KR20000010825A - Treatment of asthma with tnf-alpha - Google Patents

Abstract

PURPOSE: A treating method is provided to medicate to a patient for supplying an effective amount of a chimeric protein material for releasing the effect of asthma and to effectively prevent and delay the asthma. CONSTITUTION: The present invention is directed to a method of combating asthma with a composition containing an effective amount of a chimeric TNF-alpha-binding protein which comprises the soluble part of the p55 TNF receptor and all domains except the first domain of the heavy chain constant region of a human IgG1 or IgG3 and to the use of such a chimeric TNF-alpha-binding protein for the preparation of a medicament for the treatment of asthma.

Description

Treatment method of asthma using tumor necrosis factor receptor-immunoglobulin fusion protein

Examples of such TNFR-Ig include EP 417 563, US Pat. Nos. 5,447,851, 5,395,760, Lesslauer et al., Eur. J. Immunol. 21 (11): 2883, 1991, Loetscher et al., J. Biol. Chem. 266 (27): 18324, 1991, Ashhkenazi et al. (PNAS (USA) 88: 10535, 1991), which include proteins that can be prepared by the methods disclosed herein.

Asthma is a chronic inflammatory disease of the respiratory tract, characterized by recurring exacerbations upon exposure to certain allergens (immunoglobulin E (IgE) mediated responses), exercise, cold air or stress. Inflammation associated with asthmatic disease is characterized by the presence of activated eosinophils, increased sensitivity of the airways to nonspecific stimuli (airway hypersensitivity: AHR), edema, mucous hypersecretion and cough. This inflammatory process is thought to be mediated in part by the production and activation of Th2-type lymphocytes that secrete various cytokines. Cytokine Tumor Necrosis Factor-α (TNF-α) is a cytokine involved in the induction of asthmatic disease.

Many therapies have been used for the treatment of asthma, and inhaled β ₂ -agonists (which alleviate acute bronchial spasms) and steroids (anti-inflammatory action) have been selected. However, while the mortality rate of this disease is increasing, there is no suitable treatment. One of the most urgent medical needs is a drug that can rapidly reverse inflammation of severe progressive lungs in acute / severe asthma. In addition, no treatment is classified as a disease modifier. Therefore, there is a need for a drug that can rapidly reverse the inflammatory response seen in acute / severe asthma, as well as a true disease improver. Since the action of TNF-α promotes an asthma state, the inventors have determined that blocking the action of TNF-α may provide a means to alleviate the asthma state.

The inventors have now contemplated that at least one chimeric, each consisting of a soluble portion of the p55 TNF receptor protein fused to an IgG containing all IgG domains except the first IgG domain of the IgG H chain constant region. A) an asthma comprising administering to an asthma patient a composition comprising an agent consisting of a TNF-α binding protein and a therapeutically inert carrier, the composition providing an effective amount of a chimeric protein formulation for treating an asthma condition upon administration to a patient A method of treating the condition has been found.

The composition may be administered to the patient to provide the chimeric protein formulation in an amount sufficient to alleviate the effects of the asthma onset. The composition may be administered to an asthmatic patient in an amount effective to prevent or delay the onset of asthma prior to the onset of asthma.

The present invention relates to a method for treating asthma by administering to a patient with asthma a composition containing a formulation consisting of at least one chimeric TNF-α binding protein. The protein in the formulation consists of the soluble portion of the p55 TNF receptor protein fused to IgG, containing all domains except the first domain of the IgG H chain constant region (TNFR-Ig). The composition also contains a therapeutically inert carrier. The composition is administered to the patient to provide the patient with an amount of chimeric protein formulation effective to treat asthma.

The use of a chimeric TNF-α binding protein consisting of the soluble portion of the p55 TNF receptor protein fused to IgG, containing all domains except the first domain of the IgG H chain constant region, for the manufacture of a medicament for the treatment of asthma is another Purpose.

The composition can be effectively administered to a patient with asthma as an amount of a protein preparation sufficient to alleviate the effects of asthma onset. The composition may be administered to an asthmatic patient in an amount effective to prevent or delay the onset of asthma prior to the onset of asthma.

A chimeric TNF-α binding protein consisting of the soluble portion of the p55 TNF receptor protein fused to IgG, containing any TNFR-Ig, ie all domains except the first IgG domain of the IgG H chain constant region, was described in the asthma It can be used in the preparation of the formulations of the invention for treating asthma in patients with the condition. IgG can be human IgG1 or IgG ₃ , with IgG1 being preferred in the present invention.

Preferred preparations of the TNFR-Ig molecules of the invention are made of IgG ₁ , which contains a complex oligosaccharide terminated with one or more sialic acid residues and a protein with exposed N-acetylglucosamine. The molar ratio of sialic acid residues in the formulation is about 4 to about 7 moles, particularly about 5 to about 6 moles of sialic acid per mole of protein, and the molar ratio of exposed N-acetylglucosamine in the formulation is about 1 N-acetylglucosamine per mole of protein. To about 2 moles, and the molar ratio of sialic acid residues to N-acetylglucosamine residues in the formulation is about 0.35 to about 0.5, especially about 0.4 to about 0.45. The formulation has an isoelectric point (pi) of about 5.5 to about 7.5, which can be determined by chromatofocusing and is sensitive to neuraminidase treatment.

TNFR-Ig of the present invention can be obtained by conventional recombinant techniques or protein synthesis methods. DNA encoding the p55 TNF receptor, DNA encoding all domains except the first domain of the IgG ₁ or IgG ₃ H chain constant region, and ligating such sequences are known to those skilled in the art and Described. Suitable cloning vectors and host cells are well known and can be selected by those skilled in the art, and suitable culture conditions can be determined (Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood and Hames eds., Oxford University). Press, NY 1992).

Known protein recovery and purification methods can be used to purify TNFR-Ig. TNFR-Ig can also be recovered from host cell lysates, but is preferably recovered from the medium as a secreted polypeptide. The medium or lysate can be centrifuged to remove specific cellular debris. TNFR-Ig is then purified from contaminating soluble proteins and polypeptides, and examples of suitable methods for such purification include fractionation in an immunoaffinity column or ion-exchange column; Ethanol precipitation; Reverse phase HPLC; Chromatography on cation-exchange resins such as silica or DEAE; Chromatofocusing; SDS-PAGE; Ammonium sulfate precipitation; Gel filtration using, for example, Sephadex G-75; And Protein A Sepharose column for removing contaminants such as IgG. Protease inhibitors such as phenyl methyl sulfonyl fluoride (PMSF) may also be useful to inhibit denaturation by proteolysis during purification. Those skilled in the art will appreciate that purification methods suitable for the desired polypeptides require modifications to alter the polypeptide properties upon expression in recombinant cell culture.

TNFR-Ig of the present invention having a specific sialic acid and N-acetylglucosamine ratio and pI uses recombinant mammalian cells expressing TNFR-Ig, and TNFR-Ig is described in WO 96/39488. It can be obtained by adjusting mammalian cell culture conditions under the conditions of manufacture. The host cell selected should be able to bind N- and O-linked carbohydrates, including sialic acid, to the proteins they express. An example of such a host cell is a Chinese hamster ovary (CHO) cell. Suitable culture conditions are well known and depend on the host cell selected. Increasing cell specific productivity during glycoprotein production reduces the sialic acid content of the mature protein and vice versa. Factors affecting cell specific productivity are well known in the art and include DNA / RNA copy numbers, factors affecting RNA, such as factors that stabilize RNA, nutrients in the medium, and other supplements. Water, transcription enhancer concentration, osmotic concentration of the culture environment, temperature and pH of the cell culture, and the like. These factors can be controlled by known methods of obtaining the desired content of glycoproteins such as sialic acid. Alkanoic acid, such as sodium butyrate or salt thereof, is added at a concentration of about 0.1 to about 20 mM, or 5 to 20 mM, the osmotic pressure of the cell culture is 250 to 600 mM, and optionally by combining these conditions with each other during the transition phase. It is possible to produce proteins with different sialic acid contents. The highly sialized TNFR-Ig of the present invention is prepared at a sodium butyrate concentration of about 6.0 mM and 350-400 mM, while the less sialized TNFR-Ig of the present invention is prepared at a sodium butyrate concentration of about 12 mM. The latter concentration can be combined with the conditions of 450-550 mOsm.

Those skilled in the art will appreciate that the osmotic concentration of the medium depends on the concentration of the osmotic active particles, and the number of variables that make up the complex mammalian cell medium affects the osmotic concentration. The initial osmotic concentration of the medium is determined by the composition of the medium. Osmotic concentrations are, for example, osmotic pressure gauges sold under the trade name OSMETTE by Fisher Scientific, Pittsburgh, PA (or Precision Systems Inc., Natick, MA). Osmette Model 2007, commercially available from Presentation Systems, Inc., can be used. In order to obtain an osmotic concentration within a desired range, the concentration of various components in the medium can be adjusted. Solutes that can be added to the medium to increase the osmotic concentration of the medium include proteins, peptides, amino acids, hydrolyzed animal proteins such as peptone, non-metabolized polymers, vitamins, ions, salts, sugars (especially glucose), metabolism Substances, organic acids, lipids and the like. In one embodiment, the osmotic pressure is adjusted by adding peptone to the cell culture along with the other components of the medium during the fed batch culture process.

Three cell culture steps can be used, i.e. selecting and applying the cell culture variables as described above in relation to the desired sialic acid content of the proliferative phase and then the desired glycoprotein of mature cell growth in the selected mammalian host cell. Is a production phase that maintains selected variables in the transition phase and produces and harvests glycoprotein products. Preferred purification methods in the present invention are purification techniques and methods selective to the carbohydrates of the present invention. The desired saccharide of the present invention can be enriched for sialic acid-containing molecules, for example, by ion-exchange soft gel chromatography or HPLC using cation- or anion-exchange resins.

Preferred TNFR-Ig of the present invention has one or more of the following properties in the state of the purified composition: the pI range of the TNFR-Ig composition is about 5.5 to 7.5; The molar ratio of sialic acid to protein is about 4 to about 7, especially about 5 to about 6; Having about 1 to about 2 moles of exposed N-acetylglucosamine residues per mole of protein; The molar ratio of sialic acid to N-acetylglucosamine is about 0.35 to about 0.5, more preferably about 0.4 to about 0.45. The most suitable conditions for obtaining this preferred TNFR-Ig are sodium butyrate concentrations of about 0.1 to about 6 mM and osmotic concentrations of about 300 to 450 mM Osm and culture conditions at temperatures of about 30 to 37 ° C.

The skilled person can determine the above properties of TNFR-Ig using known methods. For example, the complex carbohydrate moiety of the glycoprotein produced by the method of the present invention can be readily analyzed by conventional carbohydrate analysis methods in some cases. Thus, methods known in the art such as, for example, lectin blotting can be used to reveal the proportion of other sugars such as terminal mannose or galactose. Fractionation of oligosaccharides by means of anhydrous hydrazine or enzymes and ion-exchange or size exclusion chromatography of the ends of mono-, non-, tri- or tetra-antennary oligosaccharides with sialic acid Or by releasing sugar from the protein using other methods known in the art. The pi of the glycoprotein can also be determined before and after treatment with neuramidase to remove sialic acid. An increase in pi after treatment with neuramidase suggests the presence of sialic acid in the glycoprotein.

Carbohydrate structures of the invention are found as N-linked or O-linked carbohydrates on expressed proteins. N-linked or O-linked carbohydrates mainly differ in their core structure. N-linked glycosylation refers to the binding of carbohydrate residues to asparagine residues in the peptide chain via GlcNAc. N-linked carbohydrates all have in common the Man1-6 (Man1-3) Manβ1-4GlcNAcβ1-4GlcNAcβ-R core structure. Thus, in the described core structure, R represents the asparagine residue of the resulting protein. The peptide sequence of the resulting protein contains asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, where X is any amino acid except proline. In contrast, O-linked carbohydrates are characterized by having a common core structure which is GalNAc bound to the hydroxyl group of threonine or serine. Among the N-linked and O-linked carbohydrates, N-linked and O-linked complex carbohydrates are the most important. Such complex carbohydrates will contain several antenna structures. The outer structure of mono-, non-, tri- and tetra- is important for the addition of terminal sialic acid. This outer chain structure provides additional sites for the specific sugars and bonds that make up the carbohydrates of the present invention.

The carbohydrate thus produced can be analyzed by any method known in the art, including the methods described above. Several methods of glycosylation analysis are known in the art and are also useful in the present invention. This method provides information regarding the identification and composition of oligosaccharides bound to peptides. Carbohydrate analysis methods useful in the present invention include lectin chromatography; HPAEC-PAD using high pH anion exchange chromatography to separate oligosaccharides based on charge; NMR; Mass chromatography; HPLC; GPC; Monosaccharide composition analysis; Including but not limited to sequential enzymatic digestion.

In addition, methods for releasing oligosaccharides are known. Such methods include (1) an enzymatic method which is typically carried out using peptide-N-glycosidase F / endo-β-galactosidase; (2) removal methods using a strong alkaline environment, mainly for releasing O-linked structures; And (3) chemical methods using anhydrous hydrazine to release both N-linked oligosaccharides and O-linked oligosaccharides.

You can perform the analysis using the following steps:

(1) The sample is dialyzed with deionized water to remove all buffered salts and then lyophilized.

(2) Release the intact oligosaccharide chain with anhydrous hydrazine.

(3) Intact oligosaccharide chains are treated with anhydrous methanolic HCl to liberate individual monosaccharides as O-methyl derivatives.

(4) N-acetylation of any primary amino group.

(5) induced with per-O-trimethylsilyl methyl glycoside.

(6) These derivatives are separated by capillary GLC (gas-liquid chromatography) on a CP-SIL8 column.

(7) Individual glycoside derivatives are identified by comparing the retention times in GLC and mass chromatography with those of known standards.

(8) Individual derivatives are quantified by FID using internal standard (13-O-methyl-D-glucose).

High performance anion-exchange chromatography can be combined with pulse current detection (HPAE-PAD Carbohydrate System, Dionex Corp.) to determine neutral and amino-sugars. For example, the sugar can be released by hydrolysis in 20% (v / v) trifluoroacetic acid at 100 ° C. for 6 hours. The hydrolyzate can then be lyophilized or dried using a speed-bag (Savant Instruments). The residue was then dissolved in a 1% sodium acetate trihydrate solution and analyzed by Anumula et al., Anal. Biochem. 195: 269-280 (1991), as described on HPLC-AS6.

Yao et al., Anal Biochem. 179: 332-335 (1989)] can be used to determine sialic acid by performing three separate replicate experiments using direct thermometry. In a preferred embodiment thiobarbaturic acid (TBA) from Warren, L. J., Biol Chem 238: (8) (1959) is used.

On the one hand, immunoblot carbohydrate analysis can be performed. According to this method, a commercial glycan detection system (Boehringer) based on the oxidative ibunoblot method described in Haselbeck and Hossel (Glycoconjugate J., 7:63 (1990)). )) Is used to detect protein-bound carbohydrates. Except that the protein is delivered to the polyvinylidene difluoride membrane instead of the nitrocellulose membrane and the blocking buffer contains 5% bovine serum albumin and 0.9% sodium chloride in 10 mM Tris buffer and has a pH of 7.4. Follow the staining method recommended by 5-bromo-4-chloro-3-indolyl-phosphate in pH 9.5 100 mM Tris buffer containing phosphate based, 0.03% (w / v) of 4-nitroblue tetrazoline chloride, 100 mM sodium chloride and 50 mM magnesium chloride Detection is performed using an anti-digoxigenin antibody coupled with alkaline phosphate conjugate (Berringer) diluted 1: 1000 in Tris buffered saline using (w / v). Protein bands containing carbohydrates are typically visualized within about 10 to 15 minutes.

Carbohydrates can also be analyzed by digesting with peptide-N-glycosidase F. According to this method, the residue is suspended in 14 μl of pH 8.6 buffer containing 0.18% SDS, 18 mM beta-mercaptoethanol, 90 mM phosphate, 3.6 mM EDTA, and heated at 100 ° C. for 3 minutes. After cooling to room temperature, the samples are equally divided into two portions. One aliquot is no longer processed and used as a control. Another aliquot is acclimated to about 1% NP-40 detergent followed by 0.2 units of Peptide-N-glycosidase F (Behringer). Both samples are warmed at 37 ° C. for 2 hours and then analyzed by SDS-polyacrylamide gel electrophoresis.

TNFR-Ig of the present invention also includes PEGylated TNFR-Ig, which refers to TNFR-Ig covalently conjugated to a polymer such as polyalkylene glycol (substituted or unsubstituted), in particular polyethylene glycol. Conjugation may be direct, but preferably various binding means known in the art, for example EP 510346, EP 593838, US Pat. Nos. 4,766,106, 4,917,888, 4,902,502, 4,847,325 , 4,179,337, 5,832,657, Veronese et al., Applied Biochem. and Biotech. 11: 141 (1985) and Monfardini et al. Bioconjugate Chem. 6:62 (1995). PEGylated TNFR-Ig can be used for the treatment and prevention of asthma as described above for TNFR-Ig.

The TNFR-Ig of the present invention is useful for treating asthma in asthmatic patients. In treating patients with asthma, the present formulations can alleviate the onset of asthma, such as reversing already developed inflammation or preventing further inflammation from occurring. When the agent is administered for prophylactic purposes, the agent may delay or prevent the onset of asthma or reduce its severity if asthma develops.

In accordance with the present invention, the formulations may be administered to the patient in any conventional manner for the purpose of treatment or prevention. Thus, the formulations of the present invention may be administered as conventional pharmaceutical compositions comprising any conventional therapeutically inert carrier. The pharmaceutical composition may contain inert as well as pharmacokinetically active additives. The liquid composition may, for example, take the form of sterile water which is miscible with water. Moreover, as well as materials commonly used as preservatives, stabilizers, humectants and emulsifiers, there may also be substances for changing the osmotic pressure, such as salts, substances for changing the pH such as buffers and other additives. If desired, antioxidants such as tocopherol, N-methyl-gamma-tocopheramine, butylated hydroxyanisol or butylated hydroxytoluene may be included in the pharmaceutical composition. Pharmaceutically acceptable excipients or carriers for the composition include saline, buffered saline, dextrose or water. The composition may also include certain stabilizers such as sugars (such as mannose and mannitol), and local anesthetics (such as lidocaine). The aforementioned carrier materials and diluents can be organic or inorganic materials such as water, gelatin, lactose, starch, magnesium stearate, talc, gum arabic, polyalkylene glycols and the like. It is essential that all auxiliaries and substances used in the preparation of the pharmaceutical composition are nontoxic.

The formulations of the present invention can be administered parenterally (eg, injected subcutaneously, intravenously, intramuscularly, intraorbital, intracavian, spinal, intrasternal) or by spray inhalation. Any conventional parenteral or spray inhalational pharmaceutical composition can be used. Examples of suitable pharmaceutical compositions are infusions or injections. Preferred route of administration is intravenous administration. Another preferred route of administration is by aerosol.

In order to alleviate or reverse the effects of asthma development or to prevent or delay the onset of asthma, the agents of the present invention can be used in any amount effective for treating asthma. A preferred dosage of the TNFR-Ig composition for parenteral (injection) administration of the present invention for treating, preventing or reversing asthma is about 0.5 mg (mg / kg) of TNFR-Ig preparation per kg body weight of the patient. A particularly preferred dosage is about 1.0 to about 3.0 mg / kg. Preferred dosages of the TNFR-Ig composition for spray administration for the same purpose are amounts which provide from 0.03 to 5.0% by weight of the TNFR-Ig formulation. In any type of route of administration, the therapeutic dose may be administered once daily, or divided into smaller doses over a period of 24 hours to reach a total constant dose.

For emergency treatment of the onset of asthma by parenteral administration, a single dose of 0.1 to 5.0 mg / kg, in particular 1.0 to 3.0 mg / kg may be administered. If asthma is to be prevented by parenteral administration, a dosage of 0.1 to 5.0 mg / kg, in particular 1.0 to 3.0 mg / kg, is preferably daily, or preferably 2 weeks 1 depending on the patient and condition. It may be administered once, once a week, once a month or at other intervals. Likewise, for emergency treatment of the development of asthma by spray administration, a single dose may be inhaled daily. For prophylaxis, such dosages may be administered daily, or preferably once a week, once a week, once a month or at other intervals, depending on the patient and condition. The exact dosage of the composition may vary depending upon the formulation, route of administration, and patient's needs as determined by the skilled artisan. The exact dosage for the patient can be specifically adjusted by the skilled person taking into account the severity of the condition, the particular combination used and any drug included.

The spray composition of the present invention may be a liquid or a powder. It may also be administered through the nose or mouth (nasal or oral administration). An example of a spray composition is a spray spray that can be sprayed toward the nose or mouth, made from a spray atomizer by mechanical action on an aqueous solution or saline solution of a TNFR-Ig formulation. Another example is a nebulized spray, which is a spray that is actively inhaled (not directly sprayed into the nose or mouth) by the patient when the mist is produced from solution. If the TNFR-Ig is in powder form, the particles should be large enough to be deposited in the airways rather than being spit out by the patient after inhalation. Depending on the treatment plan for emergency treatment or prevention, these sprays are inhaled at least once daily or less frequently as prescribed for each patient. In the case of spray administration, the TNFR-Ig of the present invention is administered by inhalation in an amount effective for the treatment or prevention of asthma as described above. Certain parts of the airways (nasal, trachea, bronchus, lower respiratory tract or bronchioles) will receive an appropriate amount of TNFR-Ig for the treatment of asthma.

Spray formulations of the present invention preferably provide about 0.03 to 5.0% by weight, more preferably about 0.03 to 0.5% by weight, in particular about 0.1 to 0.3% by weight of the formulation. Preferred formulations are aerosol formulations. In general, small dosages are preferred for aerosol formulations and higher dosages are preferred for aerosols. In the present invention, any conventional aerosol combination that can provide an effective amount, preferably the amount described above, of TNFR-Ig formulations can be used. The formulation is administered by releasing a metered dose of aerosol mist in the patient's mouth, which is inhaled by the patient, taken into the mouth and received in the lungs through the airways. As described above, the effective amount can be administered nasal or orally several times a day or divided into smaller doses, daily doses for emergency treatment, weekly or monthly intervals for prevention. do.

Suitable aerosol formulations include an appropriate amount of a pharmaceutically active compound (eg TNFR-Ig formulation) and an effective amount of any conventional aerosol propellant. Any conventional surfactant can also be included. TNFR-Ig can be combined with propellant as a liquid or powder (eg lyophilized by known methods) in solution. Propellants can be suitably liquefied.

Surfactants that are soluble or dispersible in the propellant are more effective. Surfactants that are more soluble in propellants are most effective. It is also important that the surfactant be non-irritating and non-toxic.

Any conventional propellant suitable for use in pharmaceutical compositions, known in the art, can be used, liquefied or otherwise processed. Propellants may be powders depending on whether the TNFR-Ig is in powder or liquid form, or may be suitable for use with the liquid active ingredient. Liquefied propellants used are propellants which are gases at room temperature (65 ° F.) and atmospheric pressure (760 mmHg), ie non-toxic propellants having a boiling point of less than 65 ° F. at atmospheric pressure. Among suitable liquefied propellants, lower alkanes having up to 5 carbons such as butane and pentane are usable. Examples of liquefied propellants are fluorinated lower alkanes and chlorinated lower alkanes such as those sold under the trade names such as Freon. Mixtures of the foregoing propellants can be suitably used.

Although the present invention has been described in general, the following examples will help to better understand the present invention. Unless otherwise stated, the examples are for illustration only and are not intended to limit the invention.

Example 1 Plasmids Encoding TNFR-Ig for Use in the Preparation of TNFR-Ig

A. Cell Line

The Chinese hamster ovary (CHO) used as a mammalian host cell line was derived from CHO-K1 (ATCC No. CCL61 CHO-K1). Then CHO-K1 DUX-B11 (DHFR -) - obtained from Dr. search may have noticed (L. Chasin) deficient cell line of {Columbia University (Columbia University) as called CHO-K1 mutant dihydro folate reductase (DHFR); Simonsen, CC and Levinson, AD, Proc. Natl. Acad. Sci. USA 80: 2495-2499 (1983) and Ullaub, G. and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220 (1980)] was used to transfect with a vector containing cDNA for insulin precursors to obtain cell lines with reduced insulin requirements (Sures et al. Science, 208: 57-59 (1980). Selected clones named dp12.CHO require glycine, hypoxanthine and thymidine for growth and exhibit their DHFR ^- genotype.

B. Construction of Availability Type 1 TNFR-IgG ₁ Chimeras

Soluble type 1 TNFR-IgG ₁ chimera (hereinafter referred to as TNFR-IgG ₁ ) was made by gene fusion of the extracellular domain of human type 1 TNFR with the hinge region of the IgG ₁ H chain and the C _H 2 and C _H 3 domains. Built. Alternatively, the hinge region of the IgG ₃ H chain and the C _H 2 and C _H 3 domains could be used.

Human type 1 TNFR (see Locher et al., Supra) encoding the DNA sequence was obtained from plasmid pRK-TNFR (see Schall et al., Cell 61, 361 (1990)). To construct the starting plasmid, a 2.1 kb placental cDNA clone (see Schal et al., Supra) was inserted into the mammalian expression vector pRK5 (described in EP Publication 307,247). This cDNA starts at nucleotide position 64 of the sequence reported by Rocher et al. And the starting methionine is 118 bp downstream.

IgG ₁ origin of the coding sequence is the first residue of the H chain first residues of the constant region of a cysteine residue, and then the IgG ₁ hinge associated with the C _H 2 and C _H 3 F aspartic acid 216 {H chain bonding to include the _c domain of IgG ₁ Take the amino acid 114 as a reference (see Kobat et al., Sequences of Proteins of Immunological Interest 4th edition (1987)) and mature human CD4 protein fused to a human IgG ₁ sequence ending in residue 144 ( CD4-IgG expressing plasmid pRKCD4 ₂ F _c1 (Capon, DJ) et al., Nature 337, containing a cDNA sequence encoding a hybrid polypeptide consisting of 1-180 residues of two N-terminal CD4 variable domains. 525 (1989) and Byrn et al. (Nature 344, 667 (1990)). See EP 227110. Alternatively, pCD4-Hγ3 vectors (DSM 5523, EP 394,827) may be used. CD4 cDNA is removed by SstI cleavage to obtain the desired IgG ₃ sequence.

Linkage was generated using deletion mutagenesis to generate restriction fragments of plasmids pRK-TNF-R and pRKCD4 ₂ F _c1 and to parallelize threonine residue 171 of mature TNFR to aspartic acid residue 216 of the IgG ₁ H chain. TNFR-IgG ₁ was constructed (see Kabat et al., Supra). The resulting plasmid pRKTNFR-IgG contained the full length TNFR ₁ IgG ₁ coding sequence. The plasmid was then transfected into host cells such as CHO cells by conventional methods such as calcium phosphate precipitation. The cells thus produced were cultured in a conventional manner to express TNFR-Ig, followed by isolation of TNFR-Ig by conventional methods.

Example 2: Preparation of TNFR-Ig with about 5-6 M sialic acid, 0.4-0.45 M N-acetylglucosamine, and pI 5.5-7.5 per M protein

Cell line

The gene of Example 1 encoding soluble type 1 TNFR-IgG ₁ was introduced into dp12.CHO cells by transfection. This was done using calcium phosphate technology to introduce DNA into mammalian cells. Two days after transfection, cells were trypsinized and diluted 1: 1 v / v with selective medium (glycine-hypoxanthine and thymidine-free Hams' F-12 DMEM, 2% dialysed serum) ) The isolate thus obtained was screened for the secretion of TNFR-IgG ₁ . Clones expressing TNFR-IgG ₁ are amplified in methotrexate to produce high expressing clones and acclimatized to serum free medium. The cells were placed under continuous selective pressure until transferred to non-selective medium for growth and propagation of the inoculum.

To provide cells for TNFR ₁ -IgG ₁ production medium, the cells described above were propagated by sequential passage in an increasingly bulky vessel from a medium containing methotrexate to a growth medium free of methotrexate. In this step, the non-selective growth medium may be glucose, amino acid, salt, sugar, vitamin, glycine, hypoxanthine and thymidine; Cell protective agents such as recombinant human insulin, hydrolyzed peptone (Primatone HS or Primatone RL), Pluronic F68 (Pluronic polyol) or the like; Gentamycin; Concentrations of several components such as lipids and trace elements were modified DMEM / HAM F-12-based formulations.

The pH of the culture was adjusted to 7.2 ± 0.4 using CO ₂ gas (acid) and / or Na ₂ CO ₃ (base). The temperature was adjusted to about 37 ° during the growth period. Direct saturation of air and / or oxygen gas kept the saturation of dissolved oxygen in the air at least 5%. Osmotic concentration was maintained at about 250-350 mOsm upon inoculation propagation.

After the propagation phase of each culture, there is a second stage or transition period in which the culture variable changes from optimal propagation to production. During this transition period, the temperature of the culture system was generally reduced to about 30-35 ° C. Butyric acid was added and adjusted to a specific osmotic concentration range. The product accumulated during this production period was analyzed for sialic acid content.

In a typical production schedule, about 1.2 × 10 ⁶ cells were derived from the inoculum of selected stages grown in the proliferative phase when the starting osmolarity concentration was between 300 and 450 mOsm, preferably 300 mOsm. Growth medium was supplemented with trace elements, recombinant human insulin and hydrolyzed peptone. Cells were grown under these conditions for 2 days. On the third day, the temperature of the cell culture was lowered. Simultaneously with or after the temperature change, about 1-6 mmol, preferably 6 mmol of sodium butyrate was added to the cell culture and the desired production osmotic concentration was achieved by adding various media components. Cells were grown for 9-10 days under these conditions. Cells were supplied with various media components as needed.

The pi of a composition containing a lot of sialic acid is lower than the pi of a composition containing a little sialic acid. Isoelectric focusing was performed on the composition. In a state where a pH gradient is generated by a positive electrolyte having a different pH, the isoelectric focusing gel separates the glycoproteins of the composition according to the isoelectric point (pI). Composition analysis was performed using a pH gradient of 10-4.

The composition exhibits an isoelectric point range of about 5.5 to about 7.5 as determined by chromatography focusing, wherein pI is sensitive to neuramidase treatment.

Recovery of TNFR-IgG

As described by Carpon et al., Nature 337: 525, 1989, IgG fusion proteins can be purified by centrifuging the cell culture and passing the supernatant of the culture thus obtained through a Protein A column. Thus, the cell culture thus obtained is centrifuged and the supernatant is applied to a Protein A column. The TNFR-Ig preparation was purified by affinity chromatography on immobilized Staphylococcus aureus protein A as described in Carpon et al., Supra, to have a 95% homogeneity.

Carbohydrate Analysis

A. Warren, L., J. Biol. Chem. 234: 1971-1975] can be used to analyze sialic acid content.

B. High pH anion-exchange chromatography and pulse current detection can be combined to determine the release of intact neutral and amino-sugars. The analysis was performed using the following steps:

(1) Buffer was exchanged with TNFR-IgG ₁ (about 50 μg / ml) and a suitable reference sample so that the final sample was contained in 1% acetic acid.

(2) About 90 μg of TNFR-IgG ₁ as well as reference samples were frozen in dry ice and alcohol bath and the frozen samples were lyophilized overnight.

(3) The lyophilized sample was dissolved in 500 µl trifluoroacetic acid and incubated at 120 ° C for 1 hour.

(4) After acid hydrolysis of TNFR-IgG ₁ and reference sample, the sample was cooled and evaporated to dryness.

(5) The sample was dissolved in water so that the final concentration was about 0.6 mg / ml.

(6) Pulse current detection and pH anion exchange chromatography using a Dionex CarboPac PA1 (4 × 250 mm) column (Dionex Corp., Sunnyvale, Calif.) At room temperature Monosaccharide was isolated.

(7) Individual monosaccharides were quantified by comparison with reference monosaccharides.

Example 3: Preparation of TNFR-Ig

1. Using calcium phosphate coprecipitation, CHO cell line DP12 (EP 307,247) was transfected with plasmids pSV16B.TNFR-IgG (TNFR ₁ -IgG ₁ coding) and pFD11 (DHFR coding).

2. The transfected cell clones were placed in a 9 L suspension culture using selective medium containing 2% filtered bovine serum and 100 nmol / L methotrexate. Two 9 L cultures were seeded into a 100 L fermentor using serum-free medium. 100 L cultures were inoculated into 400 L cultures using serum-free medium and cells were washed with 1000 fold production medium to reduce residual serum components and then inoculated into 1000 L production vessels. The production medium is a combination of DMEM / HAM F-12-based supplemented with glucose, amino acids, glycine, hypoxanthine, thymidine, recombinant human insulin, hydrolyzed peptone, pullulonic F68, gentamicin, lipids and trace elements It was.

3. The pH of the production medium was maintained at 7.2 ± 0.2 for 13 days using CO ₂ gas and / or Na ₂ CO ₃ . After the cell concentration reached 5 × 10 ⁶ / ml, the incubation temperature was changed to 37-30 ° C. The osmolarity of the culture was adjusted to about 450 mOsm / kg by adding media components on days 2 and 4. Sodium butyrate was added to a final concentration of 12 mM. By sparging air and / or oxygen in the culture, the dissolved oxygen concentration in the air was maintained at 60% saturation.

4. Cultures were harvested after 13 days and cells were separated from the supernatants by gradient filtration. This harvested cell culture fluid (HCCF) was concentrated about 20-fold using a 10KD Maxisette membrane. This was clarified and conditioned for Protein A chromatography by addition of 1.0 M of solid NaCl, followed by filtration through a 0.22μ Durapore filter.

5. Filtered and conditioned HCCF was passed through immobilized Protein A. The loaded material was washed several times before eluting. TNFR-IgG was eluted stepwise with 0.05M Na citrate / 10% (w / v) glycerol at pH 3.0. The pH of the eluted pool was adjusted to 5.0 by addition of 1 M Na citrate.

6. After pH adjustment, the protein A pool was diluted and added to S-Sepharose Fast Flow. It was then washed and eluted stepwise with 0.05M MOPS / 0.05M NaCl at pH 7.2.

7. After stepwise elution, the S-Sepharose Fast Flow pool was diluted and then added on a Q-Sepharose Fast Flow column. The column was eluted with a linear gradient of 0.0125M to 0.125M NaCl in 0.125M MOPS at pH 7.2.

8. After linear gradient elution, 0.1 M Na acetate / 4.0 M urea / 2.0 M ammonium sulfate at pH 5.0 was added to the Q-Sepharose fast flow pool, followed by 0.05 M Na acetate / 2.0 M urea / 1.0 M at pH 5.0. Conditioned by addition to a phenyl Toyopearl 650M column equilibrated with ammonium sulfate. Under these conditions, TNFR-Ig was flowed into the column. After addition to the column it was washed with calibration buffer. The shed and wash solution were combined to form phenyl Saturday pearl.

9. Two phenyl Toyopearl pools were combined and concentrated using a 10KD Filtron Centrassette membrane. It was passed on a G-25 column equilibrated in 0.01 M Na citrate / 0.023 M glycine / 0.023 M mannitol at pH 6.0 to obtain a TNFR-Ig composition.

Examples 4-10 that follow describe in vivo experiments showing that TNF-Ig of the present invention mitigates the effects of asthma. TNF receptor fusion protein TNFR-Ig significantly reduces or in some cases eliminates the response exhibited by antigen administration in animal models of allergic lung inflammation. The inhibitory effect by TNFR-Ig is comparable to the results obtained by extensive administration of anti-inflammatory dexamethasone.

Abbreviation

(1) TNF-α: tumor necrosis factor-alpha, (2) TNFR-Ig: recombinant soluble TNF receptor IgG ₁ fusion protein, (3) BAL: bronchoalveolar lavage, (4) OA: egg albumin, (5) R _L : lung resistance, (6) HBSS: Hanks balanced salt solution, (7) Substance P: Literature (e.g. Selig and Tocker, Eur. J. Pharmacol. 213 (3) ): 331-336 (1992), which is a neuroprotein that induces acute bronchial spasms commonly referred to as substance P.

Data analysis

Mean ± S.E.M. Was calculated for all values in each experiment. Two-way repeated measurement analysis of graded data changes followed by multiple comparisons using Student Newman-Keuls t-test or Student's t-test Statistical differences were determined by the test. P <0.05 is considered statistically significant. Microsoft Excel 5.0 (Microsoft EXCEL 50) (Softmart, Exton, PA) and SigmaStat (Jandel Scientific, San Rafael, CA) software packages The calculation was performed by running on a PC.

drugs

All agents were administered at a dose of 1 ml / kg. TNFR-Ig was prepared, purified as described in Example 3, and stock solutions were prepared in buffers of sodium citrate (10 mM), glycine (23 mM) and mannitol (230 mM) at pH 6. Aliquots of stocks were stored at −20 ° C. until use and dilutions were made with sterile saline. Egg albumin, aluminum hydroxide, urethane, substance P acetate, (+/-) propanolol hydrochloride, dexamethasone 21-phosphate and Evans blue were obtained from Sigma Chemical Co. ( From St. Louis, Missouri, USA, and prepared in sterile saline.

Example 4 Asthma Symptoms of Guinea Pigs Alleviated with TNFR-Ig (Hypersensitivity)

This example uses guinea pigs that are allergic to OA, which, when exposed to OA, causes asthma symptoms called airway hyperresponsiveness. This symptom is alleviated by TNFR-Ig and compared to dexamethasone, a steroid known to relieve asthma.

Male guinea pigs were sensitized to OA (a mixture of 1 mg Al (OH) ₃ and 10 μg OA in 0.5 ml saline subcutaneously) on the day of the experiment, and the same amount of OA was boosted on day 14 . On day 21, animals were dosed with 0.1% OA aerosol for 30 minutes. This sensitization induced asthma-like symptoms, including airway hypersensitivity to substance P in guinea pigs. To determine the effect of TNFR-Ig on these symptoms, TNFR-Ig was administered prior to antigen administration and the symptoms were found to be reduced when compared to non-administered animals. The effect on airway hypersensitivity was comparable to dexamethasone (10 mg / kg, intraspinal administration 1 hour before and 4 hours after challenge). Dexamethasone is known as a treatment for asthma.

Guinea pigs were sensitized, challenged and treated as experimental for the following:

Sensitization

A single subcutaneous injection of egg albumin (OA) on days 0 and 14 (10 μg OA and 0.5 ml) to a male guinea pig (Hartley, Kingston Charles River, NY) weighing 250-300 g A mixture of 1 mg of aluminum hydroxide in sterile saline), sensitized to OA and used in the study between 21 and 28 days.

Challenge

Antigen administration protocols are already described in O'Donnell et al. (1994). The sensitized animals were placed in a Plexiglas chamber and challenged with 0.1% (w / v sterile saline) OA aerosol for 30 minutes. The aerosol is powered by a built-in fan, De Vilbiss Ultra-Neb 100 ultrasonic nebulizer with an air flow rate of 30 L / min (Breathing Services, Eprata, PA, USA) Is delivered by). To minimize the mortality caused by anaphylaxis, the nebulizer is cycled every 60 seconds for the first five minutes of exposure. Under these conditions, the mortality rate of the animals was about 5%.

Medication administration

Groups of animals received either excipients (see “Pharmaceuticals”) or TNFR-Ig (1 or 3 mg / kg, spinal cord administration) 18 hours and 1 hour before OA aerosol. For experiments with dexamethasone, another group of animals was given an excipient (saline) or dexamethasone (30 mg / kg, spinal cord administration) 1 hour prior to OA aerosol and 4 hours after administration. Dosing parameters for TNFR-Ig and dexamethasone were determined to be optimal based on preliminary observations.

From the results of this example, it can be seen that OA-sensitized guinea pigs exposed to OA showed an increased airway response to substance P (1-10 μg / kg, intravenous) 6 hours after OA challenge. Increased airway response to substance P is a characteristic of asthma-like sensitization, indicating that guinea pigs suffer from asthma symptoms of hypersensitivity caused by these experimental conditions. Hypersensitivity was inhibited by TNFR-Ig (3 mg / kg intraspinal administration 18 hours and 1 hour prior to antigen administration).

The method used was as follows: the airway response to substance P was challenged 6 hours after OA aerosol exposure according to the sensitized and non-administered guinea pig, and the previously described method (Celieg and Talker, 1992). Analysis was performed on both sensitized guinea pigs. Animals were anesthetized with urethane (2 g / kg, spinal cord administration) and cannulated PE-50 tubes into the carotid artery (blood pressure) and jugular vein (pharmaceutical administration). A 15-gauge needle was cannulated into the trachea and the animal was placed into a systemic flow meter (Modular Instruments, Malvern, PA). Spontaneous breathing was stopped with succinylcholine chloride (1.2 mg / kg, intravenous) and the animals were mechanically breathed using a 1 mL / 100 g body weight and 60 breaths per minute (Harvard, South Natick, USA). Aperateus model (Harvard Apparatus Model 683). The flowmeter had a total volume of 2 L with a respiratory conduit volume of 10 ml between the dols and the respiratory tract. The flowmeter is equipped with a Fieisch respiratory recorder (model number 0000) connected to a Balidyne differential pressure transducer (DP 45-14) for measuring airflow. The pressure through the lungs was measured through a second ballidine transducer (DP 45-28) connected between the side branches of the tracheal cannula and the 16-gauge spinal cord needle inserted between the fifth and sixth intercostals. Airflow and lung pressure were recorded with a Modular Instruments (Melbourne, PA) M-3000 data acquisition system operated by an IBM 486DX2PC. The computer used commercially available software (BioReport, Modular Instruments) for calculating lung resistance R ₁ , based on the methods of Amdur and Mead (1958). Continue reading and averaged at 10 second intervals. The R _L value was corrected for internal resistance of the suction conduit (0.11 cmH ₂ O / ml / sec). Animals were administered propanolol (1 mg / kg, intravenous) and stabilized for 10 minutes prior to administration of substance P.

The dose-response effect of substance P (1, 3, 5 and 10 μg / kg, intravenous) was obtained by round injection at about 5-10 minute intervals. For each dose a maximum change in R _L was obtained and expressed as a percentage of the default value determined prior to administration of substance P. The ED ₂₀₀ value, defined as the dose of substance P that increases R _L by 200%, was determined by linear regression of the log dose-response curve for each animal.

The default value of R _L was 0.30 cmH ₂ O / ml / sec and was not significantly different between groups (P> 0.05). The sensitized and non-administered guinea pigs were relatively insensitive to substance P and required a dose of 30 μg / kg to increase R _L by at least 200% (Table 1). In contrast, the response of airways to substance P was markedly increased after OA challenge. ED ₂₀₀ increased by about 10-fold (Table 1) and the maximum R _L obtained by the highest dose of substance P increased by about 5-fold.

Effect of TNFR-Ig and Dexamethasone on the Airway Response of OA-Sensitized Guinea Pigs to Substance P ^a Treatment ^b -logED ₂₀₀ ^c Response ^d (% increase in R _L ) n ^e Antigen treated ^f 4.86 ± 0.08 139 ± 28 6 TNFR-Ig Excipient 5.82 ± 0.04 1811 ± 228 7 1mg / kg 5.87 ± 0.01 1344 ± 195 6 3 mg / kg 5.48 ± 0.05 ^* 714 ± 69 ^* 5 Dexamethasone Excipient 5.87 ± 0.05 935 ± 91 5 30 mg / kg 5.35 ± 0.04 ^* 596 ± 66 ^* 6 ^The dose-response effect of substance P was determined 6 hours after 30 min challenge with 0.1% OA aerosol. Animals were pretreated with propanolol (1 mg / kg, intravenous) 10 min before testing airway response to substance P. ^b Animals were administered intramuscularly with 1 ml / kg of excipients, TNFR-Ig (18 hours and 1 hour before OA challenge) or dexamethasone (1 hour and 4 hours after OA challenge), respectively. ^c This value represents the negative logarithm (mean ± SEM) of the dose (g / kg) of substance P required to increase lung resistance (R _L ) by 200% of the default value. ^d This value (mean ± SEM) represents the increase in baseline R _L represented by substance P (10 μg / kg, intravenous). ^e Number of animals in each group. ^The dose-response effect of ^f substance P was examined in a group of sensitized animals not challenged with OA aerosol. ^* Statistically significant (P <0.05) difference in values between each carrier and drug treated group

Administration of TNFR-Ig (3 mg / kg) to sensitized animals significantly inhibited OA-induced airway hypersensitivity to substance P (P <0.05). The ED ₂₀₀ value for substance P decreased about threefold and the maximum R _L value decreased about 60% (Table 1). Lower amounts of TNFR-Ig (1 mg / kg) did not affect sensitivity to the material (Table 1). Treatment with dexamethasone (30 mg / kg) significantly inhibited (P <0.05) OA-induced airway hypersensitivity in guinea pigs sensitized to a lesser extent than that obtained with higher amounts of TNFR-Ig. It became.

Example 5 Asthma Symptoms (Inflammatory Cell Influx) of Guinea Pigs Alleviated with TNFR-Ig

This example uses the guinea pig of Example 4, which is allergic to OA, when exposure to OA causes an asthma symptom of inflammatory cell influx. This symptom is alleviated by TNFR-Ig and compared to dexamethasone, a steroid known to relieve asthma.

As in Example 4, male guinea pigs were sensitized to OA and challenged to cause asthma symptoms including airway inflammatory cell accumulation 6, 24, 48 and 72 hours after challenge. As in Example 4, to determine the effect of TNFR-Ig on these symptoms, TNFR-Ig was administered prior to challenge and the symptoms were found to be reduced when compared to non-administered animals. The effect on inflammatory cell accumulation at 6 and 24 hours was comparable to dexamethasone (30 mg / kg, intraspinal administration 1 hour before and 4 hours after challenge).

The method used was as follows: airway inflammatory cell influx was analyzed 6 and 24 hours after OA-antigen administration by BAL according to the method previously described (Celrig and Talker, 1992). Briefly, animals were anesthetized with urethane (2 g / kg, spinal cord administration) and tracheotomy with a 15-gauge catheter. Lungs were washed with 3 × 5 ml sterile Hanks Balanced Salt Solution (HBSS) containing no Ca ²⁺ and Mg ²⁺ (Gibco, Grand Island, NY). Samples were centrifuged at 200 g at 200 g for 10 minutes and red blood cells were hemolyzed from the pellet with distilled water (1 ml for 30 seconds) before the osmotic concentration was restored by adding 10 ml of HBSS. The sample was centrifuged again (200 g, 10 minutes, 25 ° C.) and the resulting pellet was resuspended in 1 ml HBSS. Total cell number was determined by Trypan Blue (Sigma Chemical, St. Louis, MO) and aliquots of the cell suspension were taken using a hemocytometer. For differential cell counts, aliquots of the cell suspension were centrifuged in Cytospin (5 min, 1300 rpm; Shandon Southern Instruments, Siwicki, PA), and the slices were fixed and modified light. (Wright's) dye (Leukostat; Fisher Scientific, Pittsburgh, PA). More than 300 cells were observed by light microscopy using standard morphological criteria. Data was expressed as BAL cells × 10 ⁶ / animals.

TNFR-Ig (1 to 3 mg / kg, spinal cord administration) at 6 and 24 hours after OA-antigen administration for 18 hours and 1 hour for BAL and 1 hour pre-administration at 6 and 24 hours The accumulation of neutrophils and total cells in BAL of significantly inhibited (P <0.05), respectively. TNFR-Ig (3 mg / kg, spinal cord administration) also significantly reduced (P <0.05) the number of eosinophils in BAL at both time points, while lower amounts (1 mg / kg, spinal cord administration) ) Did not show any effect. The results obtained from this study show that the neutrophil component of the allergic-induced inflammatory response is mediated by TNF. Most notably, neutrophil influx in BAL of guinea pigs sensitized 24 hours after TNFR-Ig challenge was almost suppressed and the number of neutrophils became about 2% of the total cell number of BAL. In addition, treatment with TNFR-Ig rather than dexamethasone resulted in substantially reduced neutrophils in guinea pigs 6 hours after challenge. Blocking TNF by TNFR-Ig or similar antagonists may indirectly reduce the factors contributing to the influx of neutrophils into the lungs.

The cellular composition of BAL in sensitized and non-administered guinea pigs has already been described (Celrig and Talker, 1992). The total cell number in such animals is on average about 1 × 10 ⁶ / animal, of which about 2-3% are eosinophils and neutrophils. Six hours after OA-antigen administration, eosinophils and neutrophils constituted at least 40 and 20% of total cell numbers in animals treated with excipients for TNFR-Ig or dexamethasone, respectively. The percentage of eosinophils in BAL remained constant at 24 hours but the eosinophil count decreased to about 10% of the total cell number. Inflammatory cell number and total cell number were not different between each group of excipients for TNFR-Ig and dexamethasone (P <0.05).

Inflammatory cell influx of sensitized and challenged guinea pigs to BAL was also inhibited by dexamethasone (30 mg / kg). Six hours after OA-antigen administration, neutrophils and total cell numbers in BAL were significantly reduced (P <0.05), similar to the results by TNFR-Ig. At 6 hours there was no effect on neutrophil counts. 24 hours after OA-antigen administration, eosinophils, neutrophils and total cell numbers in BAL were significantly reduced (P <0.05) by dexamethasone. In contrast to TNFR-Ig, animals treated with dexamethasone had about 5 times less eosinophils (P <0.05). In contrast, treatment with dexamethasone reduced the number of neutrophils in BAL and the proportion of these cells did not change at about 8% of the total cell number (P <0.05).

The ability of TNFR-Ig to reverse the progressive inflammatory response was examined in sensitized guinea pigs. Animals were sensitized to OA and then challenged as described above. TNFR-Ig (3 mg / kg, spinal cord administration) was administered 30 minutes after OA-antigen administration. Inflammation was determined by BAL at 24, 48 and 72 hours post challenge. At 48 and 72 hours, TNFR-Ig was administered daily. Antigen administration following administration of TNFR-Ig significantly reversed the influx of inflammatory cells in BAL in sensitized guinea pigs.

Example 6: Asthma Symptoms (Pulmonary Edema) in Guinea Pigs Alleviated with TNFR-Ig

This example uses the guinea pig of Example 4, which is allergic to OA, when exposure to OA causes asthma symptoms called pulmonary edema. This symptom is alleviated by TNFR-Ig and compared to dexamethasone, a steroid known to relieve asthma.

As in Example 4, male guinea pigs were sensitized to OA and challenged to induce asthma symptoms including lung edema 6 hours after challenge. As in Example 4, to determine the effect of TNFR-Ig on these symptoms, TNFR-Ig was administered prior to challenge and the symptoms were found to be reduced when compared to non-administered animals.

The method used was as follows: airway microvascular leakage, a symptom of pulmonary edema, was examined by infiltrating Evans blue dye 6 hours after OA-antigen administration using the previously described method (Come et al. See Prostaglandin Thromboxane Leukotriene Res. 23: 271-273, 1995). Animals were anesthetized with urethane (2 g / kg, spinal cord administration) and catheter inserted in the jugular vein. The animals were then dosed with Evans blue dye (30 mg / kg, intravenous) for 60 seconds. After 10 minutes the ribcage was opened and a catheter (PE-240 tube) was inserted through the left ventricle into the aorta. Cross-pinch the left ventricle, dissecting the right atrium, and using a cartridge pump (Masterflex; Cole-Parmer, Chicago, Ill.) At a rate of 100 ml / min with 100 ml saline. Blood was drained by perfusing the animal with water. The pulmonary circulatory system was perfused with an additional 50 ml saline by inserting a catheter into the pulmonary artery and dissecting the left ventricle. The entire lung was removed and the trachea (distal 5 mm) and main bronchus were detached, blotted between filter papers and weighed. Quantification by extracting Evans blue dye from formamide (37 ° C., 24 hours) and measuring absorbance at 620 nm using a spectrophotometer (Beckman Instruments Model DU-64, Somerset, NJ) It was. Tissue dye content was interpolated from a standard curve of Evans blue dye concentration (0.5-10 μg / ml) and expressed as tissue wet weight (μg / ml).

Basal levels in the trachea and main bronchus of guinea pigs sensitized and challenged with Evans blue dye as a symptom of airway microvascular leakage were on average 10-20 μg / ml tissue wet weight. After 6 hours of OA-antigen administration, the content of Evans blue dye in the trachea and main bronchus increased about 5 fold. Treatment with TNFR-Ig (1 or 3 mg / kg) or dexamethasone (30 mg / kg) reduced OA-induced airway leakage 6 h after challenge in both trachea and main bronchus (P <0.05).

TNFR-Ig (1 or 3 mg / kg, spinal cord administration) eliminated OA-induced airway edema (quantified by tissue content of Evans blue dye) in trachea and main bronchus in sensitized guinea pigs.

Example 7: Asthma Symptoms in Brown Norwegian Rats Alleviated with TNFR-Ig

As in Example 4 with guinea pigs, the asthma symptom of inflammatory cell accumulation was induced in rats and treated with TNFR-Ig. This model differs from the guinea pig model in that allergic reactions in Brown-Norway rats are mediated by IgE.

The method used was as follows: Male Brown-Norway rats were sensitized to OA (mixture of 1 mg OA and 100 mg Al (OH) ₃ in 0.5 ml saline, intra spinal administration) on days 0, 1 and 2. On day 21, animals were challenged with 1% OA aerosol for 30 minutes. Inflammatory cell accumulation was quantified by BAL 24 hours post challenge. Protocols for sensitization, challenge and BAL were similar to those described above for guinea pigs with the following exceptions. OA by spinal injection of OA once on Days 1, 2 and 3 in male Brown-Norway rats (Kingston Charles River, NY) weighing 200-250 g (a mixture of 1 mg OA and 100 mg aluminum hydroxide in 1 ml saline) Activity was sensitized and used in the study on day 21 (Elwood et al., 1992). One hour prior to OA aerosol administration, separate groups of rats were dosed with excipients, TNFR-Ig (1 or 3 mg / kg, spinal cord administration) or dexamethasone (0.3 mg / kg, spinal cord administration), respectively.

On the day of the experiment, rats were challenged with 1% OA aerosol for 30 minutes. BAL was performed 24 hours after challenge by washing the lungs with 2 × 1 mL / 100 g of HBSS. Erythrocytes were lysed with 0.5 ml of distilled water and the osmotic pressure was restored with 5 ml of HBSS.

The rats were treated with TNFR-Ig (3 mg / kg intrathecal administration 1 hour prior to antigen administration) and the accumulation of neutrophils in BAL was visually eliminated and eosinophils and total cell numbers were significantly reduced. Similar results were obtained with treatment with dexamethasone (0.3 mg / kg intraspinally administered 1 hour prior to antigen administration). Administration of a lower amount of TNFR-Ig (1 mg / kg intrathecal) significantly reduced the number of eosinophils in BAL, but did not change eosinophils and total cell numbers.

More specifically, sensitized and non-administered Brown-Norway rats have a basal total cell number in the BAL of about 1 × 10 ⁶ / animal, of which about 1-2% are eosinophils and neutrophils. After 24 hours of OA-antigen administration, the total cell number in BAL of the animals treated with excipients increased about threefold, and eosinophils and neutrophils constituted more than 40 and 25% of the cell numbers, respectively. Inflammatory and total cell numbers in BAL did not differ between groups treated with each excipient (P> 0.05).

Treatment with TNFR-Ig (3 mg / kg, spinal cord administration) or dexamethasone (0.3 mg / kg, spinal cord administration) significantly increased the accumulation of eosinophils, neutrophils, and total cells in BAL (P <0.05). ) Decreased. Treatment with a lower amount of TNFR-Ig (1 mg / kg, spinal cord administration) significantly reduced the number of neutrophils in BAL (P <0.05), but did not affect the accumulation of eosinophils or total cell numbers in BAL. There was no impact.

Example 8: Reduction of Neutrophils in Impaired Rat Lungs

The rat model of Sephadex particle-induced pulmonary inflammation shows an increase in the number of eosinophils and neutrophils in the lung as well as granulation similar to the symptoms seen in chronic asthma. This model is used to show that TNFR-Ig reduces these chronic symptoms.

The method used was as follows: Inflammatory cell accumulation into the lung was induced by administering a suspension of Sephadex beads to Sprague-Dawley rats. At 24 and 72 hours after Sephadex administration, the total number of leukocytes in BAL fluid increased significantly. After 24 hours, the neutrophil count reached about 50% of the total white blood cell count, but after 72 hours the neutrophil count decreased to about 10%. Eosinophil count was maintained at about 10% of total leukocyte count.

TNFR-Ig (administered 1 and 3 mg / kg in the spinal cord 1 hour prior to antigen administration) or dexamethasone (0.1 and 0.3 mg / kg in the spinal cord) inhibited neutrophils 24 hours after Sephadex administration. Had no effect on the total cell number. After 72 hours of Sephadex administration, TNFR-Ig (administration of 1 and 3 mg / kg daily in the spinal cord) significantly reduced neutrophil influx into BAL fluid but had no inhibitory effect on eosinophil count. In contrast, dexamethasone (0.1 and 0.3 mg / kg daily in the spinal cord) eliminated infiltration of neutrophils and eosinophils into BAL fluid. TNFR-Ig (administration of 1 and 3 mg / kg in the spinal cord) or dexamethasone (0.1 and 0.3 mg / kg in the spinal cord) significantly reduced the total cell count 72 hours after dexamethasone administration.

Example 9: Attenuation of Atopic Asthma in Primates

Using a primate model of atopic asthma using wild live cynomolgus monkeys that innately express airway hyperresponsiveness to the Ascaris suum antigen, we used TNFR-Ig to treat asthmatic symptoms of airway hypersensitivity. I will show mitigation. Repeated exposure to antigens leads to asthma symptoms in the airways, especially increased lung resistance (R _L ). Monkeys receiving TNFR-Ig have a decreased R _L.

Wild captured cynomolgus monkeys that are innately ascaris-sensitized increase airway response to inhaled methacholine and show airway eosinophilia after repeated exposure to the Ascaris breath antigen. Thus, the antigen was used in these monkeys to induce an allergic reaction and then to induce airway hypersensitivity, an asthma symptom. The following protocol was used to examine the effect of TNFR-Ig on airway hypersensitivity.

1 day Bronchospasm-inducers such as substance P are used to determine the dose of methacholine (MCh), which increases lung resistance (R _L ) by 100% (PC ₁₀₀ ). 3 days Monkeys are challenged by inhaling a dose of Ascaris breath antigen that increases R _L by at least 2 fold. 5 and 8 days Repeat the process on day 3. 10 days Repeat the process on day 1. Determine the change in logPC ₁₀₀ value between 1 and 10 days.

Administration of TNFR-Ig (3 mg / kg, spinal cord) on days 1, 3, 5 and 8 resulted in attenuation of airway hypersensitivity in cynomolgus monkeys.

Example 10 Allergic Airway Inflammation Alleviated by TNFR-Ig in Mice

A mouse model of OA-induced allergic lung inflammation in mice is used to show that TNFR-Ig relieves asthma symptoms of inflammatory cell influx. Sensitized mice repeatedly exposed to OA developed progressive airway hyperresponsiveness and increased eosinophil influx in BAL. This model is associated with an increase in IgE levels and showed a typical Th2 cytokine profile (ie, an increase in the levels of IL-4 and IL-5). In this model, the ability of TNFR-Ig to attenuate the progressive inflammatory response was evaluated.

The mouse model of allergic lung inflammation is similar to the primate model in that multiple exposure of sensitized animals to antigen leads to airway hyperresponsiveness and inflammatory cell influx. Female C57BL / 6 mice were sensitized with OA on day 0 (gel of 10 μg OA and 1 mg Al (OH) ₃ , 0.1 mL, intrathecal administration) and treated with 1% OA aerosol for 30 minutes daily on days 14-20. Challenge. Airway hypersensitivity to MCh and BAL was performed 24 hours after the last OA challenge. Some of the lungs of sensitized and challenged mice were fixed for histology and some were homogenized for determination of TNF levels.

Inflammatory cell accumulation and TNF levels in sensitized and challenged mice peaked after the last challenge (20 days) with OA aerosol. In this study, TNFR-Ig (3 mg / kg, spinal cord administration) was immediately administered followed by final challenge to OA. TNFR-Ig attenuated OA-induced hypersensitivity but had no effect on inflammatory cell influx in BAL. Daily administration of TNFR-Ig during the challenge period had no effect on BAL cell numbers. However, administration of TNFR-Ig (3 mg / kg, spinal cord administration, 20 days) significantly reduced the number of eosinophils in lung tissue of sensitized and challenged mice and eliminated TNF in lung tissue.

Example 11: Aerosol Composition

Examples of aerosol compositions containing a TNFR-Ig formulation of the present invention are as follows:

Example A: Aerosols

TNFR-Ig (particle size 1-5 μ) 3.0% Span (registered trademark) 85 (sorbitan trioleate) 1.0% Freon 11 (trichloromonofluoromethane) 30.0% Freon 114 (registered trademark) (dichlorotetrafluoroethane) 41.0% Freon 12 (Dichlorodifluoromethane) 25.0%

Example B: Aerosols

TNFR-Ig 0.5% Span 85 0.5% Propellant B (consisting of 10% Freon 11, 50.4% Freon 114, 31.5% Freon 12 and 8.0% Butane) 99.0%

Example C: Aerosols

TNFR-Ig 1.00% Span 85 0.25 Freon 11 5.0% Freon W (consisting of 61.5% Freon 114 and 38.5% Freon 12) 93.75%

Example D: Aerosols

TNFR-Ig 0.50% Span 85 0.50% Propellant C (consisting of 30.0% Freon 11 and 70% Freon W) 99.0%

Example E: Aerosols

TNFR-Ig 0.88% Sodium Sulfate (anhydrous, finely divided) 0.88% Span 85 1.00% Propellant (consists of 50% Freon 12, 25% Freon 11 and 25% Freon 114) 97.24%

Example F: Aerosols

TNFR-Ig 0.06% Span 85 0.05% Freon 11 20.0% Freon 12 / Freon 114 (20/80) 78.9%

Example 12 Injectable Compositions

The following is an example of an injectable composition containing a TNFR-Ig preparation of the present invention.

ingredient Content per ml TNFR-IgG1 * Citric Acid Anhydride (USP) 1.92mg Glycine (USP) 1.70mg Mannitol (contains no heating material, USP) 41.90mg Sodium hydroxide Required amount to reach pH 6.0 Hydrochloric acid Required amount to reach pH 6.0 Water for Injection (USP) Required amount to be 1.0ml ^{** *} The concentration of TNFR-Ig used in this formulation may be 1.0㎎ / ㎖ to 20㎎ / ㎖. The final amount of TNFR-Ig in this formulation is chosen based on the formulation concentration and volume per vial. ^** Removed by lyophilization.

1mg vial Use 1 ml of 1.0 mg / ml blend 2.5mg vial Use 1 ml of 2.5 mg / ml blend 5.0mg vial Use 1 ml of 5.0 mg / ml blend 10mg vial Use 2 ml of 5.0 mg / ml blend 10mg vial Use 1 ml of 10.0 mg / ml blend 20mg vial Use 2.5 ml of 8.0 mg / ml blend 20mg vial Use 4 ml of 5.0 mg / ml blend 50mg vial Use 2.5 ml of 20 mg / ml blend

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Claims (11)

One or more chimeric, each consisting of a soluble portion of the p55 tumor necrosis factor (TNF) receptor protein fused to an IgG containing all IgG domains except the first IgG domain of the immunoglobulin GH chain constant region. An asthma comprising administering to an asthma patient a composition comprising an agent consisting of a TNF-α binding protein and a therapeutically inert carrier, the composition providing an effective amount of a chimeric protein formulation for treating asthma when administered to an asthmatic patient. Treatment method. The method of claim 1, When the patient is suffering from asthma, the chimeric protein preparation is administered in an amount sufficient to mitigate the effect of asthma. The method of claim 1, A method of administering said composition to an asthma patient prior to the onset of asthma in an amount effective to prevent or delay the onset of asthma. The method according to any one of claims 1 to 3, The fused IgG is human IgG ₁ . The method according to any one of claims 1 to 4, Wherein the protein in the protein preparation has a complex oligosaccharide terminated with one or more sialic acid residues and exposed N-acetylglucosamine, the molar ratio of sialic acid residues in the preparation is about 4 to about 7 moles of sialic acid per mole of protein, The molar ratio of exposed N-acetylglucosamine in the drug is about 1 to about 2 moles of N-acetylglucosamine per mole protein, the molar ratio of sialic acid residues to N-acetylglucosamine in the formulation is about 0.35 to about 0.5, and the formulation is about 5.5 to A method having an isoelectric point (pi) of about 7.5. The method of claim 5, The molar ratio of sialic acid to N-acetylglucosamine is about 0.4 to about 0.45 and the molar ratio of sialic acid to protein is about 5.0 to about 6.0. The method according to any one of claims 1 to 6, A method of administering a formulation to a patient by injecting the formulation at a dosage of 0.1 to 5.0 mg per kg body weight of the patient per day. The method of claim 7, wherein The dosage is 1.0 to 3.0 mg / kg body weight of the patient per day. The method according to any one of claims 1 to 8, Method of administering the formulation by oral or nasal spray. The method of claim 9, The spray is a liquid spray composition containing from about 0.03% to 5.0% by weight of the formulation. Use of a chimeric TNF-α binding protein consisting of a soluble portion of a p55 TNF receptor protein fused to IgG, containing all domains except the first IgG domain of an IgG H chain constant region, for the preparation of a therapeutic agent for asthma.