor by QAE-52 column. As an anion exchanger, QAE-52 has the ability to separate IL-1α, which is pI 5.5, from IL-1 inhibitors at pH 7.4.

To isolate IL-1 inhibitors, peritoneal exudates cells 3.0 × 10 ⁸ are cultured in 10 mm tissue culture dishes at a cell density of 2.3 × 10 ⁵ / cm 2 (approximately 1.8 × 10 ⁷ cells / plate) in complete medium. After warming for 2.0 hours, the plates are washed twice with onajeon medium, 5.0 ml (1.0 μg / ml) of M-CSF-3-153 per plate are added and then warmed for 20 hours. After this pretreatment step the liquid is aspirated and the plates are washed twice and 5.0 ml RPMI 1640 containing 0.5 mg / ml bovine serum albumin and 1.0 pg / ml LPS per plate is added. Incubate the plate for 40 hours and collect the control medium, then centrifuge for 15 minutes at 100 x g at 4 ° C. The resulting pellet is diluted with the same amount of ice cold 20 mM HEPES buffer (pH 7.4). The contents are loaded on a 1.5 × 10 cm 2 column filled with quaternary ammonium cellulose (QAE-52, Whatman).

Samples not attached to the QAE-52 are flowed through the column, collected and ordered with fluid. The sample attached to QAE-52 is released in the presence of 1 M NaCl, collected and named as eluent. Both fractions of the fluid and the eluate are filtered and concentrated in the same amount in Amicon chamber on the membrane with 10 kd cutting point.

IL-1 pleural cell assay, IL-1 radioreceptor assay, TNF bioassay, and IL-2 lunch assay are performed with the fluid and eluate samples.

1. IL-1 Thoracic Cell Analysis

The fluid and eluate samples are subjected to IL-1 pleural cell analysis as described above to determine the bioactive IL-1 level compared to the activity of the IL-1 sample introduced for analysis. The result is as shown in FIG.

IL-1 activity in the eluate is much greater than that of IL-1 samples because thymic cells were supplemented with submaximal doses of IL-1. In contrast, IL-1 activity is significantly reduced in fluids. Thus, it can be seen that the eluate exhibiting IL-1 activity contains IL-1 and possibly IL-6, and the fluid with reduced IL-1 activity contains an IL-1 inhibitor.

2. IL-1 Radioreceptor Analysis

Two samples of fluid and eluate are analyzed by IL-1 radiocontainer analysis as shown in Scilche 1. As shown in FIG. 7, both fractions can substitute ¹²⁵ IL-1α bound to EL-4.6 cells, although the fluid exhibits greater substitution than the eluate. This suggests that IL-1 and IL-1 inhibitors bind to IL-1 receptors.

3. TNF Analysis

To determine the specificity of IN-1 inhibitors present in QAE-52 fluids, fluid samples were collected from Nedwin, Ge, et al., Effect of interleukin 2, interferon gamma, and mitogens on tne production of tumor necrosis factors α and β, tested by TNF bioassay as described in J. Immunology 135: 2492 (1985). In this assay, 5.0 × 10 ⁴ cells per unit well of L929 fibroblasts are plated onto a microtiter plate at the bottom of the plate in the presence of 1.0 u / ml of actinomycin D. Dilute samples of the fluid QAE-52 are added to a final volume of 0.2 ml with or without the serial dilution of TNF-α (obtained from Amgen). The monolayers are washed after 24 hours incubation at 37 ° C. under 6.0% CO ₂ . Cell disruption was determined by adding 0.5% (w / v) crystal violet solution in methanol and water at 1: 4 v / v in the mixture and quantitated by absorbance measurement at 570 nm with a Titertek multiscon ELISA reader.

As shown in FIG. 8, the curves corresponding to medium and 12.5% fluid show substantially the same pattern, indicating that the QAE-52 fluid does not inhibit or stimulate the L929 indicator cell disruption of TNF-α. will be. In other words, the study shows that IL-1 inhibitors are specific for IL-1 and have no effect on TNF activity.

4. IL-2 Biological Testing

To determine the specificity of an IL-1 inhibitor in a QAE fluid, both the QAE fluid and the QAE eluate were treated with Gillis, S, et al., T cell growth factor parameters of production and a quantitative microassay for activity, J. Immunol. 120: Tested by bioassay of IL-2 as described in 2027 (1978). As shown in FIG. 9, neither fluids nor eluents can affect the activity of IL-2. Both samples show almost horizontal lines and do not inhibit or promote IL-2 proliferation.

B. Purification of IL-1 Inhibitors

After determining that the IL-1 inhibitor is resistant to acid and heat but not to trypsinization, the IL-1 inhibitor is purified on off-HPLC using acetonitrile as the eluent for the C-A column.

In this purification, the material in the fluid collection is lyophilized and resuspended in 10% (v / v) acetyl00.1% (v / v) trifluoroacetic acid / water and connected to reverse phase HPLC. Inject into 4 columns (Nest group). After applying acetonitrile gradient (25% -50%) (v / v) for 60 minutes at a flow rate of 1.0 ml / min, the fractions are collected, lyophilized and resuspended in the corresponding buffer. Samples are tested by the IL-1 thymocyte biopsy described above, the IL-1 receptor assay described in Example 1 and the thymic cell assay described in Example 2.

As shown in FIG. 10, the highest inflorescence of IL-1 inhibition determined by IL-1 biopsy (solid line) is found in fractions 33-38. These fractions correspond approximately 37% acetonitrile. Additional tests with IL-1 radioreceptor binding assays show that only these fractions can substitute for IL-1 (* line). Similarly, in IL-1 thymic cell assays stimulated with ConA and IL-1 as indicated above, only these fractions can inhibit thymic cell proliferation (triangle). Therefore, IL-1 inhibitors stimulated with M-CSF can be separated by reverse phase HPLC.

Hereinafter, the anti-allergic composition of the present invention will be described in detail as a reference example showing an M-CSF derivative preparation of the present invention and an example showing anti-allergic properties.

Here, the generated human M-CSF derivative is defined as follows. That is, M-CSF generated by the expression vector encoding the amino acid sequence from Val at position 3 to Thr at position 153 defined in formula (1) in host E. coli. Named Coli [3-153] M-CSF, and similarly generated by a transfection vector encoding the amino acid sequence from Val in position 3 to Pro in position 214. Collie [3-214] is named M-CSF.

Also a host. The M-CSF generated by the transfection vector encoding the amino acid sequence from Ser of position 4 to Thr in position 153 in E. coli. Calli [4-153] M-CSF, similarly, a transgenic vector that coats the amino acid sequence from Ser at position 4 to Pro at position 214 is used. Colli [4-214] M, -CSF.

M-CSF derivative obtained by the expression expression vector obtained in Reference Example 1 and encoding an adult M-CSF protein comprising a signal peptide comprising 32 amino acid sequences and 552 amino acid sequences in a host CHO cell. Is named CHO [-32-522] M-CSF.

The samples obtained in the examples are subjected to CSF activity analysis by the following method.

How to Measure CSF Activity

Fetal calf serum (FCS, 20 ml), 30 ml of α-biz and 20 ml of 2-fold α-medium are mixed together and maintained at 37 ° C. 23.3 ml of the solution is mixed with 10 ml of a 1% agar (difco Laboratories) solution already kept at 50 ° C. and then the mixture is kept at 37 ° C.

Separately, bone marrow cells (BMC) collected from femoral bones of BALB / c mice were washed twice with Hanks' solution, resuspended in α-medium to a concentration of 10 ⁷ cells / ml, and suspension 1nl maintained at 37 ° C. Agar medium is added. The mixture is stirred thoroughly and then maintained at 37 ° C. 0.5 ml of the mixture is placed in each well already containing 5 μl of the test sample (Tissue Culture Cluster 12, manufactured by Costar Corporation), and the resulting mixture is stirred quickly and prevented at room temperature. Upon solidification of the agar in each well, the well is placed high in a carbon dioxide gas incubator and warmed at 37 ° C. for 7 days.

In this way, the number of generated colonies is counted by a stereomicroscope to provide CSF active resin. CSF activity in units of U / ml is the value calculated from the colony numbers mentioned above according to the following formula (a).

CSF activity in U / ml = (number of colonies) × (dilution factor) ÷ 1.5 (a)

In morphological and enzymatic observations, the colonies formed above are almost macrophage colonies.

Reference Example 1

Preparation of CHO [-32-522] M-CSF

Microcarrier-cultured CHO cell clone No. 1 in OPTI-MEN (manufactured by Gibco Laboratories) containing 1% FCS. Using the culture supernatant of 2, 3-8 [JP-A-1-104176], the desired uniform CHO [-32-522] M-CSF is obtained by the following purification procedure. In the following procedure, a target protein is detected by western blotting. The western blotting method mentioned is carried out using Transbollot cells from Bio-Rad Laboratories. After the transition, the nitro cellulose membrane was blocked with PBS-containing 1% skim milk, reacted with rabbit antiserum against M-CSF, and further with peroxidase-labeled goat anti-rabbit antibody (manufactured by Bio-Rad Laboratories). React. In order to detect the M-CSF band, the nitrocellulose membrane thus obtained is reacted with a solution of chromogen substrate 4-chloro-1-naphthol.

(1) ConA-Sepharose Chromatography

The above-mentioned CHO cell culture supernatant (69.3 L) is concentrated by ultrafiltration and fractionated with ammonium sulfate using 650 ml of the concentrate (842 ml) as starting material. The precipitate fraction obtained by saturating 35 to 65% with ammonium sulfate is dissolved in distillation (1, 120 ml) to obtain a sample solution.

Separately, a column (5 × 25 cm) filled with about 500 ml of Cona-Sepharose gel is equilibrated with 20 mM sodium phosphate buffer (pH 7.4) containing 0.15 M NaCl, and then the sample solution is applied to the column. After complete washing with the same buffer, it is eluted with the same buffer containing 0.5 M methyl α-D-mannoside. The total eluate is concentrated by ultrafiltration using a YM-10 membrane, and the buffer is exchanged with 20 mM sodium phosphate buffer (pH 7.4), and the resulting solution is subjected to anion exchange high performance liquid chromatography under 7 partial samples under the following conditions. .

(2) anion exchange high performance liquid chromatography

Column: TSK gel DEAE-5PW (21.5 mm I.D × 15 cm, manufactured by Tosoh Corporation)

Eluent A: 40 mM sodium phosphate buffer (pH 7.4)

Eluent B: 40 mM sodium phosphate buffer with pH 1.0 M NaCl (pH 7.4)

Flow rate: 3.0 ml / min

Fraction Volume: 65ml / Tube / 2min

Concentration gradient: time (minutes) B%

0 0

10 0

20 10

95 30

100100

110100

115 0

130 0

As a result of the elution, the desired M-CSF is eluted in fractions 26 to 38 (0.18 to 0.25 M NaCl). The active fractions are combined, concentrated by ultrafiltration (using YM-10 membrane) and purified as follows.

(3) gel filtration high performance liquid chromatography

The concentrated sample obtained in the step (2) is subjected to gel filtration high performance liquid chromatography under the following conditions into five partial samples.

Column: TSK Gel G 3000 SWG (21.5 mm I.D × 15 cm, manufactured by Tosoh Corp.)

Eluent: 5 mM sodium phosphate buffer with 0.3 M NaCl (pH 7.4)

Flow rate: 3.0 ml / min

Fraction volume: 6ml / Tube / 2 minutes

As a result of the gel filtration high performance liquid chromatography, M-CSF was detected in the fractions of Nos. 20 to 27, and the fractions of Nos. 24 to 27 were combined, concentrated by ultrafiltration, and treated by the following purification process.

(4) TSK Gel Ether-5PW High Performance Liquid Chromatography

The concentrated sample (12 ml) obtained in the above step (3) was purified into eight partial samples under the following conditions.

Prior to injection into the tube, the above mentioned pesticide is mixed and chromatographed with 80% ammonium sulfate-saturated 20 mM sodium phosphate buffer (pH 7.4).

Column: TSK gel ether 5-PW (7.5 mm I.D × 75 mm, manufactured by Tosoh Corp.)

Eluent A: 20 mM sodium phosphate buffer (pH 7.4)

Eluent B: 40% ammonium sulfate-saturated 20 mM sodium phosphate buffer (pH 7.4)

Flow rate: 1.0 ml / min

Fraction Volume: 2ml / Tube / 2min

Concentration gradient: time (minutes) B%

100

5100

65 0

80 0

85100

100100

As a result of the chromatography, M-CSF is detected in fractions 15-18 (22-17% saturated ammonium sulfate concentration). Fractions having the same fraction number are respectively combined and each is subjected to high performance reversed phase liquid chromatography with 3 or 4 subsamples.

(5) TSK Gel Phenyl-5PWRP High Performance Reverse Phase Liquid Chromatography

Column: TSK gel ether 5-PWRP (7.5 mm I.D × 75 mm, manufactured by Tosoh Corp.)

Eluent A: 0.1% TFA

Eluent B: n-propanol: 1% TFA (9: 1)

Flow rate: 0.8 ml / min

Fraction volume: 1.6 ml / tube / 2 minutes

Concentration gradient: time (minutes) B%

0 0

5 0

10 25

50 45

60100

70100

75 0

95 0

As a result of the above chromatography, the two fraction numbers vary slightly depending on the sample and the experiment, but M-CSF is detected as two consecutive fractions in the fractions of numbers 5-8 (32-34% n-propanol).

Fractionation is performed using a tube already containing 200 μl 0.8 M disodium phosphate per tube. The M-CSF fractions are concentrated to dryness in a centrifuge tube by tube on a concentrator (manufactured by Tomy Seiko Col., Ltd), dissolved in 500 μl of distilled water per tube and the resulting solution is tested.

(6) SDS-PAGE of CHO [-32-522] -M-CSF

Laemmli sample buffer [2-mercapto ethanol containing CHO [-32-522] M-CSF obtained in step (5) according to the method of Laemmli [LaemmLi, UK Nature, 277, 680 (1970)]. 2-ME ⁺ ) and one of those containing no (2-ME ⁻ ), and then each mixture was heat treated at 95 ° C. for 10 minutes to give SDS using mini slab gel (gel concentration 12%). -PAGE Pre-stained markers (manufactured by Bio-Rad Laboratories) are used as molecular weight markers. Silver dyeing (manufactured by Wako Pure Chemical Industries) is used for dyeing.

As a result, the band dyed under non-screen conditions (2-ME-state) is detected within the molecular weight range of 62,000-115,000 and the principal component molecular weight is about 85,000. Under reducing conditions (state of 2-ME ⁺ ), dyeing bands are detected at 39,000 to 46,000 and the principal component molecular weight is 43,000.

(7) N-terminal region amino acid sequence of CHO [-32-522] M-CSF

The N-terminal region amino acid sequence of CHO [-32-522] M-CSF obtained in step (5) was determined using a gas phase sequence analyzer (manufactured by Applied Biosystems). As a result, the N-terminal ten amino acid sequence is confirmed as follows.

The amino acid (X ′) could not be identified in cycle 7, but is estimated to be Cys based on the genetic structure.

Glu-Glu-Val-Ser-Glu-Tyr-X'-Ser-His-Met-

Reference Example 2

[1] Preparation of the plasmid ptrpIL-2X-CSF101

Plasmid pcDM-CSF11-185 [prepared from plasmid pcDM-CSF11 containing the M-CSF gene (approximately 2.5 kb, λ cMll cDNA). (See Japanese Laid-Open Patent Publication No. 1-104176), the restriction enzyme ScaI And ScaI-BamHI DNA fragment (about 450 bp) is isolated after digestion with BamHI and purified by agarose gel electrophoresis. The synthetic linker (A) shown below was then ligated to the ScaI cleavage site of the obtained DNA fragment using a T4 DNA ligase to provide an XbaI-BamHI DNA fragment having an XbaI (restriction enzyme) cleavage site on the ScaI cleavage end (about 480 bp).

Synthetic Linker (A):

2nd SDter

5 '-CTA GAA CGG AGG ACT CAT TGATG GTA TCA GAA T-3'

TT GCC TCC TGA GTA ACTAC CAT TGT CTT A

XbaI to ScaI

(box)

The DNA fragment thus obtained was inserted into the human IL-2 expressing plasmid ptrpIL-2D8Δ (Digestion No. 63-12958) between the XbaI and BamHI cleavage sites, and the target plasmid ptrpIL-2X-M-CSF 101 is obtained.

The transformant obtained by transforming Escherichia coli HB101 with the plasmid was Escherichia coli under the accession number of FERM BP-2226 (E. coli [3-153] FERM BP-2226] on December 26, 1988. It was usually deposited in the Institute of Industrial Technology and Technology Microbial Industry Research Institute (FRI) under the name HB101 / ptrpIL-2X-M-CSF 101.

[2] Lee. Isolation and Purification of Coli [3-153] M-CSF

(1) Preparation of M-CSF Fragments from Escherichia coli

E. coli with the plasmid ptrpIL-2X-M-CSF 101 obtained in the above procedure [1]. To 1.5 g (wet amount) of Coli strain SG21058, 50 ml of 50 mM trishydrochloride buffer (pH 7.0) containing 0.5 M sucrose is added and the mixture is stirred thoroughly. Then, 6 ml of 2 mg / ml lysozyme is added, 4 ml of 0.14 M EDTA is further added, stirred at 4 ° C. for 15 minutes, and the mixture is centrifuged at 10,000 revolutions / min for 20 minutes.

Discard the supernatant and wash the pellet with the same buffer (mM 7.0 tris-hydrochloride at pH 7.0 containing 0.5 M sucrose) and then centrifuge similarly at 10,000 revolutions per minute for 20 minutes to obtain spheroplasts as pellets. The pellet is suspended in 50 ml of 50 mM tris-hydrochloride (pH 7.0) and the suspension is sonicated at 20 KHz for 10 minutes and centrifuged at 10,000 revolutions / minute for 20 minutes. The pellet is washed with the same buffer (50 mM Trishydroclyde, pH 7.0) and centrifuged again under the same conditions to yield an M-CSF fraction as a pellet.

(2) refolding of M-CSF in M-CSF fraction

To the M-CSF fraction obtained in the above step (1), 100 ml of 50 mM tris-hydrochloride (pH 7.0) containing 7.0 M guanidine hydrochloride was added and the mixture was stirred for 1 hour at 4 ° C. to dissolve. This solution was slowly added dropwise (be stirred with a stirrer) to a beaker already containing 300 ml of 10 mM trishydrochloride (pH 8.5) and after the addition was completed the mixture was thoroughly brought to 10 mM tris-hydrochloride (pH 8.5) at 4 ° C. After dialysis, centrifuge at 10,000 revolutions / minute for 20 minutes. Discard the precipitate and recover the supernatant.

Refolded M-CSF is present in the supernatant thus obtained.

(3) Purification of M-CSF

The M-CSF obtained in the above (2) is purified as follows.

(3-1) Gel Filtration High Performance Liquid Chromatography

The supernatant obtained in the above step (2) was concentrated by a single filter (membrane: YM-10 membrane, manufactured by Amicon), and the concentrated solution was passed through a 0.45 μm Millipore filter to perform high performance gel filtration liquid chromatography under the following conditions. do.

Column: TSK gel G3000 SW (60 cm x 21.5 mm I.D., manufactured by Tosoh Corporation)

Eluent: 50 mM sodium phosphate buffer containing 0.3 M NaCl (pH 6.8)

Flow rate: 3.0ml / min

Fraction volume: 6ml / Tube / 2 minutes

In this process, the standard protein for gel filtration HPLC (manufactured by Oriental Yeast Co., Ltd), namely glutamate dehydrogenase (molecular weight 290,000), lactate hydrogenase (molecular weight 142,000), enolase (molecular weight 67,000) and ade Judging from the elution position of the nilate kinase (molecular weight 32,000), the M-CSF molecular weight is estimated to be 32,000.

The active fractions are collected and exchange buffer for 40 mM sodium borate (pH 8.0) with the above-mentioned ultrafiltration unit.

(3-2) TSK Gel DEAE-5PW Ion Exchange High Performance Liquid Chromatography

The eluate fraction obtained in step 93-1) is subjected to TSK gel DATE-5PW ion exchange high performance liquid chromatography under the following conditions.

Column: TSK gel DEAE-5PW (7.5 mm I.D x 75 mm, manufactured by Tosoh Corp.)

Eluent A: 40 mM sodium borate buffer (pH 8.0) containing 5% methanol

Eluent B: 40 mM sodium borate buffer (pH 8.0) containing 1.0 M NaCl and 5% methanol

Flow rate: 1.0ml / min

Fraction Volume: 1.0ml / Tube / Min

Concentration gradient: time (minutes) B%

0 0

7 0

42 30

47100

52100

57 0

62 0

Judging from the TSK gel DEAE-5PW ion exchange high performance liquid chromatography result (elution pattern), the peaks observed in No. 42 to 43 fractions (NaCl concentration 0.23 to 0.25 M) correspond to M-CSF, and the peaks were collected. do. C-CSF (E. coli [3-153] M-CSF) thus purified is obtained from Escherichia coli.

Reference Example 3

CHO [-32-522] M-CSF Preparation

930 ml of the concentrated culture supernatant obtained by culturing CHO cells in the same manner as in Reference Example 1 were fractionated with ammonium sulfate. A fraction obtained by saturation of 25-65% with ammonium sulfate is obtained. Then, the fraction is dissolved in distilled water (1180 ml) and purified by the following method. M-CSF detection is performed as in Reference Example 1 by Western blotting.

(1) ConA-Sepharose Chromatography

Equilibrate with 20 mM sodium phosphate buffer (pH 7.4) containing curum (5 × 25 cm) DMF 0.15 M NaCl filled with about 500 ml ConA-Sepharose gel and apply the solution to the column. After complete washing with the same buffer, it is eluted with the same buffer containing 0.5M methyl α-D-mandoside. The total eluate is concentrated by ultrafiltration using a YM-10 membrane and the buffer is exchanged with 20 mM sodium phosphate buffer (pH 7.4). The resulting solution is subjected to high performance anion exchange liquid chromatography with five aliquots.

(2) Anion Exchange High Performance Liquid Chromatography

Column: TSK gel DEAE-5PW (21.5 mm I.D × 15 cm, manufactured by Tosoh Corp,)

Eluent A: 40 mM sodium phosphate buffer (pH 7.4)

Eluent B: 40 mM sodium phosphate buffer containing 0.1M NaCl, pH 7.4

Flow rate: 3.0 ml / min

Fraction Volume: 6ml / Tube / 2min

Concentration gradient: time (minutes) B%

0 0

10 0

20 10

95 30

100100

110100

115100

115 0

130 0

As a result of the elution, the desired M-CSF is eluted into Nos. 26 to 43 fractions. The main M-CSF fractions, ie fractions 30-39, are combined and concentrated by ultrafiltration (using YM-10 membrane) (15 ml) and purified into four aliquots as follows.

(3) TSK Gel Phenyl-5PWRP High Performance Reverse Phase Liquid Chromatography

Column: TSK gel phenyl-5PWRP (21.5 mm I.D × 15 cm, manufactured by Tosoh Corp,)

Eluent A: 0.1% TFA

Eluent B: n-propanol: 1.0% TFA (9: 1)

Flow rate: 3 ml / min

Concentration gradient: time (minutes) B%

0 0

10 0

20 20

100 45

120100

130100

140 0

180 0

The M-CSF fractions of the elution fractions were combined, concentrated by ultrafiltration (using YM-10 membrane), further concentrated by Centricon 30 (manufactured by Amiocon) (1 ml), and then gel-filtered high performance liquid chromatography with three aliquots. do.

(4) Gel Filtration High Performance Liquid Chromatography

Column: Super Ross 12 HR 10/30 (10 mm I.D × 30 cm, manufactured by Pharmacia LKB)

Eluent: 20 mM sodium phosphate buffer containing 0.3 M NaCl, pH 7.4

Flow rate: 0.8 ml / min

Fraction volume: 0.8 ml / tube / 2 minutes

As a result of the chromatography, M-CSF was eluted in fractions 15-17, and the fractions 16 were combined and tested (5.6 mg, specific activity: 3.8 × 10 ⁷ units / mg protein).

Reference Example 4

Improved Isolation and Purification of M-CSF Derivatives

(E. coli [3-153] -M-CSF).

(1) Lee. Preparation of M-CSF Fraction from Collie

E. carrying a plasmid ptrpIL-2X-M-CSF 101. To 7.5 g (wet weight) of Coli strain SG 21058, 1.0 ml of 50 mM Tris-hydrocride buffer (pH 7.0) is added and the mixture is stirred thoroughly. Then 6 ml of 4 mg / ml lysozyme and an amount of EDTA to produce a final concentration of 10 mM were added and the mixture was stirred at 4 ° C. for 15 minutes, then sonicated (20 KHz, 10 minutes, 200 W) and 10,000 × g Pellets are obtained by centrifugation at / min for 20 minutes. It is washed with washing buffer (500 mM Tris-hydrochloride, pH 7.0 containing 2% Traton × 100) and centrifuged again under the same conditions. This method is repeated twice to obtain the M-CSF fraction (pellet).

(2) refolding of M-CSF from M-CSF fraction

To the M-CSF fraction obtained according to the above method (1) was added 20 ml of 50 mM tris-hydrochloride buffer (pH 7.0) containing 7M guanidine hydrochloride and 25 mM 2-mercapto ethanol and the mixture at room temperature. Stir for dissolution for at least 4 hours. The solution is slowly added dropwise into a beaker already containing 2,000 ml of 50 mM tris-hydrochloride buffer (pH 8.5) containing 0.5 mM reduced glutathione, 0.1 mM oxidized glutathione and 2 M urea. (With stirring with a stirrer). The resulting mixture is then left at 4 ° C. for at least 2 days.

The solution is centrifuged at 10,000 x g / min for 30 minutes. Remove the precipitate and recover the supernatant.

There is M-CSF refolded in the supernatant thus obtained.

(3) Purification of M-CSF

The supernatant obtained by the method (2) is purified by the following method.

(3-1) Concentration by ion exchange chromatography

The refolded product solution obtained by method (2) was subjected to QAE-Zeta Prep 100 (product of Pharmacia-LBK) previously equilibrated with 50 mM Tris-hydrochloride buffer (pH 8.5) and then completely with the buffer. Wash and elute the M-CSF fractions with the above buffer containing 0.5M NaCl.

(3-2) Hydrophobic High Performance Liquid Chromatography

Ammonium sulfate is added to the fraction obtained by the above method to obtain a 30% -saturated solution and the solution is centrifuged at 10,000 x g / min for 20 minutes. Remove the sediment and recover the supernatant. This supernatant is passed through a 0.45 μm Millipore filter and subjected to hydrophobic high performance liquid chromatography under the following conditions.

Column: TSK gel phenyl 5PW (21.5 mm I.D × 150 mm, product of Tosoh Corpration).

Eluent A: 30% ammonium sulfate-saturated 40 mM sodium phosphate buffer (pH 7.4)

Eluent B: 40 mM sodium phosphate buffer (pH 7.4)

Flow rate: 3 ml / min

Fraction Volume: 3.0ml / Tube / Min

Concentration gradient: time (minutes) B%

0 0

7 0

47100

52100

57 0

As a result of this, M-CSF activity was eluted in the fraction corresponding to ammonium sulfate concentration of 6 to 3%. The active fractions are collected and buffer exchanged with 40 mM sodium phosphate (pH 7.4) using ultrafiltration.

(3-3) Anion Exchange High-Performance Liquid Chromatography

Fractions obtained by the above method (3-2) are subjected to anion exchange high-performance liquid chromatography under the following conditions.

Column: TSK Gel Phenyl-5PW (21.5mm I.D × 15mm, Tosoh Corpation)

Eluent A: 40 mM sodium phosphate buffer (pH 7.4)

Eluent B: 40 mM sodium phosphate buffer containing 1.0 M NaCl, pH 7.4

Flow rate: 3.0ml / min

Fraction Volume: 3.0ml / Tube / Min

Concentration gradient: time (minutes) B%

0 0

5 0

43 30

48100

53100

58 0

From the results of the anion exchange high-performance liquid chromatography (elution pattern), it was found that the peaks observed in fractions 35 and 36 (NaCl concentration 0.28-0.29M) correspond to M-CSF. The peaks are collected to obtain purified human M-CSF derivative (E. coli [3-153] -M-CSF).

Reference Example 5

(1) Preparation of ptrpIL-2X-M-CSF 201

Plasmid ptrpIL-2X-M-CSF 101 was digested with restriction enzymes BstEII and SalI to obtain 4.7 kb DNA fragment (I) containing IL-2 and M-CSF DNA portions.

Individually, plasmid pcDM-CSF 11 is digested with restriction enzyme EcoRI. The digested ends were blunt ends using DNA polymerase I Klenw fragments and then ligated to SalI linkers (5'-pGGTCGACC _OH- 3 ') (New England Biolabs). This ligation product was cleaved with restriction enzymes SalI and BstEII to yield about 1.1 Kb of BstEII-SalI fragment (II).

Said DNA fragments (I) and (II) were ligated together using T4 DNA ligase and E. coli using ligation products. Competent cells of Coli HB 101 were transformed to carry E. coli carrying the target transformant, the plasmid ptrpIL-2X-M-CSF201. One obtains a strain of Coli HB 101.

The plasmid ptrIL-2X-M-CSF201 thus obtained was E. coli. Two polypeptides are encoded in a transcription unit under the control of the coli tryptophan promoter. One is a 65 amino acid polypeptide consisting of methionine (initiation of translation), 60 amino acids of human termination of IL-2 in the human body and 4 amino acids coated by a synthetic DNA linker, and the other is Met (initiation of translation) and formula ( A polypeptide comprising a human M-CSF derivative consisting of 520 amino acids comprising position-3 aminoline (Val) to position-522 amino acid (Val) of the amino acid sequence defined by 1).

In the 2-cistron expression system, translation of the second cistron is initiated by binding ribosomes to a second SD sequence located in a synthetic DNA linker.

(2) Preparation of ptrpIL-2X-M-CSF 202

Plasmid ptrpIL-2X-M-CSF 201 was digested with restriction enzymes NcoI and SalI and the NcoI-SakI DNA fragment of about 4.8 kb was recovered. The two ends of this DNA fragment are ligated with T4 DNA ligase using synthetic oligodeoxynucleotides [5'-CATGGCCTGATAAG-3 'and 5'-TCGACTTATCAGGC-3'] with each other. this. Competent cells of Coli HB101 were transformed with ligation products and the desired transformants carrying the plasmid ptrpIL-2X-M-CSF 202. Obtain Collie HB 101 strain.

The obtained ptrpIL-2X-M-CSF 202 is E. coli. 182 amino acids, including position-3 amino acids (Val) to position-184 amino acids (Ala), of the amino acid sequence defined by formula (1) in the second cistron in the transcription unit under the control of the coli tryptophan promoter and Met (initiation of translation) Encoding a polypeptide comprising a human M-CSF derivative composed of

(3) Preparation of ptrpIL-2X-M-CSF 203

Plasmid ptrpIL-2X-M-CSF 201 was digested with restriction enzymes BamHI and SalI to recover about 4.9 kb of BamHI-SalI DNA fragment. Both ends of this DNA fragment were ligated with T4 DNA ligase using synthetic oligodeoxynucleotides [5'-GATCCATGATAAG-3 'and 5'-TGCACTTATCATG-3'] to each other and the ligation product was extracted. A desired E. coli carrying the plasmid ptrpIL-2X-M-CSF 203 used to transform Coli HB 101 competent cells. Obtain Coli HB 101 transformant.

The plasmid ptrpIL-2X-M-CSF 203 thus obtained is E. coli. 212 amino acids, including position-3 amino acids (Val) to position-214 amino acids (Pro), of the amino acid sequence defined by formula (I) in the second cistron in the transcription unit under the control of the coli tryptophan promoter and Met (initiation of translation) The polypeptide comprising a human M-CSF derivative consisting of

E. coli carrying the plasmid ptrpIL-2X-M-CSF 203. The strain Collie HB 101 was designated as Escherichia coli HB 101 / ptrpIL-2X-M-CSF 203, and was assigned to the Institute of Microbiological Technology (FRI) of the Ministry of Trade, Industry and Technology under the accession number FERM-P 11053 on October 18, 1989. I got stuck.

(4) Preparation of ptrpIL-2X-M-CSF204

Plasmid ptrpIL-2X-M-CSF201 was digested with restriction enzymes SmaI and SalI and the SmaI-SalI DNA fragment of about 5.0 kb was recovered. Both ends of this DNA fragment were ligated with T4 DNA ligase using synthetic oligodeoxynucleotides [5'-GGGTGATAAG-3 'and 5'-TCGACTTATCACCC-3'] to each other and the ligation product was then cloned. Desired E. coli with the plasmid ptrpIL-2X-M-CSF204 used to transform competent cells of Coli HB 101. Obtain Coli HB 101 transformant.

The resulting plasmid ptrpIL-2X-M-CSF204 yielded E. coli. 256 amino acids, including position-3 amino acids (Val) to position-258 amino acids (Gly), of the amino acid sequence defined by formula (1) in the second cistron in the transcription unit under the control of the coli tryptophan promoter and Met (initiation of translation) Encoding a polypeptide comprising a human M-CSF derivative composed of

(5) Preparation of ptrpIL-2X-M-CSF205

Plasmid ptrpIL-2X-M-CSF201 was digested with restriction enzymes SphI and SalI and the SphI-SalI DNA fragment of about 5.1 kb was recovered. Both ends of the DNA fragments were ligated with T4 DNA ligase using synthetic oligodeoxynucleotides [5'-CATGTGATAAG-3 'and 5'-TCGACTTATCACTGCATG-3'] to each other and the resulting ligation product. Desired E. coli with the plasmid ptrpIL-2X-M-CSF205 used to transform competent cells of Coli HB 101. Obtain Coli HB 101 transformant.

The plasmid ptrpIL-2X-M-CSF205 thus obtained was E. coli. 300 amino acid ALC Met comprising position-3 amino acid (Val) to position-302 amino acid (Gln) of the amino acid sequence defined by formula (1) in the second cistron in the transcription unit under the control of the coli tryptophan promoter (initiation of translation) Encoding a polypeptide comprising a human M-CSF derivative composed of

(6) Preparation of ptrpIL-2X-M-CSF206

Plasmid ptrpIL-2X-M-CSF201 was digested with restriction enzymes KpnI and SalI and the KpnI-SalI DNA fragment of about 5.2 kb was recovered. Both ends of this DNA fragment are ligated with T4 DNA ligase using synthetic oligodeoxynucleotides [5'-CGCCTGATAAG-3 'and 5'-TCGACTTATCAGGCGGTAC-3'] to each other and to the ligation product. The desired E. coli with the plasmid ptrpIL-2X-M-CSF206 was transformed by product transformation of competent cells of Coli HB 101 with the product. Coli HB 101 transformant is obtained.

The resulting plasmid ptrpIL-2X-M-CSF206 was E. coli. 332 amino acids, including position-3 amino acids (Val) to position-334 amino acids (Ala), of the amino acid sequence defined by formula (1) in the second cistron in the transcription unit under the control of the coli tryptophan promoter and Met (initiation of translation) Encoding a polypeptide comprising a human M-CSF derivative.

(7) Expression of M-CSF Derivatives

The plasmids obtained by the above methods (1) to (6) were each e. Coli strain SG 21058 [J. Bacteriol., 164, 1124-1135 (1985)] were introduced by transformation methods to transformants [E. Collie SG21058 / ptrpIL-2X-M-CSF201, Yi. Collie SG21058 / ptrpIL-2X-M-CSF202, Yi. Collie SG21058 / ptrpIL-2X-M-CSF203, Yi. Collie SG210 58 / ptrpIL-2X-M-CSF204, E. coli SG21058 / ptrpIL-2X-M-CSF205 and E. coli. Collie SG 21058 / ptrpIL-2X-M-CSF206.

These transformants were added in LB medium containing 50 μg / ml of ampicillin (T. Maniatis, EF Fritsch and J. Sambrok, Molecular Cloning, A Laboratory Manual, p. 440, (1982), Cold Spring Harbor Laboratory). Incubate overnight at 37 ° C. 0.5 ml aliquots of each culture were supplemented with 0.5 µg / ml ampicillin with a glucose concentration of 0.4%, 20 µg / ml L-cysteine, 5 µg / ml thiamine hydrochloride, and 50 ml M9 supplemented with 1% casamino acid. Add to the medium (see references cited above) and shake incubation at 37 ° C. for 8 hours. Cells are recovered by centrifugation (5,000 rpm) and the resulting protein is analyzed by SDS-PAGE and Western blotting.

Western blotting is performed using Bio-Rad's transblot cells. The passage of the nitrocellulose membrane after passage was closed with PBS ⁻ containing 1% small iron albumin, reacted with rabbit antiserum against M-CSF and also with peroxidase-labeled goat anti-rabbit antibody (product of Bio-Rad Laboratories). Let's do it. M-CSF band detection is performed by reacting the nitrocellulose membrane thus obtained with a solution of the dye substrate 4-chloro-1-naphthol.

this. Collie SG21058 / ptrpIL-2X-M-CSF202, Yi. Collie SG21058 / ptrpIL-2X-M-CSF203, Yi. Collie SG210 58 / ptrpIL-2X-M-CSF204, E. coli SG21058 / ptrpIL-2X-M-CSF205 and E. coli. For Coli SG 21058 / ptrpIL-2X-M-CSF206, M-CSFs encoded in the second cistron were each detected at positions corresponding to the predicted molecular weight ranges.

(8) Isolation and Purification of M-CSF Derivatives (E. coli [3-214] M-CSF)

10 g (wet weight) of E. g. Comprising plasmid ptrpIL-2X-M-CSF203 obtained by the above method (3). 50 mM Tris-hydrochloride buffer (pH 7.0) is added to Coli SG21058 cells to a total volume of 10 ml and then stirred thoroughly. Then 4 ml of 6 mg / ml lysozyme and 8 ml of 0.14 M EDTA are added and the mixture is stirred at 4 ° C. for 15 minutes, then sonicated (200 KHz, 2 minutes, 200 W) and centrifuged at 10,000 revolutions / minute for 20 minutes. . The resulting pellet was washed thoroughly with 50 mM tris-hydrochloride (pH 7.0) containing 2% Triton X-100 and centrifuged again under the same conditions to give the M-CSF fraction as a pellet.

To the M-CSF fraction thus obtained was added 50 mM tris-hydrochloride (pH 7.0) containing 20 ml of 7.0 M guanidine hydrochloride and 50 mM mercapto ethanol and the mixture was reduced at room temperature to reduce, denature and dissolve 4. Stir for time. To dilute this solution 100-fold, add dropwise (stirrer) into a beaker containing 2000 ml of 0.5 mM reduced glutathione, 0.1 mM oxidized glutathione and 50 mM tris-hydrochloride (pH 8.5) containing 2M urea. Shaking with). After completion of the dropping, the dilution is left at 4 ° C. for at least 2 days and then centrifuged at 3000 revolutions / minute for 30 minutes. The pellet is removed and the supernatant (2 L) is collected.

Refolded M-CSF is present in the supernatant thus obtained.

One liter of the centrifuge supernatant obtained by the above method is concentrated using an ultrafiltration apparatus (Amicon's product, YM-10 membrane (Amicon)) and the concentrate is centrifuged at 10,000 revolutions / minute for 20 minutes. The pellet is removed and the supernatant is recovered.

To this supernatant is added ammonium sulfate in an amount sufficient for 30% saturation. The resulting mixture is centrifuged again in the same manner and the supernatant is hydrophobic interaction chromatography under the following conditions.

Column: TSK gel phenyl 5PW (7.5 mm I D × 75 mm, product of Tosoh Corporation)

Eluent A: 30% ammonium sulfate-saturated 50 mM sodium phosphate buffer (pH 7.4)

Eluent B: 50 mM sodium phosphate buffer (pH 7.4)

Flow rate: 1.0ml / min

Fraction Volume: 1ml / Tube / Min

Concentration gradient: time (minutes) B%

0 0

7 0

40100

45100

50 0

60 0

As a result of this method, M-CSF activity is eluted in fractions Nos. 40-42 (saturation of ammonium antioxidant concentration of 8-6%). The active fractions are collected and subjected to buffer exchange for 50 mM sodium phosphate (pH 7.4) by the above-mentioned ultrafiltration apparatus, followed by DEAE-5PW ion exchange high-performance liquid chromatography under the following conditions.

Column: TSK gel phenyl 5PW (7.5 mm I D × 75 mm, product of Tosoh Corporation)

Eluent A: 50 mM sodium phosphate buffer (pH 7.4)

Eluent B: 50 mM sodium phosphate buffer containing 1.0 M NaCl, pH 7.4

Flow rate: 1.0ml / min

Fraction Volume: 1ml / Tube / Min

Concentration gradient: time (minutes) B%

0 0

7 0

42 30

47100

52100

57 0

62 0

In the results (elution pattern) of the DEAE-5PW ion exchange high-performance liquid chromatography, the peaks observed at fractions 31 and 32 (NaCl concentration 0.18 to 0.20 M) correspond to M-CSF. The peaks are collected and the human M-CSF derivative of the present invention is obtained.

In the purification step, the activity, protein concentration, specific activity and degree of purification of the human M-CSF derivative of the present invention are measured. The results are shown in Table 1 below.

TABLE 5

(9) SDS-PAGE of M-CSF derivatives (E. cory [3-214] -M-CSF)

Samples purified by this method according to Laemli's method [Laemmli, UK, Nature, 277, 680 (1970)] are dissolved in each of Laemry's sample buffers [2-ME ⁺ and 2-ME ⁻ ] and The solution is heat treated at 95 ° C. for 5 minutes and then electrophoresed using a small flat gel [apparatus: product from Marisol Sangyo KK or Daiichi Chemical Co., Ltd.]. Pre-dyed markers (product of Bio-Rad Laboratories) are used as molecular weight markers and Coomssie Brilliant Blue R-250 is used for staining.

As a result, this. The molecular weight of collie [3-214] -M-CSF was found to be about 52,500 in the 2-ME- state and about 26,900 in the 2-ME ⁺ state and electrophoresis showed a single band in each part.

Reference Example 6

(1) Preparation of the plasmid ptrpIL-2X-M-CSF109

Plasmid pcDM-CSF 11-185 [prepared from plasmid pcDM-CSF 11-185 containing the M-CSF gene (λcMll cDNA, about 2.5 kb) (see Japanese Patent Application Laid-Open No. 1-104176)] Digestion with ScaI and BamHI and ScaI-BamHI DNA fragment (about 450 bp) is isolated and purified by agaroosso gel electrophoresis.

Thereafter, the synthetic linker (B) shown below was ligated to the ScaI cleavage site of the DNA fragment obtained using T4 DNA ligase, so that the XbaI-BamHI DNA fragment having XbaI (restriction enzyme) cleavage site on the ScaI cleavage site (about 480 bp).

Synthetic Linker (B):

2nd SDter

5'-CTA GAA CGG AGG ACT CAT TGATG TCA GAA T-3 '

TT GCC TCC TGA GTA ACTAC AGT CTT A

Start

(Box)

The DNA fragment thus obtained was inserted between the XbaI and BamHI cleavage sites of the human IL-2 expression plasmid ptrpIL-2D8Δ (see Japanese Patent Application Laid-Open No. 63-12958), and the desired plasmid, ptrpIL-2X-M -CSF109 is obtained.

The plasmid ptrpIL-2X-M-CSF109 thus obtained was E. coli. Two polypeptides are encoded in a transcription unit under the control of the coli tryptophan promoter. One is a 65-amino acid polypeptide consisting of 4 amino acids resulting from the synthetic linker DNA sequence and 60 amino termini of human IL-2 and Met (translation initiation), and the other is Met (translation initiation) and formula ( A polypeptide consisting of 150 amino acids comprising position-4 amino acids (Ser) to position-153 amino acids (Thr) of the amino acid sequence defined by 1).

(2) Preparation of ptrpIL-2X-M-CSF402

Plasmid ptrpIL-2X-M-CSF109 was digested with restriction enzymes BetEII and ScaI to recover a DNA fragment of about 1.5 kb.

Individually, the plasmid ptrpIL-2X-M-CSF202 is digested with restriction enzymes BstEII and ScaI in the same manner to recover a DNA fragment of about 3.3 kb.

The two DNA fragments were ligated with each other using T4 DNA ligase and the ligation mixture was extracted. E. coli with the desired plasmid ptrpIL-2X-M-CSF402, which was used to transform competent cells of Coli HB101. Obtain Coli HB101 transformant.

The plasmid ptrpIL-2X-M-CSF402 thus obtained was E. coli. From the second cistron in the transcription unit under the control of the coli tryptophan promoter to 181 amino acids from position-4 amino acid (Ser) to position-184 amino acid (Ala) of the amino acid sequence defined by Met (translation initiation) and formula (1) It encodes a polypeptide comprising a constructed human M-CSF derivative.

(3) Preparation of ptrpIL-2X-M-CSF403

Plasmid ptrpIL-2X-M-CSF109 was digested with restriction enzymes BstEII and ScaI to recover a DNA fragment of about 1.5 kb.

Individually, the plasmid ptrpIL-2X-M-CSF203 was digested with restriction enzymes BstEII and ScaI in the same manner to recover a DNA fragment of about 3.4 kb.

The two DNA fragments were ligated with each other using T4 DNA ligase and the ligation mixture was extracted. E. coli with the desired plasmid ptrpIL-2X-M-CSF403, which was used to transform competent cells of Coli HB101. Obtain Coli HB101 transformant.

Thus obtained polamide ptrpIL-2X-M-CSF403 is E. coli. 211 amino acids of position-4 amino acids (Ser) to position-214 amino acids (Pro) of the amino acid sequence defined by Met (translation initiation) and formula (1) in the second cistron in the transcription unit under the control of the coli tryptophan promoter It encodes a polypeptide comprising a constructed human M-CSF derivative.

(4) Preparation of ptrpIL-2X-M-CSF404

Plasmid ptrpIL-2X-M-CSF109 was digested with restriction enzymes BetEII and ScaI to recover a DNA fragment of about 1.5 kb.

Individually, the plasmid ptrpIL-2X-M-CSF204 is cleaved with restriction enzymes BstEII and ScaI in the same manner to recover a DNA fragment of about 3.5 Kkb.

The two DNA fragments were ligated with each other using T4 DNA ligase and the ligation mixture was extracted. E. coli with the desired plasmid ptrpIL-2X-M-CSF404 used to transform competent cells of Coli HB101. Obtain Coli HB101 transformant.

The plasmid ptrpIL-2X-M-CSF404 thus obtained was E. coli. Consists of 255 amino acids from position-4 amino acids (Ser) to position-258 (Gly) of the amino acid sequence defined by Met (translation initiation) and formula (1) in the second cistron in the transcription unit under the control of the coli tryptophan promoter It encodes a polypeptide comprising a human M-CSF derivative.

(5) Preparation of ptrpIL-2X-M-CSF405

Plasmid ptrpIL-2X-M-CSF109 was digested with restriction enzymes BstEII and ScaL to recover a DNA fragment of about 1.5 kb.

Individually, the plasmid ptrpIL-2X-M-CSF205 is digested with restriction enzymes BstEII and ScaI in the same manner to recover a DNA fragment of about 3.6 kb.

The DAN fragments were ligated with each other using T4 DNA ligase, and the L. E. coli carrying the target plasmid ptrpIL-2X-M-CSF405 by transformation of competent cells of Coli HB 101. Obtain Coli HB 101 transformant.

The plasmid ptrpIL-2X-M-CSF405 thus obtained was E. coli. 299 amino acids from 4-position amino acid (Ser) to 302-position amino acid (Gln) of the amino acid sequence defined by Met (translation) and formula (1) in the second cistron in the transcription unit under the control of the coli tryptophan promoter A polypeptide containing a human M-CSF derivative consisting of.

(6) Preparation of ptrpIL-2X-M-CSF406

Plasmid ptrpIL-2X-M-CSF109 was digested with restriction enzymes BstEII and ScaI and DNA fragments of about 1.5 kb were recovered.

Separately, the plasmid ptrpIL-2X-M-CSF206 is cleaved with restriction enzymes BstEII and ScaI in the same manner and a DNA fragment of about 3.7 kb is recovered.

The DNA fragments were ligated with each other using T4 DNA ligase and using the ligation mixture. E. coli carrying the target plasmid ptrpIL-2X-M-CSF406 by transforming the competent cells of Coli HB 101. Obtain Coli HB 101 transformant.

The plasmid ptrpIL-2X-M-CSF406 thus obtained was E. coli. 331 amino acids from 4-position amino acid (Ser) to 334-position amino acid (Ala) of the amino acid sequence defined by Met (translation initiation) and formula (1) in the second cistron in the transcription unit under the control of the coli tryptophan promoter Encoding a polypeptide containing the constructed human M-CSF derivative.

(7) Expression of M-CSF Derivatives

The plasmids obtained in the above steps (1) to (6) were transformed into E. coli. Coli strain SG 21058 [J. Bacteriol., 164, 1124-1135 (1985), respectively, to obtain transformants [E. Collie SG21058 / ptrpIL-2X-M-CSF109, Yi. Collie SG21058 / ptrpIL-2X-M-CSF402, Yi. Collie SG21058 / ptrpIL-2X-M-CSF403, Yi. Collie SG21058 / ptrpIL-2X-M-CSF404, Yi. Collie SG21058 / ptrpIL-2X-M-CSF 405 and Yi. Collie SG21058 / ptrpIL-2X-M-CSF406.

These transformants were transformed into LB medium containing 50 μg / ml ampicillin [T. Maniatis, E. F. Fritsch and J. Sambrook, Molecuar Cloning, A Laboratory Manual, p. 440 (1982), Cold Spring Harbor Laboratory]. 0.5 ml of each culture was added to M9 medium with 1% cassanoic acid, 5 μg / ml thiamine hydrochloride, 20 μg / ml L-cysteine and 50 μg / ml ampicillin and the glucose concentration adjusted to 0.4% (see cited above). ) Was added to 50 ml, and the cells were recovered by centrifugation (5,000 rpm) after 8 hours of shaking culture at 37 ° C, and the resulting protein was subjected to SDS-PAGE and Western blotting in the same manner as in Reference Example 5- (7). Analyze

As a result, the second, M-CSF respectively encoded in the cistron. Collie SG21058 / ptrpIL-2X-M-CSF109, Yi. Collie SG21058 / ptrpIL-2X-M-CSF402, Yi. Collie SG210 58 / ptrpIL-2X-M-CSF403, Yi. Collie SG21058 / ptrpIL-2X-M-CSF404, Yi. Collie SG2 1058 / ptrpIL-2X-M-CSF405, and E. Detects at positions corresponding to the respective estimated molecular weights of Coli SG21058 / ptrpIL-2X-M-CSF406.

(8) Isolation and Purification of M-CSF Derivatives (E. cory [4-153] -M-SCF)

[1] this. Preparation of M-CSF Fraction from Collie

E. coli carrying the plasmid ptrpIL-2X-M-CSF109 obtained in step (1) above. 50 g of Tris-hydrochloride buffer (pH 7.0) was added to 7.5 g (humidity) of Coli SG 21058 strain to make 100 ml of total volume, followed by stirring. Next, 6 ml of 2 mg / ml lysozyme is added followed by 4 ml of 0.14 M EDTA. The mixture was stirred at 4 ° C. for 15 minutes, then subjected to sonication (20 KHz, 10 minutes, 200 W) and centrifuged at 10,000 revolutions / minute for 20 minutes to obtain pellets. It is further washed with washing buffer (50 mM Tris-hydrochloride buffer containing 2% Triton X-100, pH 7.0) and re-isolated under the same conditions as above. This process is repeated twice to obtain an M-CSF fraction (pellet).

[2] refolding of M-CSF in M-CSF fraction

In the M-CSF fraction obtained in [1], 7M guanidine hydrochloride and 25mN 2-mercaptoethanol are stirred at room temperature for 4 hours or more to reduce, denature and dissolve protein. This solution was slowly added dropwise (with stirring in a stirrer) to a beaker containing 2000 ml of 50 mM Tris-hydrochloride (pH 8.5) containing 0.5 mM reduced glutathione, 0.1 mM oxidized glutathione and 2 M urea and the resulting mixture was stirred 4 Leave at least 2 days at ℃. The solution is then centrifuged at 3500 revolutions / minute for 30 minutes, the pellet is removed and the supernatant is recovered.

Refolding M-CSF is present in the supernatant thus obtained.

[3] purification of M-CSF

The centrifugation supernatant obtained in the above [2] was purified in the following manner.

[3-1] Hydrophobic High Performance Liquid Chromatography

The centrifuged supernatant was concentrated with an ultrafiltration apparatus (manufactured by Amicon, using a YM-10 membrane (icon)) and ammonium sulfate was added to the concentrate to form a 30% saturated solution. The resulting solution is centrifuged for 20 minutes at 10,000 revolutions per minute. Remove the pellet and recover the supernatant. This supernatant is passed through a 0.45 μm Millipore filter and subjected to hydrophobic interaction chromatography in two portions under the following conditions.

Column: TSK gel phenyl 5PW (7.5㎜ ID × 75㎜, manufactured by Toso Kabukishi Kaisha)

Eluent A: 30% ammonium sulfate-saturated 40 mM sodium phosphate buffer (pH 7.4)

Eluent B: 40 mM phosphate natrum buffer (pH 7.4)

Flow rate: 1.0ml / min

Fraction Volume: 1ml / Tube / Min

Concentration gradient: time (minutes) B%

0 0

7 0

40100

45100

50 0

As a result, M-CSF activity elutes in fractions corresponding to ammonium sulfate concentrations of 11-6%. The active fractions are collected and 40 mM sodium phosphate (pH 7.4) buffer exchange is carried out using the ultrafiltration apparatus.

[3-2] Anion Exchange High Performance Liquid Chromatography

The fraction obtained by the above step [3-1] is subjected to DEAE-5PW anion exchange high performance liquid chromatography in four portions under the following conditions.

Column: TSK Gel DEAE-5PW (7.5mm ID × 75mm, manufactured by Toso Kabushiki Kaisha)

Eluent A: 50 mM phosphate natrum buffer (pH 7.4)

Eluent B: 50 mM sodium phosphate buffer containing 1.0 M NaCl, pH 7.4

Flow rate: 1.0ml / min

Fraction Volume: 1ml / Tube / Min

Concentration gradient: time (minutes) B%

0 0

5 0

55 30

60100

65100

70 0

In the DEAE-5PW ion exchange high performance liquid chromatography results (elution pattern), the peaks observed at fractions 41 and 42 (NaCl concentration 0.21 to 0.22M) were found to correspond to M-CSF. These peaks are collected to obtain the purified human M-CSF derivative (E. coli [4-153] -M-CSF) of the present invention.

(9) M-CSF Derivatives (N-terminal amino acid sequence determination of E. coli [4-153] -M-CSF

The N-terminal amino acid sequence of the M-CSF derivative (E. coli [4-153] -M-CSF) obtained in the above step (8)-[3-2] was subjected to a gas phase sequencer (Applied Biosystems). Determine using

As a result, the sequence Ser-Glu-Tyr-X'-Ser and the sequence Met-Ser-Glu-Tyr-X'-Ser are observed at a ratio of about 7-8: 1.

The amino acid X 'in this sequence could not be identified but is assumed to be Cys based on the genetic structure.

Bioavailability

Bioavailability serves as an indicator of whether the administered agent is useful for the living body. Here, the bioavailability is expressed as the ratio of the blood half-life in the case of intravenous injection and the blood half-life in the case of subcutaneous injection.

Blood half-life is measured by RIA. The starch of blood half-life is measured by the trapezoidal rule and the bioavailability is calculated according to the following equation.

Bioavailability (%) = AUCsc / AUCiv × 100

(1) blood half-life measurement

Physiological saline containing MSA (mouse serum albumin) containing M-CSF derivative (E. coli [3-153] -M-CSF) and CHO [-32-522] -M-CSF obtained in Reference Example 1 (30 µg / ml), each was diluted to a concentration of 20 µg / ml and administered intravenously (iv) in an amount of 0.25 ml / mouse (200 µg / kg) to a group of 4 mice (BALB / c male, 7 weeks old). After administration, the amount of M-CSF in the blood is measured by RIA at regular intervals.

Blood half-life measurement by this RIA is performed by the following method. That is, 100 μl of a test sample (serum) or standard M-CSF (M-CSF derivative (E. coli [3-153] -M-CSF) obtained in Reference Example 2), Na ¹²⁵ I-labeled M-CSF ( 100 μl of a solution containing 10,000 cpm (per 100 μl) derived from the standard M-CSF by labeling ¹²⁵ I by the iodogen method, and anti-M-CSF rabbit antiserum (polyclonal antibody obtained by the method described below) 200 μl of a mixture of OCT 511 diluted 40,000 times with 0.1% BSA / PBS / 0.05% thimerozal) is left at room temperature for 20 hours or 48 hours at 4 ° C. to give an antigen-antibody reaction. After completion of the reaction, the labeled M-CSF-binding product and unbound product are separated by the 2-antibody method. To perform this separation, 100 μl of normal rabbit serum diluted 400-fold with 0.1% BSA / PBS / 0.05% thimerozal, 100 μl of 40-fold diluted anti-rabbit IgG serum and 12.5% polyethylene glycol solution in PBS 200 μl is added first, the reaction proceeds at 4 ° C. for 30 minutes, centrifuged at 3,000 rpm for 15 minutes, and then decanted to obtain labeled M-CSF antibody conjugate as a precipitate. The precipitate is counted with a γ counter for 1 minute. A standard curve was prepared using a standard solution, the same procedure was repeated for the test sample containing the M-CSF derivative of the present invention, and the similarly measured number was plotted on the standard curve, and the M-CSF in the test sample was measured. Measure the concentration.

The results thus obtained are shown in Table 6 below.

TABLE 6

The unit in the table is ng / ml.

CHO [-32-522] -M-CSF and E. Bioavailability of Coli [3-153] -M-CSF was calculated from the results in Table 6 above to give the following results.

CHO [-32-522] -M-CSF:

(5876.6 / 85087.5) × 100 = 6.63%

this. Collie [3-153] -M-CSF:

(24519.9 / 20966.5) × 100 = 116.95%

this. Collie [3-214] -M-CSF:

(44602.0 / 267107.0) × 100 = 16.70%

As is evident from the above, the M-CSF derivative of the present invention. Coli [3-153] -M-CSF is better transported to the blood by subcutaneous administration, which indicates better bioavailability by subcutaneous administration than CHO [-32-522] -M-CSF. It is considered to be particularly suitable as an anti-allergic agent usually administered subcutaneously.

Other cloned antibodies against M-CSF used in the RIA have the following characteristics.

1) The polyclonal antibody used in the above process is prepared by the following method.

To prepare other cloned antibodies, the same amount of Freund's complete adjuvant with a female New Zealand white rabbit weighing 2.5-3.0 kg with 20 μg / animal CHO [-32-522] -M-CSF (dissolved in PBS) purified sample After intramucosal immunization, the cells were immunized every month with 20 µg of the same sample and Freund's incomplete supplement. The total number of immunizations is seven, including the first. One week after the end of the immunization process, the rabbits are harvested for whole blood and antiserum obtained.

One of the three antibodies obtained is named OCT 511 and stored at -80 ° C.

2) The polyclonal antibody OCT 511 used for the blood half-life measurement of each M-CSF derivative has the following characteristics.

[1] antibody titers

CHO [-32-522] -M-CSF can be labeled with ¹²⁵ I by the iodogen method and antibody titers can bind 50% of this ¹²⁵ I-labeled CHO [-32-522] -M-CSF 10 kpm. Defined as the dilution factor for antiserum.

As a result, the antibody titer of OCT511 is 80,000.

[2] neutralization activities

Neutralization activity is determined by clonal assay using mouse bone marrow cells.

The neutralizing activity of OCT511 is to neutralize OCT511 1 ml CHO [-32-522] -M-CSF 1 to 2 × 10 ⁶ units.

[3] cross-reactivity

This OCT 511 does not cross react at all with mouse CSF (L-cell culture supernatant) or human GM-CSF (Amersham). In addition, human body 1L-1α (digestion 63-164899), IL-1β (digestion 63-152398), IL-2 (amersham) or TNF-α (armor) Does not cross react at all).

Example 5

E. coli for Pycryl chloride-induced delayed-type hypersensitivity skin reaction (PC-DTH) in mice used in cellular immune assays. Investigate the effect of Coli [32-153] -M-CSF.

The following materials are used.

Mice: Male BALB / c mice (SL C) at 8 weeks of age, 5 mice in each group

Antigen solution: As a naïve antigen, a solution prepared by dissolving picryl chloride (PC) (manufactured by Nakarai Kagaku) in ethanol at a concentration of 0.5% (w / v).

As a challenge antigen, a solution of picryl chloride dissolved in olive oil at a concentration of 1% (w / v) was used.

Measuring device: Use dial thickness gauge (Ozaki Seisakusho)

Sensitization: Apply to mouse ears to perform sensitization. Application is carried out by the method of Tajima et al. [Shiogeru, TAKIMA. EiKO IMAI, Keizo ITO and Takashi NOSE, Simplified method in sensitization to picryl chloride for induction of delayed type hypersensitivity in mice, Igaku no Ayumi, Vol. 122, No. 4, pp 240-242, 1982.

That is, the antigen-inducing antigen is applied to the right ear in the front and the rear in an amount of 101 μl using a micropipette. Four days after sensitization, the thickness of the left ear is measured. This is used as the prereaction value. Subsequently, the challenge antigen solution is applied to the front and rear of the left ear in an amount of 5 μl, respectively, and after 24 hours, the thickness of the left ear is measured, and the difference (ΔT) between the thickness and the pre-reaction value is expressed as an increase in ear thickness. .

Inhibition of the control can be measured by the following equation.

% Inhibition = [1- (ΔT of M-CSF treated group / ΔT of control)] × 100

this. Coli [32-153] M-CSF is administered in the following manner.

this. Coli [32-153] is administered subcutaneously after adjustment to a concentration of 10 or 100 μg / kg / mouse using physiological saline containing mouse serum albumin (MSA). Treatment groups are as follows.

The group containing the solvent (physiological saline containing 30 μg / ml MSA) was used as a control.

This group is treated in the same manner as Treatment group 1.

Treatment group

1: group of drug sensitization and administration for 3 consecutive days and attack on day 4

2: group administered after the sensitization of the drug, three consecutive days, and performing a pore on the fourth day

3: group of the drug administered for 3 consecutive days before sensitization and 4 consecutive days after sensitization again for 4 consecutive days

4: group of the drug administered for 3 consecutive days before sensitization, 3 consecutive days after sensitization again, and a single administration after 4 days of attack

The results are shown in Table 7 below.

TABLE 7

Ear thickness increase is expressed in terms of mean ± SE and its unit is x10 ⁻² mm.

As a result of this example, DTH induction is significantly suppressed in the treated group compared to the control.

Comparable inhibition was observed in treatment group 1 administered with 10 or 100 μg / kg of M-CSF. In treatment groups 2 and 3 an inhibition of about 50% was observed at 10 μg / kg than at 100 μg / kg.

The results suggest that M-CSF is administered before sensitization, followed by sequential seawater administration after sensitization, followed by an attack and then strongly inhibited the induction of DTH by M-CSF in a single dose group.

Example 6

this. Dosing time of Coli [3-153] M-CSF is checked for effect on mouse PC-DTH using the method of Example 5.

this. The subcutaneous route (sc) was diluted with the colli [3-153] M-CSF diluted with physiological saline containing MSA (30 µg MSA / ml) and containing the solvent (physiological saline containing MSA) as a control. Doses 1, 10 and 100 μg / kg at the administration times shown below.

Treatment group

I: Group that attacked on day 4 after sensitization of drugs

II: The drug was sensitized and administered two days in a row, and the attack was performed on the fourth day.

III: Group that administered the drug after sensitization, and administered it continuously for 3 days, and attack and drug administration on the 4th day

IV: group treated with the same method as group III except that the drug was administered twice a day

V: Group administered 3 times of attack without attack before attack, 4 hours before attack, simultaneous with attack and 4 hours after attack

The results are shown in Table 8 below.

TABLE 8

The numerical value represents the increase in ear thickness (flat group ± SE) and the unit is x10 ⁻² / mm ² .

Numbers in parentheses indicate inhibition rates.

*: p 0.05

**: p 0.01

As a result, significant inhibitory effects were observed at doses of 10 μg / kg or more in Groups II to IV, in which the drug was administered three or five times, or five times twice daily for four days from primary to secondary sensitization, respectively. do. In addition, a significant inhibitory effect is observed in group V administered with the drug after establishment of DTH.

Example 7

Three M-CSFs, i. Collie [3-153] M-CSF, 2. Coli [3-214] M-CSF and CHO [32-522] are tested for mouse PC-DTH inhibition.

Each M-CSF is diluted with physiological saline containing MSA (30 μg / ml) and administered subcutaneously (SC) and intravenously (IV) at doses 1, 10 and 100 μg / kg.

Dosing time is the same as in Treatment Group III of Example 6. That is, the drug is sensitized and administered, followed by three consecutive days, followed by attack and administration on the fourth day. Other processes are the same as in Examples 5 and 6.

The results are shown in Tables 9 and 10 below.

For intravenous administration, CHO (-32-522) M-CSF and E. coli. Coli [3-214] M-CSF shows a dose-dependent inhibition trend and 100 μg / kg apparent inhibition is observed. this. Coli [3-153] M-CSF exhibited inhibition at the more verbal doses (1 µg / kg, 10 µg / kg), while its inhibition was weak.

In the case of subcutaneous administration, three M-CSFs show a tendency to suppress, and only this collie [3-153] shows a strong suppression. As is apparent from the above, DTH inhibition is common to M-CSF. Coli [3-153] M-CSF is superior for subcutaneous administration. This result is consistent with the results of bioavailability obtained by subcutaneous administration (Table 6). This is this. It is strongly suggested that collie [3-153] M-CSF is a derivative having excellent properties, in particular as a subcutaneous M-CSF.

Table 9 Intravenous Administration

TABLE 10 Subcutaneous Administration

The numerical value represents the increase in ear thickness (mean ± SE).

The unit is x10 ⁻² / mm ² .

Numbers in parentheses indicate inhibition rates.

* p 0.05,

** p 0.01

Other embodiments of the invention are apparent to those skilled in the art by the description and examples of the invention described herein. It is intended that the description and examples be exemplary only and that the scope of the invention be defined by the claims.

The numerical value represents the increase in ear thickness (mean ± SE).

The unit is x10 ⁻² / mm ² .

Numbers in parentheses indicate inhibition rates.

* p 0.05

** p 0.01

Toxicity Test Results

About LD ₅₀ of M-CSF

LD ₅₀ , which is part of the toxicity test of M-CSF, was investigated. M-CSF used the same 151 Thr-M-CSF as before, and at 2, 3, 5 and 7 days after SC administration to mice at doses of 1, 3, 10, 30 and 100 mg / kg. Body weight was measured to investigate weight change. One group of mice was four.

result

The weight change is shown in Table 11 below. No deaths were seen in any of the groups and no significant weight loss was seen. Therefore, LD ₅₀ of M-CSF may be considered to be 100 mg / kg or more.

TABLE 11

M-CSF LD ₅₀ inch

Other embodiments of the invention are apparent to those skilled in the art by the description and examples of the invention described herein. It is intended that the signature and embodiment be exemplary only and that the scope of the invention be indicated by the claims.

Claims (8)

Therapeutic composition for the treatment of allergy and treatment of IL-1 related inflammatory diseases characterized by containing M-CSF as an active ingredient. The therapeutic composition of claim 1, wherein the M-CSF is recombinant M-CSF. The therapeutic composition of claim 1, wherein the M-CSF is native M-CSF. The therapeutic composition of claim 1, wherein the M-CSF is derived from L292 fibroblast cell line. The M-CSF according to claim 1, wherein the M-CSF has an amino acid primary sequence from Val at position 2 or Ser at position 4 to Thr at position 153 in formula (1): Val or position 4 The therapeutic composition is M-CSF having an amino acid primary sequence from Ser of Pro to position 214, or M-CSF having an amino acid primary sequence from Met of position-32 to Val of position 522: Wherein X is Tyr or Asp The therapeutic composition according to claim 1, wherein the therapeutic composition contains M-CSF having an amino acid primary sequence from Val at position 3 or Ser at position 4 to Thr at position 153 in formula (1). The therapeutic composition according to claim 1, wherein the therapeutic composition contains M-CSF having an amino acid primary sequence from Val at position 3 or Ser at position 4 to Pro at position 214 in formula (1). The therapeutic composition of claim 1, wherein the allergy is type I allergy and type IV allergy.