CN1233189A - Treatment of Asthma with TNFR-Ig - Google Patents

Abstract

The present invention relates to a composition comprising an effective amount of a chimeric TNFα-binding protein comprising a soluble portion of the p55 TNF receptor and all domains of human IgG1 or IgG3 except the first domain of the heavy chain constant region. A method for asthma, and an application of the chimeric TNFα-binding protein for preparing a medicine for treating asthma.

Description

Treatment of Asthma with TNFR-Ig

Asthma is a chronic inflammatory disease of the airways characterized by relapses and exacerbations by exposure to specific allergens (IgE-mediated response), exercise, cold air or stress. Inflammation associated with asthma is characterized by the presence of activated eosinophils, increased sensitivity of the airways to nonspecific stimuli (anaphylaxis of the respiratory tract: AHR), edema, mucus hypersecretion and coughing. This inflammatory process is believed to be mediated in part by the generation and activation of Th2-type lymphocytes that secrete various cytokines. Cytokine TNF[alpha] is one cytokine involved in causing asthma symptoms.

Combination therapy is used in the treatment of asthma using an inhaled beta ₂ stimulant (to relieve acute bronchospasm) and a steroid as an agent of choice (anti-inflammatory). However, none of the treatment options are believed to be sufficiently effective, yet the mortality rate from the disease is steadily increasing. One of the most pressing medical needs is the need for an agent that can rapidly reverse the severe episodes of pneumonia seen in acute/severe asthma. Additionally, none of the therapeutics could be classified as disease-modifying agents. There is a clear need for a drug that can rapidly reverse the inflammatory response seen in acute/severe asthma as well as an agent that acts as a true disease modifying agent. The role of TNF[alpha] is to initiate the symptoms of asthma, and Applicants have determined that inhibiting the action of TNF[alpha] provides a way to alleviate these symptoms.

Applicants have now discovered a method of combating asthma in a patient suffering from asthmatic symptoms comprising administering to said patient a composition comprising one or more chimeric TNFα-binding proteins, said Each protein in the preparation consists of a soluble portion of the p55TNF receptor protein fused to an IgG containing all IgG domains except the first IgG domain of the IgG heavy chain constant region, so Said composition comprises a therapeutically inert carrier, and said composition is administered to said patient so as to provide said chimeric protein preparation to the patient in an effective amount to counteract said asthmatic symptoms.

The composition can be effectively administered to a patient suffering from asthma, wherein the chimeric protein preparation is administered in an amount sufficient to alleviate the effects of said disease. The composition may also be administered to an asthma patient prior to the onset of asthma in an amount effective to prevent or delay the onset of said disease.

The present invention relates to methods of combating asthma in a patient suffering from asthmatic symptoms by administering to the patient a composition comprising a preparation comprising one or more chimeric TNFα binding proteins. The protein in this preparation consists of the soluble portion of the p55 TNF receptor protein (TNFR-Ig) fused to an IgG (Immunoglobulin G) containing in addition to the first domain of the IgG heavy chain constant region all structural domains. The composition also contains a therapeutically inert carrier. The composition is administered to said patient to provide the patient with an effective amount of the chimeric protein preparation to counteract said asthmatic symptoms.

It is another object of the present invention that the chimeric TNFα-binding protein composed of the soluble part of the p55 TNF receptor protein fused to IgG is used for the preparation of a drug for the treatment of asthma, wherein said fusion IgG contains the IgG heavy chain constant region All IgG domains except the first IgG domain.

The composition is effectively administered to a patient during an asthma attack in an amount of the protein preparation sufficient to alleviate the effects of an asthma attack. The composition can also be administered to an asthma patient before the onset of asthma in an amount of the protein preparation effective to prevent or delay the onset of the disease.

Any TNFR-Ig, i.e. chimeric TNFα binding protein consisting of a soluble portion of the p55 TNF receptor protein fused to an IgG, can be used in the preparations of the invention to combat asthma in patients with asthmatic symptoms as described in the paragraph above, wherein The fusion IgG contains all IgG domains except the first IgG domain of the IgG heavy chain constant region. IgG can be human IgG1 or IgG3, IgG1 is preferred in the present invention.

Examples of the TNFR-Ig are included in EP417563; U.S. Pat. Nos. 5,447,851 and 5,395,760; Lesslauer et al., European Journal of Immunology, 21(11):2883, 1991; Loetscher et al., J. Biological Chemistry, 266(27):18324, 1991; Ashkenazi et al., Proc. National Academy of Sciences USA, 88:10535, 1991, which can also be obtained by the methods disclosed in these documents.

Preferred preparations of TNFR-Ig molecules of the invention are prepared with IgG1 and comprise proteins having complex oligosaccharides terminated with one or more sialic acid residues and having exposed N-acetylglucosamine, the sialic acid residues in the preparation being The molar ratio is about 4 to about 7 moles of sialic acid per mole of protein, especially about 5 to about 6, and the molar ratio of N-acetylglucosamine exposed in the preparation is from about 1 to about 2 moles of N-acetylglucosamine per mole of protein. For acetylglucosamine, the molar ratio of sialic acid residues to N-acetylglucosamine residues in the preparation is from about 0.35 to about 0.5, especially about 0.4 to about 0.45. The isoelectric point (pi) of the preparation is from about 5.5 to about 7.5, as determined by chromofocusing and is sensitive to neuraminidase treatment.

The TNFR-Ig of the present invention can be obtained by recombinant technology or conventional methods of protein synthesis. DNA encoding the p55 TNF receptor, DNA encoding all domains except the first IgG1 or IgG3 heavy chain constant region, and linking these sequences together for expression in a suitable vector is within the skill of the artisan. known and described in the literature. Suitable cloning vectors and host cells are well known and can be selected by the skilled artisan, and suitable culture conditions have been determined (see Animal Cell Culture: A Practical Exploration, 2nd Edition, edited by Rickwood and Hames, Oxford University Press, NY 1992 ).

TNFR-Ig can then be purified using known protein recovery and purification methods. TNFR-Ig is preferably recovered from the culture medium as a secreted polypeptide, although it can also be recovered from host cell lysates. Cell culture media or lysates can be centrifuged to remove particulate cell debris. Subsequent purification of TNFR-Ig from soluble proteins and peptides with impurities, the following procedures are given as examples of suitable purification procedures: separation on immunoaffinity or ion exchange columns; ethanol precipitation; reverse HPLC; Chromatography on a DEAE cation-exchange resin; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, eg, Sephadex G-75, and Protein A Sepharose columns to remove contaminants such as IgG. Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) can also be used to inhibit proteolytic degradation during purification. Those skilled in the art will appreciate that purification methods suitable for a polypeptide of interest will need to be modified as the characteristics of the polypeptide expressed in recombinant cell culture vary.

The TNFR-Ig of the present invention having a specific pI and ratio of sialic acid and N-acetylglucosamine can be expressed by using recombinant mammalian cells to express TNFR-Ig and controlling the production of TNFR-Ig by mammalian cells as described in, for example, WO 96/39488 Culture conditions are obtained. The host cells chosen should be able to attach N- and O-linked carbohydrates, including sialic acid, to the proteins they express. An example of such a host cell is a CHO cell. Suitable culture conditions are well known and depend on the chosen host cell. Increased cell-specific yields during glycoprotein production lead to decreased sialic acid content in mature proteins and vice versa. Factors affecting cell-specific yield are well known in the art and include, but are not limited to, factors affecting DNA/RNA copy number, factors affecting RNA, such as factors that stabilize RNA, media nutrients and other supplements, transcription enhancement subconcentration, osmolality of the culture environment, temperature and pH of the cell culture, etc. These factors can be adjusted by well-known methods to obtain preferred levels of glycoproteins such as sialic acid. By adding an alkanoic acid, such as sodium butyrate or a salt thereof, to the cell culture at a concentration of about 0.1 mM to about 20 mM, or 5 to 20 mM, and ensuring that the osmolality of the cell culture is between about 250 to 600 mOsm In between, changes in cell-specific yields during the production phase of cell cultures are best combined with each other during the transition to produce proteins with different amounts of sialic acid. A concentration of about 6.0 mM sodium butyrate and conditions of 350-400 mOsm provide highly sialylated TNFR-Ig of the invention, while a concentration of about 12 mM sodium butyrate provides a less sialylated TNFR-Ig of the invention. The latter concentration can be combined with conditions of 450-550 mOsm.

The skilled artisan will recognize that the osmolality of the culture medium is dependent on the concentration of osmotically active particles in the culture medium, and that many variables in formulating mammalian complex cell culture media affect the osmolality. The composition of the medium determines the starting osmolality of the medium. Osmolality is measured using an osmometer, such as that sold under the trade name OSMETTE by Fisher Scientific, Pittsburgh, Pennsylvania (or model Osmette 2007 available from Precision Systems, Inc., Natick MA). The concentration of each component in the medium can be adjusted in order to obtain the desired range of osmolality. Solutes that may be added to the medium to increase its osmolality include proteins, peptides, amino acids, hydrolyzed animal proteins such as peptone, non-metabolizable polymers, vitamins, ions, salts, sugars (especially glucose ), metabolites, organic acids, lipids, etc. In one embodiment, osmolality is controlled by adding peptone, along with other media components, to the cell culture during the fed-batch culture procedure.

Three phases of cell culture can be used, the growth phase: in which the cells of the selected mammalian host cells grow fastest; followed by the transition phase, in which the sialic acid content required for the mature glycoprotein is selected and applied as described above. Cell culture parameters; followed by a production phase where the parameters selected for the transition phase are maintained, glycoprotein product is produced and harvested. With respect to preferred purification in the context of the present invention, there may be the purification techniques and methods selected for use in the saccharides of the present invention. Enrichment of the desired sugar forms of the invention for sialic acid-containing molecules can be achieved, for example, by ion exchange soft gel chromatography or HPLC using cation or anion exchange resins, wherein the more acidic fractions are collected.

Preferred TNFR-Igs of the invention have one or more of the following characteristics in purified compositions: TNFR-Ig compositions have a pI in the range of about 5.5 to about 7.5 and a molar ratio of sialic acid to protein of about 4 to about 7 , especially about 5 to about 6, with about 1 to about 2 moles of exposed N-acetylglucosamine residues per mole of protein, with about 0.35 to about 0.5 and more preferably about 0.4 to about 0.45 sialic acid Molar ratio to N-acetylglucosamine. The most suitable conditions for obtaining the preferred TNFR-Ig are to use a sodium butyrate concentration of about 0.1 mM to about 6 mM, maintain an osmolality between about 300 to 450 mOsm, and a temperature between about 30° C. and 37° C. cultivation conditions in between.

The above characteristics of TNFR-Ig can be determined by the skilled artisan using well-known techniques. For example, complex carbohydrate moieties of glycoproteins produced by the methods of the invention are readily analyzed, if desired, using conventional techniques for carbohydrate analysis. Thus, for example, techniques well known in the art such as lectin blotting reveal the ratio of terminal mannose or other sugars such as galactose. Mono-, di-, tri-, tetra-branched oligomerization can be demonstrated by releasing sugars from proteins using anhydrohydrazide or enzymatic methods and isolating oligosaccharides by ion exchange or size exclusion chromatography or other methods well known in the art. The sugar is terminated by sialic acid. The pi of glycoproteins can also be determined before and after treatment with neuraminidase to remove sialic acid. An increase in pi after neuraminidase treatment indicates the presence of sialic acid on the glycoprotein.

The carbohydrate structures of the invention occur on proteins expressed as N-linked or O-linked carbohydrates. N-linked and O-linked carbohydrates mainly differ in their core structure. N-linked glycosylation refers to the attachment of a sugar moiety to an asparagine residue of a peptide chain via GlcNAc. N-linked carbohydrates also contain a common core structure Man1-6(Man1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-R. Thus, in the core structure described, R represents the asparagine residue of the producer protein. The peptide sequence of the produced protein will contain 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 a common core structure, which is GalNAc attached to the hydroxyl groups of threonine or serine. Among the N-linked and O-linked carbohydrates, the most important are the N- and O-linked complex carbohydrates. This complex carbohydrate contains some branched structures. Mono-, di-, tri- and tetra-outer structures are important for increasing terminal sialic acid. Such outer chain structures provide sites for the attachment of specific sugars and linkages comprising the sugars of the invention.

The resulting carbohydrates can be analyzed by any method known in the art, including those described herein. Several methods for glycosylation analysis are known in the art and are useful in the context of the present invention. This method provides information about the identity and composition of the oligosaccharides attached to the peptide. Carbohydrate analysis methods useful in the present invention include, but are not limited to, lectin chromatography; HPAEC-PAD separation of oligosaccharides based on charge using high pH anion exchange chromatography; NMR; mass spectrometry; HPLC; GPC; Analysis of sugar composition; sequential enzymatic digestion.

In addition, methods for releasing oligosaccharides are known. These methods include 1) enzymatically, typically using peptide-N-glycosidase F/endo-β-galactosidase; 2) using a strongly alkaline environment to release the major O-linked structure elimination; and 3) using A chemical method for the dehydration of hydrazides to release N- and O-linked oligosaccharides.

Analysis can be performed using the following steps:

1. Samples were dialyzed against deionized water to remove all buffer salts and then lyophilized.

2. Intact oligosaccharide chains are released with anhydrohydrazide.

3. Treatment of intact oligosaccharide chains with anhydrous methanolic HCl releases individual monosaccharides as O-methyl derivatives.

4. N-acetylation of any first amino group.

5. Derivatization to provide high-O-trimethylsilylmethyl glycosides.

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

7. Individual glycoside derivatives were identified using retention times from GLC and mass spectrometry compared to known standards.

8. Individual derivatives were quantified by FID with an internal standard (13-O-methyl-D-glucose).

Neutral and amino sugars were determined by high performance anion exchange chromatography combined with pulsed amperometric detection (HPAE-PAD sugar system, Dionex). For example, carbohydrates can be released by hydrolysis in 20% (v/v) trifluoroacetic acid at 100°C for 6 hours. The hydrolyzate was then dried by lyophilization or with a Speed-Vac (Savant Instruments). The residue was then dissolved in 1% sodium acetate trihydrate solution and analyzed on a HPLC-AS6 column as described by Anumula et al. (Annual Biochemistry 195:269-280 (1991)].

Sialic acid can be determined separately in triplicate samples using the direct colorimetric method of Yao et al. (8) (1959) Thiobarbituric acid (TBA).

Alternatively, immunoblot carbohydrate analysis can be performed. According to this method, protein-bound carbohydrates are detected using a commercial glycan detection system (Boehringer) according to the oxidative immunoblotting method described by Haselbeck and Hosel [Hasebeck et al., J. Glycoconjugates, 7:63 (1990)]. Following the manufacturer's recommended staining protocol, except that proteins were transferred to polyvinylidene fluoride membranes instead of nitrocellulose membranes, the blocking buffer contained 5% bovine serum albumin. Detection was performed with an anti-digoxigenin antibody linked to a basic phosphate linker (Boehringer) diluted 1:1000 in tris-buffered saline, using 100 mM Tris-buffered saline (pH 9.5) containing 100 mM sodium chloride and 50 mM magnesium chloride. ), 0.03% (w/v) 4-nitroblue tetrazolium chloride, and 0.03% (w/v) 5-bromo-4-chloro-3-indole-phosphate. Glycoprotein bands are usually observed within about 10 to 15 minutes.

Carbohydrates can also be analyzed by digestion with peptide-N-glycosidase F. According to this method, the residue was suspended in 14 μl of buffer containing 0.18% SDS, 18 mM β-mercaptoethanol, 90 mM phosphate, 3.6 mM EDTA, pH 8.6, and heated at 100° C. for 3 minutes. After cooling to room temperature, the sample was divided into two equal portions. One aliquot was not processed further and served as a control. The second portion was adjusted to contain approximately 1% NP-40 detergent, followed by the addition of 0.2 units of Peptide-N-glycosidase F (Boehringer). Duplicate samples were incubated at 37°C for 2 hours and then analyzed by SDS-polyacrylamide gel electrophoresis.

The TNFR-Ig of the present invention comprises PEGylated TNFR-Ig, which means covalently linked to polymers such as poly(alkylene) glycols (substituted or unsubstituted), especially polyethylene glycol TNFR-Ig molecules. Linkage can be direct, but is preferably achieved by means of various coupling agents known in the art, for example in EP510346, EP593838, U.S. Pat. and Biotechnology, 11:141 (1985) and those coupling agents disclosed in Monfardini et al., Bioconjugation Chemistry 6:62 (1995). PEGylated TNFR-Ig can be used for asthma treatment and prevention as described for TNFR-Ig.

The TNFR-Ig preparations of the present invention are useful against asthma in asthmatic patients. In the treatment of patients with asthma, these preparations can reduce the effects of the disease, such as reversing the inflammation that has already occurred and preventing further inflammation. When provided in a prophylactic manner, these preparations can delay or prevent the onset of asthma, or ensure that the severity of the disease is reduced when it does occur.

According to the invention, the preparation may be administered to a patient in any conventional manner for therapeutic or prophylactic purposes. The preparations of the present invention may thus be administered in conventional pharmaceutical compositions comprising any conventional therapeutically inert carriers. The pharmaceutical composition can contain both inert and pharmacokinetically active additives. Liquid compositions may take the form, for example, of sterile solutions for admixture with water. In addition, substances customary for protecting, stabilizing, moisturizing and emulsifying agents as well as substances for changing the osmotic pressure, such as salts, substances for changing the pH value, such as buffers and other additives, are also present. Antioxidants such as tocopherol, N-methyl-y-tocopherol, butylated hydroxyanisole or butylated hydroxytoluene may be included in the pharmaceutical composition if desired. Pharmaceutically acceptable excipients or carriers for compositions include saline, buffered saline, dextrose or water. The composition may also include certain stabilizers, such as sugars, including mannose and mannitol, and for indictable compositions, local anesthetics including, for example, lidocaine hydrochloride. The aforementioned carrier substances and diluents may be organic or inorganic substances, for example, water, gelatin, lactose, starch, magnesium stearate, talc, gum arabic, poly(alkylene) glycols and the like. The prerequisite is that all adjuvants and substances used to produce the pharmaceutical composition are non-toxic.

The preparations of the invention may be administered parenterally (eg, by subcutaneous, intravenous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal injection) or by inhalation spray. Any conventional pharmaceutical preparations for parenteral or aerosol inhalation administration may be used. Examples of suitable pharmaceutical compositions are solutions for infusion or injection. The preferred mode of administration is by intravenous injection. Another preferred mode of administration is by aerosol.

Any effective amount of an anti-asthmatic preparation of the invention may be used to lessen or reverse the effects of the disease or to prevent or delay the onset of the disease. A preferred dose of the TNFR-Ig composition for parenteral (injection) administration of the present invention for treating, preventing or reversing asthma is about 0.1 to about 5.0 mg of TNFR-Ig preparation per kilogram body weight of the patient (mg/kg ). A particularly preferred dosage is from about 1.0 to about 3.0 mg/kg. The preferred dosage of the TNFR-Ig composition for the same purpose and in the spray administration is 0.03% to 5.0% by weight of the TNFR-Ig preparation. In either mode of administration, the therapeutic dose may be administered once per treatment day, or in smaller doses administered over a period of about 24 hours to give the total administered dose.

For the rapid treatment of asthma by parenteral administration, a single dose of 0.1 to 5.0, especially 1.0-3.0 mg/kg per patient per day may be given. For prophylaxis of asthma by parenteral administration, depending on the patient and state, from daily intervals, but preferably from twice a week to once a week to once a month or other intervals preferably from 0.1 to 5.0, especially 1.0-3.0 mg/kg of this dose. Likewise, for the rapid treatment of asthma by nebulization, a single daily dose can be inhaled. For prophylaxis, the dose is inhaled at daily intervals, but preferably at intervals ranging from twice weekly to weekly to monthly or other intervals depending on the patient and condition. The precise dosage of the composition administered will vary as determined by the skilled artisan, depending on the type of use, mode of use and patient requirements. The precise dosage for a patient can be specifically modified by the skilled artisan taking into account the severity of the symptoms, the particular formulation employed and other drugs that may be involved.

The spray compositions of the present invention may be liquids and powders. They can be administered nasally or orally (intranasally or buccal). An example of a spray composition is a nasal and oral spray produced from a nebuliser by mechanical action on water or saline solution of the TNFR-Ig preparation. Another example is an aerosolized spray, where a mist is created from the solution and then effectively inhaled by the patient (rather than sprayed directly into the nose and mouth). If TNFR-Ig is in powder form, the particles should be large enough to remain in the respiratory tract and not be exhaled by the patient after inhalation. The spray may be inhaled one or more times per day or less frequently as directed by the patient, depending on the medical regimen for rapid treatment or prophylaxis. For nebulized administration, the TNFR-Ig of the present invention can be administered to a patient by inhalation of an amount effective for treating or preventing asthma as described above. For asthma, the affected part of the airway (nasal cavity, trachea, lower airways or bronchioles) receives an appropriate amount of TNFR-Ig in this way.

The spray formulations of the invention preferably provide from about 0.03% to 5.0%, more preferably about 0.03 to 0.5%, especially about 0.1 to 0.3% by weight of the preparation. Preferred spray formulations are aerosol formulations, in general lower dosage ranges are preferred for sprays, eg nebulized formulations, and higher dosage ranges are preferred for aerosols. Any conventional aerosol formulation may be used in the present invention to provide an effective amount of the preparation of TNFR-IG, preferably the amount of preparation described above. The preparation is most effectively administered by releasing a measured dose of an aerosol mist in the mouth of the patient as the patient breathes in, drawing the mist into the mouth and through the respiratory tract into the lungs. As noted above, an effective amount may be administered orally or intranasally in smaller doses one or several times a day, in daily doses for rapid treatment, and at weekly to monthly intervals for prophylaxis.

Suitable aerosol formulations include an appropriate dose of a pharmaceutically active compound (eg, a TNFR-Ig preparation) and an effective amount of any conventional aerosol propellant. Any conventional surfactants may also be included. TNFR-Ig can be combined with a propellant either as a liquid in solution or as a powder (eg, lyophilized by well-known methods). The propellant may be suitably liquefied.

Those surfactants that are soluble or dispersible in the propellant are more effective. Surfactants that are more soluble in propellants are most effective. Also more importantly, the surfactant should be non-irritating and non-toxic.

Any conventional propellant known to those skilled in the art to be acceptable for pharmaceutical compositions, liquefied or non-liquefied, may be used. Depending on whether the TNFR-Ig is in powder or liquid form, the propellant may be a propellant suitable for use with a powder or a propellant suitable for use with a liquid active ingredient. The liquefied propellant employed may be a propellant that is a gas at room temperature (65°F) and atmospheric pressure (760 mm Hg), ie, it may have a boiling point below 65°F at atmospheric pressure and be non-toxic. Suitable liquefied propellants which may be employed include lower alkanes containing up to 5 carbon atoms, such as butane and pentane. Examples of liquefied propellants are fluorinated and fluorinated lower alkanes such as those sold under the trademark "Freon". Mixtures of the above propellants may suitably be employed.

Having now generally described the invention, it will be more readily understood by reference to the following examples which are given by way of illustration and are not intended to be limiting unless otherwise indicated.

Example

Example 1: Plasmids Encoding TNFR-Ig for Production of TNFR-Ig

A． cell line

The Chinese hamster ovary (CHO) cell line used as a mammalian host cell line was derived from CHO-K1 (ATCC number: CCL61 CHO-K1 ). A CHO-K1 mutant dihydrofolate reductase (DHFR-)-deficient cell line designated CHO-K1DUX-B11 (DHFR-) (obtained from Dr. L. Chasin, Columbia University; Simonsen, CC and Levinson, AD, (1983) Proceedings of the National Academy of Sciences of the United States of America 80:2495-2499; UrlaubG., and Chasin, L., (1980) Proceedings of the National Academy of Sciences of the United States of America 77:4216-4220) by using the vector containing the proinsulin precursor cDNA (Sures et al. (1980) Science, 208:57-59] Transfected to obtain a cell line with reduced insulin requirement. Growth of selected clones designated dp12.CHO required glycine, hypoxanthine and thymidine, confirming that they were DHFR ^- genotype.

B． Construction of Soluble Type 1 TNFR-IgG ₁ Chimera

Soluble type 1 TNFR _- IgG 1 hybrids were constructed by genetically fusing the extracellular domain of human type 1 TNFR to the IgG ₁ heavy chain hinge region and the _CH 2 and _CH 3 domains (hereafter referred to as TNFR1-IgG ₁ ). fit. Alternatively, the hinge region and CH2 and CH3 domains of IgG3 heavy chains can also be used.

The DNA sequence encoding human type 1 TNFR (see Loetscher et al., supra) was obtained from the plasmid pRF-TNFR [Schall et al., Cell, 61, 361 (1990)]. To construct this starting plasmid, a 2.1 kb placental cDNA clone (Schall et al., supra) was inserted into the mammalian expression vector pRK5, the construction of which is described in EP Publication No. 307,247. The cDNA starts at nucleotide 64 of the sequence reported by Loetscher et al., and the starting methionine is located 118 bp downstream.

The source of the IgG ₁ coding sequence is the CD4-IgG expression plasmid pRKCD4 ₂ F _c1 [Capon, DJ et al., Nature, 337, 525 (1989); Byrn et al., Nature, 344, 667 (1990)], which contains the coding sequence fused to the human IgG ₁ sequence. The cDNA sequence of the hybrid polypeptide composed of 1-180 residues of the mature human CD4 protein (2 N-terminal CD4 variable domains), the IgG ₁ sequence starts at the 216th aspartic acid (the 114th amino acid is taken as 1st residue of heavy chain constant region) [Kabat et al., Protein Sequences of Immunological Interest, 4th Edition (1987)], IgG ₁ after the cysteine residue involved in heavy-light chain binding 1st residue of the hinge) and terminated at residue 441 to include the _CH2 and _CH3 Fc domains of _IgG1 . See also EP227110. Alternatively, the pCD4-Hγ3 vector (DSM5523, EP394,827) can be used. The CD4 cDNA was removed by SstI digestion to obtain the desired IgG3 sequence.

Deletion mutagenesis was used to align the threonine residue 171 of the mature TNFR's with the aspartic acid residue 216 of the IgG ₁ heavy chain by generating restriction fragments of the plasmids pRK-TNFR and pRKCD4 ₂ F _c1 and ligating them [ Kabat et al., supra] to construct TNFR1- _IgG1 . The resulting plasmid pRKTNFR-IgG contains the full length of the TNFR ₁ -IgG ₁ coding sequence. This plasmid can then be transfected into host cells such as CHO cells by conventional methods such as calcium phosphate precipitation. The resulting cells can be cultured to express TNFR-IgG by a conventional method, and then isolated by a conventional method.

Example 2: Production of TNFR-Ig with approximately: 5-6 M. sialic acid per protein, 0.4-0.45 M N-acetylglucosamine per protein, 5.5-7.5 pI

cell culture

The gene encoding soluble type 1 TNFR-IgG ₁ in Example 1 was introduced into dp12.CHO cells by transfection. Transfection is accomplished using the calcium phosphate technique for introducing DNA into mammalian cells. 2 days after transfection, cells were trypsinized and replated onto selection medium (Ham's F-12 DMEM without glycine-hypoxanthine and thymidine supplemented with 2% dialyzed serum, 1:1 v/v) . Subsequently, isolates secreting TNFR1- _IgG1 were screened. Clones expressing TNFR1- _IgG1 were expanded in methotrexate to generate high expressing clones and subsequently adapted to serum-free medium. These cells are under continuous selection pressure until transferred to non-selective media for growth and expansion of the inoculum.

To provide TNFR1- _IgG1 producing cell cultures, the above cell populations were expanded from methotrexate-containing medium to methotrexate-free growth medium by serial subculturing in vessels of increasing volume. For these steps of the method, the non-selective growth medium is a DMEM/HAMF-12 based formulation (see, e.g., U.S. Pat. , sugars, vitamins, glycine, hypoxanthine, and thymidine; recombinant human insulin, hydrolyzed peptone (Primatone HS or Primatone RL), cytoprotective agents such as Pluronic F86 (polyol) or equivalent; gentamicin; lipid classes and trace elements.

The culture was controlled at pH 7.2 +/- 0.4 by using CO ₂ gas (acid) and/or Na ₂ CO ₃ (base). During cell growth, the temperature was controlled at about 37°C. Dissolved oxygen was maintained above 5% of air saturation by direct injection of air and/or oxygen. Osmolarity was maintained between approximately 250 mOsm and 350 mOsm during inoculum expansion.

The growth phase of each culture is followed by a second or transition phase in which culture parameters are changed from optimal growth to production conditions. The temperature of the culture system is generally reduced to between about 30 and 35°C during this transition period. Butyrate is added to ensure a certain osmolality range. The products accumulated during this production period were analyzed for their sialic acid content.

In a typical production procedure, approximately 1.2 x ¹⁰⁶ cells are expanded from an inoculum in the selection phase with an initial osmolality of 300-450 mOsm, preferably approximately 300, grown in the growth phase. The growth medium was supplemented with trace elements, recombinant human insulin and hydrolyzed peptone. Cells were grown under these conditions for two days. The temperature of the cell culture was lowered at the beginning of day 3. Simultaneously with or after the temperature change, approximately 1 to 6 mmol of sodium butyrate, preferably 6 mmol, is added to the cell culture and the osmolality required for production is ensured by addition of various media components. Cells were cultured for 9-10 days under these conditions. Cells were supplied with various media components as needed.

The pi of the highly sialylated composition is lower than the pi of the low sialylated composition. The composition is subjected to isoelectric focusing. Isoelectric focusing gels separate the glycoproteins of the composition according to their isoelectric points pi using a pH gradient created by ampholytes of different pH. Composition analysis was performed using a pH gradient from 10 to 4.

The above compositions exhibit isoelectric points ranging from about 5.5 to about 7.5 as determined by chromofocusing, with the pI being sensitive to neuraminidase treatment.

Recovery of TNFR-IgG

IgG fusion proteins can be purified by centrifuging cell cultures and passing the resulting culture supernatant through a protein A column as described by Capon et al., Nature, 337:525, 1989. Therefore, the cell culture obtained above was centrifuged and the supernatant was passed over a protein A column. This affinity chromatography was purifiable to greater than 95% homogeneity by TNFR1-Ig preparations on immobilized S. aureus protein A as described by Capon et al., supra.

Sugar analysis

A. The content of sialic acid can be analyzed by the method of Warren, L. (1959), Journal of Biochemistry 234:1971-1975.

B． The release of intact neutral and amino sugars can be determined by high pH anion exchange chromatography combined with pulsed amperometric detection. Use the following steps to analyze:

1. Buffer exchange was performed with TNFR1-IgG1 (approximately 50 [mu]g/ml) and an appropriate reference so that the final sample was contained in 1% acetic acid.

2. Approximately 90 μg of TNFR1- _IgG1 and reference samples were frozen in a dry ice and ethanol bath, and frozen samples were lyophilized overnight.

3. Lyophilized samples were reconstituted in 500 μl trifluoroacetic acid and incubated at 120° C. for 1 hour.

4. After acid hydrolysis TNFR1- _IgG1 and reference samples were cooled and evaporated to dryness.

5. Samples were reconstituted with water to a final concentration of approximately 0.6 mg/ml.

6. Separation of monosaccharides was performed at room temperature by high pH anion exchange chromatography with pulsed amperometric detection using a Dionex CarboPac Pal (4 x 250 mm) column (Dionex Corporation, San Nivale, CA, USA).

7. The amount of simple sugar per serving is determined by comparison to a reference simple sugar.

Embodiment 3. Preparation of TNFR-Ig

1. The CHO cell line DP12 (EP307,247) was transfected with plasmids pSV16B.TNFR-IgG (encoding TNFR1-IgG1) and pFD11 (encoding DHFR) using the calcium phosphate co-precipitation method.

2. Cell clones obtained by transfection were expanded to 9 liters of suspension cultures with selection medium containing 2% diafiltered bovine serum and 100 nmol/L methotrexate. Two 9 liter cultures were inoculated into 100 liter fermentors with serum-free medium without methotrexate. After washing with 1000-fold production medium to reduce residual serum components, 100-liter cultures were inoculated into 400-liter cultures with serum-free medium before inoculating cells into 1000-liter production culture vessels. The production medium is a DMEM/HAM F-12 based formulation supplemented with glucose, amino acids, glycine, hypoxanthine, thymidine, hydrolyzed peptone, pluronic F68, gentamicin, lipids and trace elements.

3. Production cultures were maintained at pH 7.2 + or _-0.2 for 13 days with _CO2 gas and/or _Na2CO3 . After the cell concentration reached 5×10 ⁶ cells/ml, the culture temperature was changed from 37°C to 30°C. The osmolarity of the cultures was adjusted to approximately 450 mOsm/kg on days 2 and 4 by adding media components. Sodium butyrate was added to a final concentration of 12 mM. The dissolved oxygen concentration was maintained at 60% gas saturation by sparging the culture with air and or oxygen.

4. After 13 days the culture was harvested and cells were separated from the supernatant by tangential flow filtration. The harvested cell culture fluid (HCCF) was concentrated about 20 times with a 10KD Maxisette membrane. The residue was clarified and conditioned for protein A chromatography by addition of solid NaCl to 1.0 M followed by filtration through a 0.22 micron Durapore filter.

5. After filtration, condition-depleted HCCF was passed over immobilized protein A. Loaded material was washed several times before elution. TNFR-IgG was eluted stepwise with 0.05M sodium citrate/20% (w/v) glycerol, pH 3.0. The elution pool was adjusted to pH 5.0 by adding 1M sodium citrate.

6. After pH adjustment, the Protein A pool was diluted and loaded onto the S-Sepharose Fast Flow. After the sample was washed, it was eluted step by step with 0.05M MOPS/0.05M NaCl pH7.2.

7. After step-by-step elution, dilute the S-Sepharose Fast Flow pool and load it onto the Q-Sepharose Fast Flow column. The column was eluted using a linear gradient from 0.0125M NaCl to 0.125M NaCl in 0.125M MOPS (pH 7.2).

8. After linear gradient elution, the Q-Sepharose Fast Flow pool was adjusted by adding 1 volume of 0.1M sodium acetate/4.0M urea/2.0M ammonium sulfate (pH5.0) /1.0M ammonium sulfate (pH5.0) equilibrated on the phenyl Toyopearl650M column.Under these conditions, TNFR-IgG flows through this column.The sample is then washed with the equilibration buffer, and the effluent and washing liquid are combined to form the phenyl Toyopearl Combined solution.

9. The two phenyl toyophearl pools were combined and concentrated with a 10KD Filtron Centrassette membrane. The concentrate was passed through a G-25 column equilibrated in 0.01M sodium citrate/0.023M glycine/0.023M mannitol (pH 6.0) to generate the TNFR-Ig composition.

The in vivo experiments described in Examples 4-10 below demonstrate that the TNFIg of the present invention alleviates the asthma attack effect. TNF receptor fusion protein can significantly reduce TNFR-Ig, and in some cases even eliminate the response to antigen challenge in animal models of hypersensitivity pneumonitis. The degree of TNFR-Ig inhibition was comparable to that obtained with the broad-spectrum anti-inflammatory drug dexamethasone.

Abbreviations: TNFα, tumor necrosis factor-α; TNFR-Ig, recombinant soluble TNF receptor IgG1 fusion protein; BAL, tracheoalveolar lavage fluid; OA, ovalbumin; _RL , lung resistance; HBSS, Hanks balanced salt Solution; Substance P, a neuropeptide used to induce acute bronchospasm, is commonly referred to in the literature as Substance P [see eg Selig and Tocker, Eur. J. Pharmac. 213(3):331-336 (1992)].

Data Analysis: All values are calculated as mean +/- SEM values for each experiment. Statistical differences were determined by two-method repeated measures analysis of variance for binned data, followed by multiple comparison tests using Student's Newman-Keuls t-test or by Student's t-test. P<0.05 was considered statistically significant. The calculations were performed with Microsoft EXCEL5.0 (softmart, Exton, PA, USA) and Sigma Stat (Jandel Scientific, SanRafael, CA, USA) software packages running on a PC.

Drugs: All drugs were administered at a dose of 1 mg/kg. TNFR-Ig was produced and purified as described in Example 3, and the stock solution was prepared in sodium citrate (10 mM), glycine (23 mM) and mannitol (230 mM) (pH 6 buffer. An aliquot of the stock solution was used before use in - Store at 20°C and dilute in sterile saline. The following items were purchased from Sigma Chemical Company (St.Louis, MO, USA) and formulated in sterile saline: ovalbumin, aluminum hydroxide, urethane, substance P B Acetate, (+/-) propranolol hydrochloride, dexamethasone 21-phosphate and Evans blue.

Embodiment 4: Relieve guinea pig asthma symptom (trachea hypersensitivity reaction) with TNFR-Ig

This example uses guinea pigs hypersensitized to OA such that subsequent exposure to OA will induce asthmatic symptoms of airway hypersensitivity. The symptoms were relieved by TNFR-Ig, and its effect was also compared with that of dexamethasone, a steroid known to relieve asthma.

Male guinea pigs were sensitized to OA on day 0 (10 μg + 1 mg Al(OH) ₃ in 0.5 ml saline, subcutaneously) and received the same dose of OA as a booster injection on day 14 later. On day 21 animals were challenged with 0.1% OA aerosol for 30 minutes. This sensitization caused asthma-like symptoms in guinea pigs, including airway hypersensitivity to substance P. To determine the effect of TNFR-Ig on this symptom, TNFR-Ig was administered prior to challenge, and its symptoms were compared to unchallenged animals and shown to be alleviated. Similar effects were obtained on tracheal hypersensitivity with dexamethasone (30 mg/kg, ip, 1 hour before and 4 hours after challenge). Dexamethasone is a known asthma treatment.

Guinea pigs treated by sensitization, challenge and treatment were used for the following experiments.

Sensitization: Male Hartley strain guinea pigs (Charles River, kingston, NY, USA) weighing 250-300 g were injected subcutaneously with a single dose (10 μg OA + 1 mg aluminum hydroxide in 0.5 ml sterile saline) on days 0 and 14 ) are actively sensitized to OA and were used in experiments between days 21 and 28.

Challenge: Antigen challenge protocols have been described previously (O'Donnell et al., 1994). Sensitized animals were placed in plexiglass boxes and challenged with 0.1% (w/v sterile saline) OA aerosol for 30 minutes. Aerosols were delivered with a De Vilbiss Ultra-Neb 100 ultrasonic nebulizer (Respiratory Services Center, Ephrata, PA, USA) with an airflow rate of 30 L/min driven by a built-in fan. To minimize fatalities from anaphylaxis, the nebulizer was cycled on and off at 60 second intervals during the first 5 minutes of dosing, under these conditions animal mortality was approximately 5%.

Drug treatment: 18 and 1 hour before OA spraying, animal groups received either vehicle (see Drugs) or TNFR-Ig (1 or 3 mg/kg, intraperitoneal injection). For experiments with dexamethasone, groups of animals received either vehicle (saline) or dexamethasone (30 mg/kg, ip) 1 hour before or 4 hours after OA spray, respectively. The optimal dosage parameters of TNFR-Ig and dexamethasone were determined based on preliminary observations.

The results of this example show that guinea pigs exposed to OA and hypersensitized to OA exhibit enhanced tracheal responses to substance P (1-10 ug/kg iv) 6 hours after OA challenge. The enhanced response to substance P is characteristic of asthma-like sensitization, indicating that guinea pigs have asthmatic symptoms of anaphylaxis induced by the above experimental conditions. Anaphylaxis was inhibited by TNFR-Ig (3 mg/kg, ip, 18 and 1 hour before challenge).

The method used was as follows: Tracheal responses to substance P were determined in sensitized unchallenged and challenged guinea pigs 6 hours after exposure to OA aerosols according to the previously described method (Selig and Tocker, 1992). Animals were anesthetized with urethane (2 g/kg, intraperitoneally) and carotid (blood pressure) and jugular vein (drug injection) cannulated with PE-50 catheters. The trachea was cannulated with a 15 gauge needle and the animal was placed in a whole body plethysmograph (Modular Instruments, Malvern, PA, USA). Spontaneous breathing was suppressed with succinylcholine chloride (1.2 mg/kg, iv), and the animals were mechanically ventilated (Harvard Apparatus Model 683; South Natick, MA, USA) using a tidal volume of 1 ml/100 g body weight and a rate of 60 breaths/min. ). The total volume of the plethysmograph was 2 L, and the volume of the ventilation line between the animal and the breathing apparatus was 10 ml. The plethysmograph was equipped with a Fleisch pneumotachometer (Model #0000) connected to a Validyne partial pressure transducer (DP45-14) for the measurement of airflow. Transpulmonary pressure was measured with a second Validyne transducer (DP45-28) connected between the side arm of the endotracheal tube and a 16-gauge intrapleural needle inserted between the 5th and 6th ribs. Airflow and transpulmonary pressure were recorded with a Modular Instruments (Malvern, PA, USA) M-3000 data acquisition system driven by an IBM 486DX2 PC. According to the method of Amdur Mead (1958), the calculation of lung resistance ( _RL ) was carried out by computer using conventional software (BioReport, Modular Instruments). Readings are taken continuously at intervals averaging over 10 seconds. Correct the _RL value as the internal resistance of the trachea (0.11 cm water column/ml/sec). Animals received propranolol (1 mg/kg, iv) and allowed to stabilize for 10 minutes before starting substance P administration.

Dose-response effects of substance P (1, 3, 5 and 10 [mu]g/kg, iv) were obtained by providing bolus injections at approximately 5-10 minute intervals. The maximum change in _RL was obtained for each dose and expressed as a percentage of the baseline value determined before the administration of substance P. The _ED200 value is defined as the dose of substance P that causes a 200% increase in _RL , determined by linear regression of the logarithmic dose-response curve for each animal.

The base value of _RL was about 0.30 cm water column/ml/sec, and there was no significant difference between the groups (P>0.05). Sensitized but unchallenged guinea pigs were rather insensitive to substance P, requiring a dose of 30 [mu]g/kg to raise _RL by at least 200% or greater (Table 1). In contrast, tracheal responses to substance P were significantly elevated after OA challenge. For an approximately 5-fold increase in the _RL maximum obtained with the maximum dose of substance P (Table 1), the _ED200 value increased approximately 10-fold (Table 1).

Table 1: Summary of the effects of TNFR-Ig and dexamethasone on the airway response to substance P ^a in OA-sensitized guinea pigs. treatment ^b -logED ₂₀₀ ^c maximum response response ^d n ^e

( _RL increase %) unstimulated ^f 4.86+/-0.08 139+/-28 6TNFR-Ig carrier 5.82+/-0.04 1811+/-228 71mg/kg 5.87+/-0.01 1344+/-195 63mg/ kg 5.48+/-0.05 ^* 714+/-69 ^* 5 Dexamethasone Vehicle 5.87+/-0.05 935+/-91 530mg/kg 5.35+/-0.04 ^* 596+/-66 ^* 6

^a The dose-response effect of substance P was determined 6 hours after a 30-minute aerosol challenge with 0.1% OA. Animals were pretreated with propranolol (1 mg/kg, iv) for 10 minutes before examining the tracheal response to substance P.

^b Animals were pretreated intraperitoneally with the respective vehicle, TNFR-Ig (18 and 1 hour before OA challenge) or dexamethasone (1 hour before and 4 hours after OA challenge) with a dose of 1 ml/kg.

^{The c} value represents the negative logarithm (mean +/- S.EM. value) of the dose of substance P (in g/kg) required to increase lung resistance ( _RL ) by 200% of the baseline value.

^{The d} values (mean +/- SEM values) represent the percentage increase in basal _RL caused by substance P (10 μg/kg, iv).

^eNumber of animals per group.

^f Dose-response effect curves for substance P were determined in groups of sensitized animals not challenged with OA aerosol.

^* Values are statistically significantly (P<0.05) different between the corresponding vehicle and drug-treated groups.

Administration of TNFR-Ig (3 mg/kg) to sensitized animals significantly (P<0.05) inhibited OA-induced airway hypersensitivity to substance P. The ED ₂₀₀ value of substance P was reduced approximately 3-fold and _{the RL} maximum was reduced by approximately 60% (Table 1). Smaller doses of TNFR-Ig (1 mg/kg) had no effect on substance sensitivity (Table 1). Treatment with dexamethasone (30 mg/kg) in sensitized guinea pigs also significantly (P < 0.05) inhibited OA-induced airway allergic responses to a similar extent as that obtained with higher doses of TNFR-Ig (Table 1).

Example 5: Guinea pig asthma symptoms (influx of inflammatory cells) alleviated by TNFR-Ig

This example uses OA hypersensitized guinea pigs as described in Example 4 so that subsequent exposure to OA will induce asthmatic symptoms, ie inflammatory cell influx. The symptoms were relieved by TNFR-Ig, which was also compared to dexamethasone, a steroid known to relieve asthma.

As in Example 4, male guinea pigs were sensitized to OA and challenged as described to induce asthma symptoms, including quantified airway inflammatory cell accumulation at 6, 24, 48, and 72 hours post-challenge. As in Example 4, to determine the effect of TNFR-Ig on these symptoms, TNFR-Ig was administered prior to challenge, and these symptoms and showed a reduction in symptoms compared to unchallenged animals. TNFR-Ig was also administered for inflammation prior to challenge and the results showed reversal of inflammatory cell influx. Similar effects on inflammatory cell accumulation were obtained with dexamethasone (30 mg/kg, ip, 1 hour before and 4 hours after challenge) at 6 and 24 hours.

The method used was as follows: Airway inflammatory cell influx was measured by BAL at 6 and 24 hours after OA challenge according to a method previously described (Selig and Tocker, 1992). Briefly, guinea pigs were anesthetized with urethane (2 g/kg, ip) and a tracheal incision was made with a 15-gauge cannula. The lungs were lavaged with 3 x 5 ml of sterile Hank's Balanced Salt Solution (HBSS) (Gibco, Grand Island, NY, USA) without Ca2 ⁺ and Mg2 ⁺ . Samples were centrifuged at 25°C, 200 g for 10 min, red blood cells were lysed from the resulting pellet with distilled water (1 ml, 30 min), and osmolality was restored by adding 10 ml HBSS. The samples were centrifuged a second time (200 g, 10 min at 25° C.) and the resulting pellet was resuspended in 1 ml HBSS. Total cell numbers were determined by trypan blue (Sigma Chemical Co., St. Louis, MO, USA) exclusion from aliquots of the cell suspension using a hemocytometer. For differential cell counts, an aliquot of the cell suspension was centrifuged in a Cytospin (5 minutes, 1300 rpm; Shandon Southern Instruments, Sewickey, PA, USA), smeared, fixed, and stained with modified Wright's stain (Leukostat; Fisher Scientific, Pittsburgh, PA, USA) staining. Standard morphological guidelines were employed in sorting at least 300 cells under light microscopy. Data are expressed as BAL cells x 10 ⁶ /animal.

Treatment with TNFR-Ig (1-3 mg/kg, ip, 18 and 1 hour and 1 hour pretreatment, for BAL, at 6 and 24 hours, respectively, significantly (P < 0.05) inhibited 6 and 24 hours after OA BAL accumulation of neutrophils and total cell numbers). TNFR-Ig (3 mg/kg, intraperitoneal) also significantly (P<0.05) reduced the number of eosinophils in BAL at two time points, while low dose (1 mg/kg, intraperitoneal) had no effect. The results of this experiment also indicated that the neutrophil component in the allergen-induced inflammatory response was mediated by TNF. Most notably, 24 hours after antigen challenge, TNFR-Ig nearly abolished the influx of neutrophils in the BAL of sensitized guinea pigs, where neutrophils accounted for approximately 2% of the total number of cells in the BAL. Furthermore, treatment with TNFR-Ig, but not dexamethasone, caused a massive drop in neutrophils in guinea pigs 6 hours after challenge. Blockade of TNF by TNFR-Ig or similar antagonists causes an indirect decrease in factors known to contribute to neutrophil reflux into the lung.

The cellular composition of sensitized, unstimulated guinea pig BAL has been described previously (Selig and Tocker, 1992). Total cell counts in these animals averaged approximately 1 x ¹⁰⁶ /animal, of which approximately 2-3% were eosinophils and neutrophils. Six hours after OA treatment, vehicle-treated animals with TNFR-Ig or dexamethasone contained at least 40 and 20% of total cell counts, respectively, of eosinophils and neutrophils. The percentage of eosinophils in the BAL remained constant at 24 hours, while the neutrophil count decreased to approximately 10% of the total cell number. Inflammatory cell numbers and total cell numbers were not different between the respective vehicle groups of TNFR-Ig and dexamethasone (P>0.05).

The influx of inflammatory cells into sensitized and challenged guinea pig BAL was also inhibited by dexamethasone (30 mg/kg), and 6 hours after OA treatment, there was a significant (P<0.05) decrease in the number of eosinophils and total cells in BAL, This is similar to that caused by TNFR-Ig. There was no effect on neutrophil counts at the 6-hour time point. 24 hours after OA treatment, the number of eosinophils, neutrophils and total cells in BAL was significantly (P<0.05) suppressed by dexamethasone. Compared with TNFR-Ig, eosinophils in dexamethasone-treated animals decreased about 5-fold (P<0.05). In contrast, although dexamethasone treatment decreased the number of neutrophils in the BAL, the percentage of these cells remained unchanged (P > 0.05) at approximately 8% of the total cell number among the different cells.

The ability of TNFR-Ig to reverse an active inflammatory response was examined in sensitized guinea pigs. Animals were sensitized to OA and then challenged as described above. TNFR-Ig (3 mg/kg, ip) was administered 30 minutes after OA challenge. Inflammation was detected by BAL at 24, 48 and 72 hours after challenge. TNFR-Ig was dosed daily for the 48 and 72 hour time points. Post-challenge treatment with TNFR-Ig significantly reversed the influx of inflammatory cells in the BAL of guinea pigs.

Embodiment 6: TNFR-Ig alleviates the asthmatic symptom (pulmonary edema) of guinea pig

This example uses guinea pigs hypersensitized to OA as described in Example 4 so that subsequent exposure to OA will induce asthmatic symptoms, namely pulmonary edema. The symptoms were relieved by TNFR-Ig, which was also compared to dexamethasone, a steroid known to relieve asthma.

As in Example 4, male guinea pigs were sensitized to OA and challenged as described to induce asthmatic symptoms, including quantitative edema at 6 hours post-challenge. As in Example 4, to determine the effect of TNFR-Ig on these symptoms, TNFR-Ig was administered prior to challenge, and these symptoms were compared with unchallenged animals and showed a reduction.

The method used is as follows: 6 hours after OA treatment, the method described previously (Wasserman et al., Prostaglandin, Thromboxane, Leukotriene Research Progress, 23:271-273, 1995) was quantified by the Evans blue stain extravasation method as Airway capillary leakage a hallmark of pulmonary edema. Guinea pigs were anesthetized with urethane (2 g/kg, ip) and cannulated in the jugular vein. Animals then received Evans Blue stain (30 mg/kg, iv) administered over a 60 second period. After 10 minutes the chest cavity was opened and a catheter (PE-240 tubing) was inserted into the aorta through the left ventricle. The ventricle was cross-clamped, the right atrium was incised and the blood was drained by perfusing the animal with 100 ml saline at a rate of 100 ml/min using a cartridge pump (Masterflex; Cole-Parmer, Chicago, IL, USA). The pulmonary circulation was reperfused with 50 ml of saline by cannulating the pulmonary artery and cutting the left atrium. The lungs were removed en bloc, the trachea (terminal 5mm) and main bronchi were dissected, blotted dry between filter papers and weighed. Evans blue dye was extracted in formamide (37°C, 24 hours) and quantified by measuring absorbance at 620 nm with a spectrophotometer (Beckman Instruments Model DU-64, Somerset, NJ, USA). Tissue dye content was obtained from a standard curve of Evans blue dye concentration (0.5-10 μg/ml) and expressed in ng/mg tissue wet weight.

Basal values in sensitized, unchallenged guinea pig trachea and main bronchus averaged 10-20 ng/mg tissue wet weight using Evans blue dye as a marker of tracheal capillary penetration. Six hours after OA challenge, the content of Evans blue dye in the trachea and main bronchus increased approximately 5-fold. Treatment with TNFR-Ig (1 or 3 mg/kg) or dexamethasone (30 mg/kg) attenuated (P<0.05) OA-induced airway penetration in the trachea and main bronchi 6 hours after challenge.

TNFR-Ig (1 or 3 mg/kg. ip) abrogated OA-induced airway edema (quantified as tissue content of Evans blue dye) in the trachea and main bronchi of sensitized guinea pigs.

Example 7: Alleviation of Asthma Symptoms in Brown Norway Rats with TNFR-Ig

Similar to the guinea pigs used in Example 4, accumulation of inflammatory cells (a symptom of asthma) was induced in rats and treated with TNFR-Ig. The difference between this model and the guinea pig is that the allergic reaction of the brown Norwegian rat is mediated by IgE.

The method of use was as follows: Male brown Norwegian rats were sensitized on days 0, 1 and 2 to OA (1 mg OA + 100 mg Al(OH) ₃ in 0.5 mg saline, ip). On day 21, animals were challenged with 1% OA aerosol for 30 minutes. Inflammatory cell accumulation was quantified by BAL 24 hours after challenge. The protocol for sensitization, challenge and BAL was similar to that described above for guinea pigs with the following exceptions. Male brown Norway rats (Charles River, Kingston, NY) weighing 200 to 250 g were activated on days 1, 2, and 3 with a single intraperitoneal injection (1 mg OA + 100 mg aluminum hydroxide dissolved in 1 ml sterile saline) to activate their response to OA sensitization was used to perform experiments on day 21 (Elwood et al., 1992). One hour before OA spraying, rats in each group received their own vehicle, TNFR-Ig (1 or 3 mg/kg, intraperitoneal) or dexamethasone (0.3 mg/kg, intraperitoneal).

On the day of the experiment, rats were challenged with 1% OA aerosol for 30 minutes. 24 hours after challenge, BAL was performed by lavaging the lungs with 2 x 1 ml/100 g of HBSS. Red blood cells were lysed with 0.5 ml distilled water and osmolality was restored with 5 ml HBSS.

Treatment of the above rats with TNFR-Ig (3 mg/kg, ip, 1 hour before challenge) essentially abolished the accumulation of neutrophils in the BAL and caused a significant decrease in the number of eosinophils and total cells. Similar results were obtained with dexamethasone (0.3 mg/kg, ip, 1 hour before challenge). Lower doses of TNFR-Ig (1 mg/kg, ip) also significantly reduced the number of neutrophils in the BAL, but had no effect on the number of eosinophils and total cells.

In more detail, sensitized, unchallenged brown Norwegian rats have a basal total cell count in the BAL of approximately 1 x ¹⁰⁶ /animal, of which proportionally 1-2% are eosinophils and neutrophils . Twenty-four hours after OA challenge, the total number of cells in the BAL of vehicle-treated animals was approximately 3-fold higher, with eosinophils and neutrophils accounting for at least 40% and 25% of the total number of cells, respectively. Inflammatory cell count and total cell number in BAL were not different among the respective vehicle treatment groups (P>0.05).

Treatment with TNFR-Ig (3 mg/kg, intraperitoneal) or dexamethasone (0.3 mg/kg, intraperitoneal) significantly (P < 0.05) reduced eosinophils, neutrophils, and and accumulation of total cell numbers. Treatment with a lower dose of TNFR-Ig (1 mg/kg, ip) also significantly (P < 0.05) suppressed the number of neutrophils in the BAL, but had no effect on the accumulation of eosinophils or total cell numbers in the BAL .

Example 8: Reduction of neutrophils in the lungs of injured rats

A rat model of pneumonia induced by Sephadex (dextran gel) particles showed increased numbers of eosinophils and neutrophils and granuloma formation in the lungs, similar to that seen in chronic asthma. This model was used to show that TNFR-Ig alleviates this chronic condition.

The method of use is as follows: Accumulation of inflammatory cells into the lung is induced in male Sprague-Dawley rats by administering a suspension of Sephadex (dextran gel) particles (7.5 mg/kg,.iv.). 24 hours and 72 hours after the administration of Sephadex (Sephadex), the total number of white blood cells was significantly increased in the BAL fluid. At 24 hours, the number of neutrophils accounts for about 50% of the total number of white blood cells, and then drops to about 10% of the total number at 72 hours. The eosinophil count was maintained at around 10% of the total white blood cell count.

Pretreatment with TNFR-Ig (1 and 3 mg/kg,.ip., 1 hour before challenge) or dexamethasone (0.1 and 0.3 mg/kg, intraperitoneal) 24 hours after administration of Sephadex (dextran gel) Inhibition of neutrophils, although TNFR-Ig had no significant effect on total cell count. 72 hours after administration of Sephadex (dextran gel), TNFR-Ig (1 and 3 mg/kg, ip, daily) 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, ip, daily) substantially abrogated the infiltration of neutrophils and eosinophils into the BAL fluid. TNFR-Ig (1 and 3 mg/kg, ip) or dexamethasone (0.1 and 0.3 mg/kg, ip) significantly reduced total cell counts at the 72-hour time point.

Example 9: Remission of primate atopic asthma

The primate model of atopic asthma established by wild-caught macaques with natural airway sensitivity to Ascaris suum antigen was used to demonstrate the effect of TNFR-Ig on alleviating airway hypersensitivity in asthmatic symptoms. Repeated exposure to antigens induces asthmatic symptoms in the airways, especially increased lung resistance ( _RL ). Monkeys showed a decrease in _RL when treated with TNFR-Ig.

When given repeated exposure to A. suum antigen, wild-caught wild Ascaris-susceptible macaques showed enhanced respiratory responses to inhaled methacholine and increased airway eosinophils. The use of this antigen therefore induced an allergic reaction in these monkeys and subsequent airway anaphylaxis with asthmatic symptoms. The following protocol was used to examine the effect of TNFR-Ig on respiratory allergic responses.

Day 1: The dose of methacholine (MCh), a bronchospasm inducer similar to substance P, which produced a 100% (PC ₁₀₀ ) increase in lung resistance ( _RL ) was determined.

Day 3: Monkeys are challenged with an inhaled dose of an Ascaria antigen producing at least a doubled _RL .

Days 5 and 8: Repeat the experiment of day 3.

Day 10: Repeat the experiment of Day 1, and measure the change in log PC ₁₀₀ between Day 1 and Day 10.

Administration of TNFR-Ig (3 mg/kg, iv) on days 1, 3, 5 and 8 attenuated respiratory hypersensitivity responses to MCh.

Embodiment 10: TNFR-Ig relieves mouse allergic airway inflammation

Using a murine model of OA-induced hypersensitivity pneumonitis in mice demonstrated that TNFR-Ig attenuates asthma symptoms of inflammatory cell influx. Sensitized mice repeatedly exposed to OA developed progressive airway hypersensitivity and increased eosinophil influx in the BAL. This model is associated with elevated IgE levels and exhibits typical Th2 cytokine profiles (eg, elevated IL-4 and IL-5 levels). The ability of TNFR-Ig to attenuate active allergic inflammatory responses was evaluated in this model.

Murine models of hypersensitivity pneumonitis, like primates, require multiple exposures of sensitized animals to allergens in order to induce respiratory anaphylaxis and influx of inflammatory cells. Female C57BL/6 mice were sensitized with OA (10 μg in 1 mg Al(OH) ₃ gel, 0.1 ml, intraperitoneally) on day 0, then, from day 14-20, daily with 1% OA aerosol Inspire for 30 minutes. Respiratory hypersensitivity responses to MCh and BAL were completed 24 hours after the last OA challenge. Some lungs from sensitized, challenged were fixed for histological examination and others were homogenized for determination of TNF levels.

Inflammatory cell accumulation and TNF levels in sensitized, challenged mice peaked after the last challenge with OA aerosol (day 20). In this experiment, TNFR-Ig (3 mg/kg, ip) was administered immediately after the last exposure to OA. TNFR-Ig attenuated OA-induced hypersensitivity responses but had no effect on inflammatory cell influx in the BAL. Daily administration of TNFR-Ig during challenge also had no effect on BAL cell counts. However, administration of TNFR-Ig (3 mg/kg, ip, day 20) significantly reduced the number of eosinophils in the lung tissue of sensitized, challenged mice and cleared the TNF level in the lung tissue.

Example 11: Aerosol Compositions

The following are examples of preparations containing TNFR-Ig of the present invention

Example A - Aerosol

TNFR-Ig (particle size range 1-5 microns) 3.0%

Span ^® 85 (sorbitan trioleate) 1.0

Freon® ¹¹ (trichloromonofluoromethane) 30.0

Freon® ¹¹⁴ (dichlorotetrafluoroethane) 41.0

Freon® ¹² (dichlorodifluoromethane) 25.0

Example B - Aerosol

TNFR-Ig 0.5%

^Span® 85 0.5%

Propellant B ¹ 99.0%

¹ Propellant B consists of 10% Freon 11, 50.4% Freon 114, 31.5% Freon 12, and 8.0% butane.

Example C - Aerosol

TNFR-Ig 1.00%

^Span® 85 0.25% Freon 11 5.0% Freon W ¹ 93.75% ¹ Freon W consists of 61.5% Freon 114 and 38.5% Freon 12. Example D - Aerosol TNFR-Ig 0.50% Span® ⁸⁵ 0.50% Propellant (C) ¹ 99.0% ¹ Propellant C consists of 30.0% Freon 11 and 70% Freon W. Example E - Aerosol TNFR-Ig 0.88% Sodium Sulfate (Anhydrous), Powder 0.88% Span® ⁸⁵ 1.00% Propellant Composed of 50% Freon 12, 97.24% 25% Freon 11, and 25% Freon 114 Example F-Aerosol TNFR-Ig 0.06% ^Span® 85 0.05% Freon 11 20.0% Freon 12/Freon 114 (20/80) 78.9% Example 12: Composition for Injection The following is the composition for injection containing TNFR-Ig preparation of the present invention An example of a thing.

Ingredients Contains per ml

TNFR-IgG1 *

Citric Acid Anhydrous, USP 1.92mg

Glycine, USP 1.70mg

Mannitol, pyrogen-free, USP 41.90mg

Sodium hydroxide Appropriate amount, pH6.0

Hydrochloric acid appropriate amount, pH6.0

Water for Injection, USP qs., to 1.0ml ^**

^* The concentration of TNFR-IgG1 used in this formulation can be from 1.0mg/ml to 20mg/ml. The final amount of TNFR-IgG1 in the formulation is selected based on the concentration and volume of the composition per vial.

^** Transfer by lyophilization.

1mg vial (1ml with 1.0mg/ml formula)

2.5mg vial (1ml with 2.5mg/ml formula)

5.0mg vial (1ml with 5.0mg/ml formula)

10mg vial (2ml with 5.0mg/ml formula)

10mg vial (1ml with 10.0mg/ml formula)

20mg vial (2.5ml with 8.0mg/ml formula)

20mg vial (4ml with 5.0mg/ml formula)

50mg vial (2.5ml with 20mg/ml formula)

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Claims (11)

1. One kind in suffering from the patient of symptoms of asthma the opposing asthma method, comprise to described patient and use the compositions that contains by one or more chimeric conjugated protein goods of forming of TNF α, each protein in the described goods is made up of the soluble fraction that is fused to the p55 TNF receptor protein on the IgG, the IgG of wherein said fusion contains all the IgG domains except an IgG domain of IgG CH, described compositions contains the inert carrier in the treatment, and described compositions is administered to described patient so that provide the described chimeric protein goods of effective dose to resist described symptoms of asthma to patient.
2. 2. the process of claim 1 wherein that described patient suffers from asthma, and the chimeric protein goods are used with the amount of the influence that is enough to alleviate described disease.
3. 3. the process of claim 1 wherein that described compositions is administered to asthma patient with the amount that effectively prevents or delay described seizure of disease before asthma outbreak.
4. 4. any one method in the claim 1 to 3, wherein the IgG of Rong Heing is a human IgG ₁
5. 5. any one method in the claim 1 to 4, wherein the protein in described protein articles has the composite oligosaccharide that stops with one or more sialic acid residueses and has the N-acetyl-glucosamine of exposure, in described goods the mol ratio of sialic acid residues be every mole of protein from about 4 to about 7 moles of sialic acides, the mol ratio of the N-acetyl-glucosamine that in described goods, exposes be every mole of protein from about 1 to about 2 moles of N-acetyl-glucosamines, the mol ratio of sialic acid residues and N-acetyl-glucosamine residue is from about 0.35 to about 0.5 in described goods, and the isoelectric point, IP of described goods is from about 5.5 to about 7.5.
6. 6. the method for claim 5, wherein the mol ratio of sialic acid and N-acetyl-glucosamine is from about 0.4 to about 0.45, and sialic acid and proteinic mol ratio are from about 5.0 to about 6.0.
7. 7. any one method in the claim 1 to 6 wherein is administered to described patient with every kilogram of dosage from 0.1mg to 5.0mg of every patient body weight every day through injection with described goods.
8. 8. the method for claim 7, wherein dosage be every day every kilogram of every body weight from 1.0mg to 3.0mg.
9. 9. any one method in the claim 1 to 8, wherein said goods are with the mode medication of oral cavity or nasal spray.
10. 10. the method for claim 9, wherein said spraying is to contain the liquid spray composition of weight ratio from about described goods of 0.03% to 5.0%.
11. 11. by the conjugated protein application in the medicine of preparation treatment asthma of chimeric TNF α that the soluble fraction that is fused to the p55 TNF receptor protein on the IgG is formed, wherein said fusion IgG contains all the IgG domains except an IgG domain of IgG CH.