NL8900779A - PROTEIN AGAINST THE EFFECT OF TUMOR NECROSE FACTOR, DNA CODING FOR SUCH PROTEIN, A VECTOR CONTAINING SUCH DNA, A HOST CELL TRANSFORMED WITH THAT VECTOR AND A PHARMACEUTICAL PREPARATION CONTAINING THIS PROTEIN. - Google Patents

Description

- 1- *>

Protein that inhibits the action of tumor necrosis factor, DNA encoding such protein, a vector containing such DNA, a host cell transformed with that vector, and a pharmaceutical composition containing that protein.

This invention relates to a novel protein with an inhibitory effect on the actions via tumor necrosis factor α, on the isolation and purification of such a protein from natural sources, on its preparation by DNA manipulation and the use of such protein in treating conditions associated with excessive or uncontrolled TNFa formation.

Tumor necrosis factor (TNF) is an activity exerted by a group of at least two proteins, o and 3, which are cytotoxic to tumor cells and inhibit their growth in culture 10 (E. Carswell et al in "An endotoxin-induced serum factor that causes necrosis of tumors ", Proc. Natl. Acad. Sci. USA 72 (1975) p3666. Tumor necrosis factor (TNFa) also called" cachectin "is mainly made by cells derived from the monocyte / macrophages in response to" stress signals "accompanying the invasion of bacteria, viruses, tumors and other toxins. TNF8, commonly called" lymphotoxin ", is mainly made by lymph cells.

TNF3 often has the same effects as TNFa, but it appears to be less strong, although this may also be due to difficulties in preparing pure TNF6.

20 TNFa mediates and participates in a wide variety of biological actions J_ B. Beutler et al. In "Identity of tumor necrosis factor and the macrophage-screted factor cachetin", Nature 316 (1985) 552J \ several of them have in common with interleukin 1 (IL-1) [j. Le et al. In "Tumor necrosis 25 factor and interleukin 1: cytokines with multiple overlapping biological activities", Laboratory Invest. 5i6 (1987) 234f. Increased levels of TNFa, for example caused by tumor cells, can lead to weight loss and cachexia, and TNFa has been hypothesized to be the major transducer of endo-toxin shock (septa), which can be fatal. Other biological effects of TNFa are excessive bud pressure, fever (triggered by stimulation of the hypothalamus), formation of prostaglandia (PGE ^), coagulopathy (triggered by stimulation of 8900779. ^ / - 2 - 9 cells of the vascular system). -endothelium, which secrete tissue factor, for example, and tissue destruction (for example, by stimulating the formation of a series of proteinases, including collagenase, the formation of skin fibroblasts and synovial cells 5 (~ C> Dinarello cs in "Tumor necrosis factor (cachectin)) an endogenous pyrogen and induces production of interkeulin 1 ", J. Exp. Med. 163 (1986), 1433 and J. Dayer et al. in" Cachectin / tumor necrosis factor stimulate collagenase and prostaglandin E production by human dermal fibroblasts and synovial cells " 10 J Exp Med 162 (1985) 2163 ^ 7-

Thus, there is a need for the development of a cachectin / TNFa inhibitor that prevents endotixin shock, cachexia and the other adverse effects described above. Passive immunization of animals against cachectin via TNFa-15 antibodies has been shown to prevent endotoxin induced death (B. Beutler et al. In Nature 316, supra).

A protein has now been identified which has a strong inhibitory effect on the activities exerted via TNFa, but without significant inhibition of the activity exerted by IL-1. This protein is hereinafter referred to as Tumor-Necrosis-

Factor α inhibitor (TNFaR).

Thus, one aspect of the invention is a protein that selectively inhibits the actions exerted through tumor necrosis factor α.

Here, as selective inhibition, as the protein of the invention shows, it refers to the ability to inhibit the actions exerted via TNF in the absence of the ability to inhibit other proteins that have certain but not all biological actions in common with TNF, such as IL -1.

Preferably, the tumor necrosis factor α inhibitor of the invention is in a substantially homogeneous form, substantially free of impurities and / or substantially free of other protein material.

The tumor necrosis factor α inhibitor according to the invention has been found to exhibit one or more of the following characteristics: (a) a molecular weight between 40 and 60 kDa, determined by chromatography on a molecular sieve, (b) a isoelectric point between 5.5 and 6.1, determined by chroma 8900779.

»- 3 - * graphic focusing, (c) inhibition of the standard TNF assay of the differential cytotoxicity of actinomycin D treated mouse cells L929, as described by G. Nedwin et al in" Effects of 5 interleukin 2, interferon-γ and mitogens on the production of tumor necrosis factors a and 3 ", J. Immunol. 135 (1985) 2492. This inhibition can be overcome by adding even more TNFa, indicating that the inhibition is competitive. The inhibitor also inhibits the action of TNF3, although the inhibition of TNFa in this assay is more efficient than that of TNFB, (d) inhibition of TNF-induced release of PGE1 from human fibroblasts and synovial cells, (e) disruption of the binding of TNFa to D937 cells (a culture of unicellular cancer tissue), which is evidenced by the inhibition of binding of radiolabeled TNFa (I-TNFa), (f) a temperature-dependent promotion of the dissociation of preformed complex of TNFa with U937 cells, (g) no degradation of TNF by proteolytic cleavage, and (h) no inhibition of the binding of IL-1 to its receptor, 125 indicated by the binding of I-IL-Ια to the thymoma subline EL-4-6.1. from the mouse.

The protein of the invention has been found upon further purification to have a molecular weight of about 33,000 daltons, as determined by electrophoresis on a sodium dodecyl sulfate polyacrylamide gel.

Thus, this invention also provides a protein that selectively acts through the TNFa-mediated activities, which has one or more of the following properties: (a) a molecular weight of about 33 kDa, determined by SDS-PAGE, (b) an isoelectric point between 5.5 and 6.1, determined by chromatographic focusing, (c) inhibition of the standard TNF assay of the differential cytotoxicity for actinomycin D treated mouse cells L929, as described by G. Nedwin et al in "Effects of inter- Leukin 2, interferon-γ and mitogens of the production of tumor necrosis factors a and β ", J. Immunol. 135 (1985) 2492. This inhibition can be overcome by adding even more TNFa, indicating that it is competitive. The inhibitor also inhibits the action of TNFB, although the inhibition of TNFa in this test is much more efficient than that of TNFB, 89 00771..

4 - 4 - (d) inhibition of TNF-induced release of PGE 1 from human fibroblasts and synovial cells, (e) inhibition of TNFα binding to U937 cells, 125 evidenced by inhibition of I-1 binding. TNFa, 5 (f) a temperature dependent dissociation of a preformed complex from TNFa and U937 cellett, (g) no degradation of TNF by proteolytic cleavage, and (h) no inhibition of IL-1 binding to its receptor as shown by the binding of L-IL-Ια to the mouse thymoma subline 10 EL4-6.1.

Preferably, the TNFaR has features (a) and (b) and some of features (c) to (h). Particularly preferred, the TNFaR of this invention exhibits all features (a) through (h).

The protein according to the invention has the following sequence at the amino end:

Asp-Ser-Val-Cys-Pro-Gln-Gly-Lys-Tyr-Ile-His-Pro-Gln-Cys-Asn-Ser-Ile 20 Furthermore, it is believed that the following three amino acids provide a glycosylation site and thus the sequence continues with Asn-Ser-Thr-Lys.

It will be appreciated that the inhibitor of the invention has an amino acid sequence at its amino terminus essentially identical to what has just been mentioned. If there are already omissions, insertions, substitutions, reversals or additions of allele or other origin, the sequence will still be at least 80% and preferably at least 90% homologous with the sequence in native TNFaR, while retaining the essential biological properties of that protein.

The TNFa inhibitor of the invention has been shown to be proteinaceous in that it is inactivated by heating in a Time and temperature dependent manner and in that it is destroyed by treatment with trypsin or pronase.

The TNFaR of the invention * has also been shown to be a glycoprotein since treatment with the enzyme endoglycosidase F reduces its molecular weight by 7 to 8 kDa.

Thus, the invention also relates to a TNFa inhibitor as described above, however in a substantially unglycosidated 89 00778. .

«- 5 - condition.

The inhibitors of the invention are important in the treatment of conditions in which it is desirable to suppress the TNFa inhibition, for example weight loss, shock, cachexia and chronic local inflammation, rheumatoid arthritis, metastatic intravascular coagulation and nephritis, as caused by TNFa .

Thus, another aspect of the invention is the use of the TNFa inhibitor described above or a pharmaceutically acceptable derivative thereof as a therapeutically active agent in treating conditions associated with excessive or uncontrolled TNFa formation. For this treatment, an effective amount of TNFa inhibitor or pharmaceutically acceptable derivative thereof is administered.

The invention also relates to the manufacture of a medicament for the treatment of conditions associated with excessive or uncontrolled TNFa formation.

Those skilled in the art will recognize that "treatment" is both prevention and cure here. It will further be appreciated that the amount of TNFa inhibitor required in a treatment will vary not only with the mode of administration but also with the nature of the condition treated and with the age and condition of the patient, and that is entirely at the discretion of the from the treating physician or veterinarian. Generally, however, a suitable dose will be between 5.0 and 500 µg per kg of body weight per day, for example, between 30 and 300 µg / kg per day, preferably between 50 and 150 µg / kg per day. The desired dose can be administered all at once, but also divided into two, three, four or more subdoses per day.

Although it is possible in a therapy to administer the TNFa inhibitor according to the invention as a crude protein, this preferably takes place as a pharmaceutical preparation.

The invention further relates to a pharmaceutical composition containing a TNFa inhibitor as defined above or a pharmaceutically acceptable derivative thereof together with one or more pharmaceutically acceptable carriers therefor, and optionally other therapeutic and / or prophylactic ingredients. The carrier (s) must be "acceptable" in the sense that they are compatible with ingredients of the formulation and not harmful to those 8900779.

<. - 6 - * that receives.

The inhibitors of the invention can therefore be formulated for parenteral administration (eg, by single injection, by bolus injection or by continuous infusion) and may be presented in unit doses, in ampoules, pre-filled syringes , small infusion lots and multi-dose containers with added preservative. The formulations may take up as suspensions and take solutions in aqueous carriers, and may contain formulants such as suspending, dispersing and / or stabilizing agents. Alternatively, the active ingredient may be in the form of a powder obtained by aseptic isolation or by freeze-drying a solution prepared just before use with a suitable carrier, eg sterile pyrogen-free water.

The TNFa inhibitor according to the invention can also be used in combination with other therapeutic agents, for example with other cytokines or inhibitors thereof.

Thus, the invention also provides a combination of the above-defined TNFa inhibitor or pharmaceutically acceptable derivative thereof with another therapeutically active substance, for example, with another cytokine or inhibitor thereof.

The proteins of the invention can be prepared by isolation from natural sources and purification and, where appropriate, followed by chemical modification, but they can also be prepared by methods known in the field of protein synthesis, or by recombinant DNA- techniques.

Thus, another aspect of this invention is a method of preparing the tumor necrosis factor α inhibitor according to the invention by purifying material of natural origin, in particular from urine of patients with fever.

Such purification includes, for example, the steps of concentrating the crude urine, precipitating crude TNFaR from that urine, and fractionating separating the TNFaR from other proteins in this precipitate, for example, by ion exchange chromatography, gel filtration chromatography, hydrophobicity chromatography , immunoabsorption and affinity chromatography on immobilized TNFa.

The TNFa inhibitor of the invention can also be found 8900779.

.- 7- from human tissue containing macrophages, for example from lung washings and human liver extracts, from which it can be obtained by the standard purification techniques already mentioned above.

The TNFaR according to the invention isolated from nature or made by recombinant techniques can be purified in a series of steps, as already mentioned above. After each purification, the presence and purity of the TNFaR can be measured with a cytotoxicity test in the presence of actino-10 mycine D with a TNF sensitive cell line L929, as described by G. Nedwin et al. (Loc.cit.).

In a preferred embodiment of the preparation method, the TNFaR is first isolated from untreated urine from patients with fever (> 38.5 ° C) free from urinary tract infections, using a standard concentration technique, eg ultrafiltration. A crude fraction can then be precipitated from the raw urine by adding ammonium sulfate to a concentration of 80% at 4 ° C with stirring. Preferably, the ammonium sulfate is added stepwise and the material precipitated at lower concentrations (eg 40%) is discarded. The ammonium sulfate can be removed by dialysis and the fraction obtained then purified by a variety of chromatographic techniques to remove other proteins.

For example, the TNFaR concentrate can be purified by ion exchange chromatography which separates the proteins into differences in electrical charges, reflecting the acid / base properties of those proteins. Suitable materials for anion exchange chromatography are aminoethyl cellulose derivatives, for example quaternary aminoethyl cellulose (QAE-30 cellulose) or diethylaminoethyl cellulose (DEAE cellulose), which are widely available. The anion exchange column must have been previously equilibrated with a suitable buffer such as Tris.HCl (which may also contain a metal scavenger such as EDTA). Retained material can be removed from the column with saline (for example, with the equilibrium buffer to which 0.8 M NaCl has been added).

Suitable materials for cation exchange chromatography are cellulose derivatives such as carboxymethyl (CM) cellulose or Sulphopropyl Sepharose (from Pharmacia in Uppsala, Sweden). The 8900779.

Column should be equilibrated with a suitable buffer such as sodium acetate and bound material can be eluted with this equilibrium buffer containing, for example, 0.5 M NaCl.

The active fractions are further purified by affinity chromatography on human TNFa coupled to a suitable matrix, for example Mini-Leak Agarose (Kem En Tec of the Biotechnology Corp. in Denmark). The column must be buffered, for example with a 0.8 M phosphate buffer with pH = 8.6. Active groups that do not bind to the TNFa should be blocked with an ethanolamine.HCl buffer with pH = 8.5. That column must also be equilibrated with a suitable buffer, for example with Tris.HCl containing NaCl and the TNFaR is eluted with an acidic glycine buffer (pH = 3.5). The eluted fractions must be immediately brought to a pH = 7.0, for example by adding the free base Tris.

The pooled active fractions are preferably lyophilized before undergoing final purification by rapid reverse phase liquid chromatography (FPLC). Before placing on the FPLC column, the freeze-dried TNFaR fraction must be buffered with a suitable buffer such as trifluoroacetic acid, heptafluorobutyric acid or acetic acid.

Elution of the TNFaR from the FPLC column can be done by conventional techniques, for example with the aforementioned suitable buffers, to which an alcohol such as n-propanol has optionally been added.

The eluted fractions must be buffered immediately, for example with ammonium dicarbonate, and then lyophilized. The TNFaR is then in a substantially homogeneous form, suitable for determining the biological activity and for a suitable formulation for therapeutic use.

As an alternative, the invention therefore also relates to a protein which selectively inhibits the activities exerted via TNFa and which has been obtained in the manner just described or which is identical to such a preparation.

The ability to homogeneously purify the TNFaR of this invention has made it possible to determine its amino acid sequence, at least at the N-terminus. This knowledge of the sequence aids in the cloning of a gene encoding the TNFaR, resulting in the production of large amounts of 89 00773.

- 9 - TNFaR in pure form for further biological research and ultimately for therapeutic use. In the homogeneous TNFaR of this invention, the amino acid sequence can be determined by conventional techniques, for example, with the commercially available automatic gas detection device or with ninhydrin. The first 17 amino acids from the N-terminus are:

Asp-Ser-Val-Cys-Pro-Gln-Gly-Lys-Tyr-Ile-His-Pro-Gln-Cys-Asn-Ser-Ile (determined with an automatic sequencer, model 477A of Applied 10 Biosystems).

Furthermore, it is believed that the following three amino acids provide a glycosylation site and thus the chain continues with Asn-Ser-Thr-Lys.

It will be appreciated that a possible method for obtaining large amounts of TNFaR in pure form is by the recombinant DNA techniques known in the art. However, the successful application of such techniques requires not only accurate measurement of the recombinant TNFaR or its activity, but also that both the natural and the recombinant product can be homogeneously purified.

Yet another aspect of this invention relates to a method for making the tumor necrosis factor inhibitor of the invention or a derivative thereof by expressing a DNA sequence encoding this inhibitor in a suitable transformed host. Such a method involves culturing that host transformed with recombinant DNA molecules containing DNA sequences encoding this inhibitor and incorporated into a suitable vector.

Suitable eukaryotic and prokaryotic hosts can be, for example, strains of bacteria, yeasts, fungi and animal cells (including insect cells) and also plant cells in tissue. Particularly preferred host cells are those of yeasts, E. coli and those of animals.

Expression to a protein inhibiting tumor necrosis factor is achieved by culturing the transformed host in an appropriate medium. Typically, such a medium will contain a nitrogen source such as ammonium sulfate, a source of carbon and energy such as glucose or glycerol, trace elements and the fac 8900779 required for growth of those special host cells.

- 10 - contain tower. The exact culture conditions will depend on the host selected; for example, for E. coli, aerobic submerse fermentation at about 37 ° C is preferred.

In addition, expression can be elicited, for example, by adding some substance or by setting conditions under which the promoter system of this vector is activated.

Depending on the host, the TNFa inhibitor can be formed in granular inclusion bodies 10 which, after lysis of the cells, can be recovered by differential centrifugation; they can then be solubilized in the usual manner and purified by the methods described here for purifying TNFaR from urine. But the TNFa inhibitor can also be in solution in the cytosol, secreted in the periplasmic space or quite simply in the culture medium.

The host cells are transformed by recombinant DNA molecules containing a DNA sequence encoding a TNFa inhibitor inserted into an expression vector. Such expression vectors can consist of segments of chromosomal, non-20 chromosomal and synthetic DNA strings, such as the various ones. known derivatives of SV-40 and known bacterial plasmids, for example the "natural" plasmids such as ColEl, pSCIOI or pRSF2124 and phage DNA, or "artificial" plasmids (built in vitro) such as pBR322, pMB9 and pAT153. Phage DNA includes, for example, the numerous derivatives of phage λ and other DNA phages, eg M13 and other filamentous single stranded DNA phages. The vectors useful in yeast include the 2β plasmid and those useful in eukaryotic cells such as animal cells include those containing the adenovirus SV-40 and retrovirus.

Such expression vectors may also be characterized by at least one sequence controlling expression which may be operably linked to the TNF inhibitor DNA sequence to control expression of the cloned DNA sequence. Examples of useful sequences for controlling expression are the lac, trp, tac and trc systems, the major operator and promoter regions of phage λ (such as the P promoter shown below

Control of the thermolabile ts cl857 repressor state), the control region of the fd coat protein, the glycolytic promoters of yeast (e.g. the promoter of 3-phosphoglycerate kinase), the 40 promoters of yeast acid phosphatase (e.g. Pho 5), promoters 8800773.

- 11 - of the α-factors controlling yeast pairing, and the promoters derived from polyoma, adenovirus, retrovirus and monkey virus.

In addition, such expression vectors may contain various sites for inserting DNA strings for the TNFa inhibitor 5 of this invention. These sites are characterized by the specific restriction endonuclease that cleaves them there. Such splitting sites are recognized by those skilled in the art. The expression vector, and in particular the site selected for inserting the selected DNA fragment and linking it operatively to a sequence controlling expression, is determined by a variety of factors including the number of susceptible enzyme sensitive to a particular restriction enzyme. placement, size of the desired protein, contamination or binding of the desired protein by the host cell proteins which may be difficult to remove during purification, location of start and stop codons, and other factors known to those skilled in the art. The choice of a vector and insertion site for a DNA sequence is therefore determined by considering all these factors, which in a given case do not all count equally.

Likewise, not all host / vector combinations will work equally efficiently in expressing the DNA strings of this invention. The choice is made depending on a range of factors, including host and vector compatibility, the ease with which the desired protein is obtained, expression properties of the DNA strings and the operatively linked control strings, and any necessary modifications of the protein after that expression.

The DNA arrays of the invention encoding proteins with TNFα inhibitory activity can be isolated by searching various DNA libraries for such DNA arrays using a series of DNA probes. The DNA probes can be made from the purified natural protein used to establish the amino acid sequence. For example, the purified natural protein can be prepared from the urine of fever patients as indicated above. Degenerate DNA arrays encoding various parts and fragments of the amino acid arrays, e.g. in combination with span probes, are used to design the DNA probes.

Various DNA libraries are therefore searched for DNA 900779.

Sets encoding the TNFa inhibitors of the invention.

Such libraries include the chromosomal gene banks and the DNA and cDNA libraries made from cell lines or tissues that have been shown to make TNFα inhibitors, such as alveolar macrophages and liver tissue. The search can be done by direct immune expression, for example in Xgtll or the like systems, or, in case the TNFaR-making cell has been identified, by identification of the TNFaR-specific mRNA by direct expression in Xenopus oocytes.

A variety of conventional cloning and selection techniques can be used to detect and identify DNA arrays encoding the TNFα inhibitors of this invention upon expression in a suitable eukaryotic or prokaryotic host cell. These read DNA strings can themselves serve as probes for the search for other DNA strings encoding TNFa inhibitors and can be used in appropriate recombinant DNA molecules to transform appropriate eukaryotic or prokaryotic host cells so that they then encode the encoded Start producing TNFaR.

An aspect of the invention also relates to the single and double stranded DNA sequences encoding TNFaR, vectors containing such sequences and suitable for transformation into a host, and host cells transformed with such DNA sequences.

Also within the scope of this invention is any protein with selective TNFα inhibitory activity made by expressing a DNA sequence encoding such a protein in a host transformed therewith. TNF inhibitors according to the invention thus made will thus be identical to the native TNFaR or contain one or more omissions, substitutions, insertions, reversals or additions of allele or other origin, but at least 80% and preferably for be at least 90% homologous to the native TNFaR and have essentially the same biological properties. In particular, a TNF inhibitor according to the invention can have an N-terminal methionine. Also, for example, the DNA sequence of the invention encoding TNFaR in an expression vector can be fused with a portion of a DNA sequence encoding a eukaryotic or prokaryotic polypeptide to support expression of 6900779.

- the DNA sequence encoding TNFaR or for its secretion, maturation or purification; the fused polypeptide can be removed intra- or extracellularly by known techniques or the TNFaR can be used in conjunction with the fused polypeptide.

The TNFaR made by culturing eukaryotic or prokaryotic host cells transformed with TNFaR-encoding DNA sequences can then, after purification, be used in pharmaceutical compositions of the invention.

It will be appreciated that the TNFaR of the invention will be a glycoprotein in animal cell production. Pro-karyotic expression systems, however, will yield a protein in an unglycosidated state. In addition, the glycosidated protein can be substantially completely glycosidated by techniques known in the art, for example, using endoglycosidases. The invention is further illustrated by the following non-limiting examples. All temperatures are in ° C and all concentration percentages are by weight by volume.

Determination of TNFa inhibition The percentage of TNFaR activity in the fractions described in Examples 20 was determined assuming that the absorbance of actinomycin D (abbreviated to "act D") mouse cells L929 corresponds to 100% inhibition, while the absorbance of cells cultured with actinomycin D and TNFa corresponds to maximum mortality, i.e. 0% inhibition by TNFa. The TNFa used in these experiments was recombinant human TNF (rhTNF) made in E.coli as described by A. Marmenout et al. In "Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor", Eur. J. Biochem. 152 81985), 515. The percentage of TNFα-30 inhibition was thus calculated in the determination of cytotoxicity according to the formula (I) (ext, with act. D + rhTNFa + TNFaR) χ - (ext, with act D + rhTNFa) _ (ext. with act D) - (ext. with act D + rhTNFa) 35

Example 1

Preparation of TNFaR from urine a) Concentrating the protein from human urine * 900779.

- 14 -

15 liters of urine were obtained from a group of five patients who had not yet undergone any treatment. Two patients suffered from small cell carcinoma, one from malignant histocytosis, one from polymyocitis and one from sepsis. All had a high fever (> 38.5 ° C) but were free from urinary tract infections. The urine was concentrated at 4 ° C in a hollow fiber ultrafiltration device from Amicon with a retention threshold of approximately 5 kDa.

b) Precipitation of the protein from the concentrated urine. To the concentrated batch of urine, solid ammonium sulfate was added at 4 ° C with continuous stirring to a degree of saturation of 40%. The resulting precipitate was removed by centrifugation and discarded, and the saturation degree was brought to 80% in the supernatant by adding more ammonium sulfate. By centrifugation, a sediment was obtained which was resuspended in 150 ml of 20 mM sodium phosphate (pH = 7.2) and 150 mM NaCl. The ammonium sulfate was removed by dialysis at 4 ° C against 10 mM Tris.HCl containing 2 mM EDTA and 5 mM benzamidine dHCl (pH 7.4).

C) Identification of the TNFaR action.

The semi-purified concentrate of 1 (b) was tested for cytotoxicity with the TNF-sensitive cell line L929 in the presence of actinomycin D. With a 1:20 dilution of the semi-purified solution, a total inhibition of the cytotoxic effect of rhTNFa was determined; the absorbance at 570 nm was equal to that measured in the presence of actinomycin D alone, i.e. 1.5.

Furthermore, an inhibitory effect was found at dilutions up to 1: 160 (E ^ q = 0.83), while the blank value with rhTNFa at a final concentration of 0.2 ng / ml in the presence of actinomycin D was lower (E57q = 0.73), so that 50% inhibition was observed at a dilution of about 1: 100 (E ^ q = 1.10). The TNFaR had no effect on cell viability when tested without actinomycin D.

D) Comparison of the effect of TNFaR on the cytotoxicity caused by TNFa and β

A cytotoxicity test was performed on the TNF-sensitive cell line L929 in the presence of actinomycin D at a range of TNFa and TNFB concentrations. The semi-purified solution of 1 (b) was tested in dilutions 1:20, 1:50 and 1:80. Blank tests were performed by omitting TNFa and TNF8 in the absence of the inhibitor. The results are shown in Table 1 and show that the inhibitor of the invention has the TNF8-acting cytotoxicity ranging from about 50% to only 2% inhibition of the TNFa with increasing TNFB concentration.

Table 1

Final concentration of TNF Es7n form of L929 cells 10 (a or 8) (pg / ml) cytokine

Added to L929 cells treated with actinomy that was treated in the Final Dilution Cine D cells were 1/1 1/20 1/50 1/80 0 0> 1.90> 1.90> 1.90> 1.90

15 10 α NB NB NB NB

8 1.16 1.66 1.46 1.39 20 α 1.30> 1.90 1.72 1.68 β 1.02 1.51 1.21 1.22 50 α 1.02> 1.90 1.72 1.68 20 0 0.65 1.03 0.84 0.68 100 α 0.73 1.78 1.69 1.51 β 0.37 0.71 0.59 0.52 250 α 0 .38 1.70 1.70 1.33 0 0.24 0.44 0.31 0.24 25 500 α 0.30 1.52 1.49 1.11 8 0.17 0.30 0.26 0 , 18 1250 α 0.19 1.09 1.10 0.76 8 0.11 0.13 0.19 0.13 2500 α 0.06 1.03 0.80 0.47 30 _._ 8_NB NB NB ΝΒ

Example 2

Gel filtration of TNFaR from urine

The semi-purified TNFaR of Example 1 (b) was purified by gel filtration chromatography at 4 ° over a column

Sephacryl S-200 (from Pharmacia de Uppsala, Sweden) measuring 0.9 x 60 cm, which was equilibrated with 50 mM Tris.HCl buffer (pH = 7.4) containing 100 mM NaCl. 0.8 ml (20 l of protein) of this fraction was applied to the column and eluted with the same buffer at a flow rate of 5.4 ml / hour. 1.35 ml fractions were collected and examined for TNFaR activity. This inhibitory effect appeared in a single peak. The inhibitory activity had an apparent molecular weight of 40 to 60 kDa (see Figure 1).

8800779.

- 16 -

Example 3

Chromatographically focusing TNFaR from urine

The semi-purified TNFaR of Example 1 (b) was chromatographed at 4 ° on a pre-packed column of 5 Mono-P (HR 5/20, 5 x 200 mm) (from Pharmacia in Uppsala, Sweden) containing a 25 mM Bis Tris buffer, the pH of which was equilibrated with imino-diacetic acid (from Fluka of Buchs, Switzerland). A sample with 30 mg of protein was applied to the column and eluted with "poly buffer 74" containing iminodiacetic acid (pH = 4.0). 1 ml fractions were tested after dilution 1:10 for their effect on the cytotoxicity of 0.2 ng / ml rhTNFa in the presence of 1 x xg / ml actinomycin D. The actual pH of each fraction from the column was measured with a pH meter; the largest amount of TNFaR activity was in the fractions released with a pH between 5.5 and 6.1 (see Figure 2). This therefore corresponds to the isoelectric point of the TNFaR protein.

Example 4

Ion Exchange Chromatography of TNFaR from Urine The semi-purified TNFaR of Example 1 (b) was purified by anion exchange chromatography at 4 ° over a 2.6 x 20 cm column of DEAE-Sephadex (from Pharmacia in Uppsala, Sweden) which had been equilibrated with a 10 mM Tris.HCl buffer (pH = 8.0) containing 2 mM EDTA. Bound material was eluted from the column with the same buffer containing 0.8 M NaCl.

8.0 ml fractions were collected and tested for TNFaR activity and the inhibitory fractions were pooled (160 ml) and dialyzed against 4x2 liters of 10 mM sodium acetate buffer with pH = 5.0.

The TNFaR was further purified by cation exchange chromatography at 4 ° over a 0.8 x 15 cm Sulphopropyl-Sephadex column (also from Pharmacia in Uppsala, Sweden) equilibrated with a 10 mM sodium acetate buffer with pH = 5 , 0. Bound material was eluted from the column with the same buffer containing 0.5 M NaCl. 7.5 ml fractions were collected and tested for TNFaR activity and the inhibitory fractions were pooled and concentrated 20-fold in an Amicon ultrafiltration device with a separation threshold of approximately 10 kDa.

8306779.

- 17 -

Example 5

Gel filtration of the TNFaR from urine

The TNFaR concentrate of Example 4 was further purified by gel filtration chromatography at 4 ° over a column of Sephacryl S-200 measuring 2.6 x 100 cm (also from Pharmacia in Uppsala,

Sweden) which had been equilibrated with a 50 mN Tris.HCl buffer with pH = 7.4. 200 mg of the product of Example 4 was loaded onto the column and eluted with the equilibrium buffer at a flow rate of 27 ml / h. 9.0 ml out-10 fractions were captured and tested for TNFaR activity, and the inhibitory fractions were pooled. As shown in Figure 4, the column was calibrated with dextran blue (DB) of 2000 kDa, bovine serum albumin (BSA) of 67 kDa, ovalbumin (OA) of 43 kDa, α-chymo-trypsinogen-A (aCT) of 25 kDa and rubonuclease A (RNase) at 13.5 kDa.

Example 6

Affinity chromatography of TNFaR from urine

An affinity column for TNFa was prepared by coupling 1.0 mg of recombinant human TNFa in 0.8 M potassium phosphate buffer 20 with pH = 8.6 to agarose "Mini Leak" (from Kern En Tec

Biotechnology Corp. in Denmark). The remaining active groups were blocked by incubation in 0.1 M ethanolamine. HCl buffer of pH = 8.5. The gel was washed with 3 x 50 ml 50 mM Tris.HCl buffer with pH = 7.4 containing 100 mM NaCl.

15 ml of the TNFaR fraction from Example 5 was added to this column and eluted with a 0.2 M glycine.HCl buffer of pH = 3.5.

1.0 ml fractions were collected, the pH of which was immediately adjusted to 7.0 with 1 M Tris (5 to 40 µl) and then assayed for TNFaR activity.

30 Example 7

Reverse phase chromatography of TNFaR from urine

The TNFaR fractions of Example 6 were lyophilized, taken up in 2.0 ml 0.1% trifluoroacetic acid and applied to a 5 x 20 cm column ProRPC for reverse phase chromatography (also from Pharmacia in Uppsala, Sweden), which had 0 1% trifluoroacetic acid was equilibrated. Bound material was eluted with a gradient from 0 to 100% acetonitrile in 0.1% trifluoroacetic acid solution, at a flow rate of 0.3 ml / min. 10 µl 0.5 M ammonium bicarbonate 8900778 was added to each 0.75 ml fraction.

- 18 - and then lyophilized.

In reversed phase liquid chromatography, only one major peak appeared corresponding to the TNFaR activity. The lyophilized fractions containing this activity were dissolved together in 10 mM Tris.HCl buffer with pH = 7.4 containing 2 mM EDTA and by electrophoresis on sodium dodecyl sulfate polyacrylamide gel according to U. Laemmli et al. method described in Nature 277 (1970) 680. The TNFaR was shown to elute with a molecular weight of 33 kDa (see Figure 4). Samples run under 10 non-reducing conditions were tested for dilution of 1:10 in the presence of 0.15 mg / ml rhTNFa for TNFaR activity for L929 cells. The activity against the rhTNFa ran at an apparent molecular weight similar to that of the 33 kDa band under reducing conditions.

15 Example 8

Establishing the amino acid sequence in TNFaR

The TNFaR fraction isolated from reverse phase chromatography was concentrated in vacuo and placed on a conditioned sequence filter. The protein was tested with a Model 477A sequencer from Applied Biosysterns.

Fractions from the sequence cycles were evaporated to dryness and suspended in Ν, Ν-diisopropylethyl ammonium acetate / acetonitrile before being injected into an HPLC column for identification.

The first 17 amino acid residues from the N-terminus 25 were identified; these were: Asp-Ser-Val-Cys-Pro-Gln-Gly-Lys-

Tyr-Ile-His-Pro-Gln-Cys-Asn-Ser-Ile. Furthermore, it is believed that the following three amino acids form a glycosylation site and that the sequence thus continues with Asn-Ser-Thr-Lys. This sequence is not significantly homologous to any amino acid sequence in the NBRF's amino acid sequence database (November 1988).

Example 9

Demonstrate that the TNFaR is a protein (a) Time and temperature dependent denaturing

The TNFaR of Example 2 purified over Sephacryl S-200, obtained by pooling the active fraction tubes, was heated to 56 °, 75 °, and 95 °. TNFaR activity was measured after 10, 20 and 60 minutes and compared to that of untreated samples. The percent activity was calculated according to formula I. The results shown in Table 2 below show 8900779.

- 19 - that the operation of the TNFaR decreases in a time and temperature dependent manner.

Table 2

Inactivation by heating 5 Temperature (° C) Time (min) Percentage TNFaR activity 56 10 100 20 100 60 93 75 10 60 10 20 26 60 15 95 10 27 20 10 __60_ 13_ 15 (b) Trypsin sensitivity

To the pooled fractions of TNFaR purified over Sephacryl S-200 from Example 2, 500 µg of trypsin (from Sigma of St. Louis, MO) was placed in 0.2 M Tris.HCl buffer (pH = 8.0). After incubation at 37 ° C for 4 hours, an additional 500 µg of trypsin 20 was added and filtration was continued for an additional 20 hours. Now the reaction was stopped by adding 2 mg of soybean trypsin inhibitor (also from Sigma of St. Louis, MO). The TNFaR activity of this trypsin-digested lot and of a blank lot were determined in a final dilution of 1:20 on rhTNFa in the presence of actinomycin D on L929 cells; the result was calculated according to formula I. The results are shown in Table 3 below.

Table 3

Inactivation by trypsin

Conditions Percentage E_7_ 30 _TNFaR operation_

Buffer only 0 0.71

Trypsin + trypsin inhibitor 0 0.70 from soybeans in buffer

Partially purified over 61 1.46 35 Sephadex S-200

Partly over 23 1.03

Sephadex S-200 purified and trypsin digested 40 (c) Urea treatment

The solution of the TNFaR purified over S-200 of Example 2 was made 2 M urea and then dialyzed extensively at 4 ° against phosphate buffered saline 8900779.

- 20 - which was also 2 M of urea. This dialysis was repeated just before the biological assay. The TNFaR activity was found to be unaffected, indicating that no protein-bound low molecular substance is involved in this inhibition.

5 Example 10

Demonstration of competitive inhibition

The TNFaR of Example 2 purified over Sephacryl S-200 was used in a 1:10 dilution against increasing amounts of rhTNFa with L929 cells. There was an inverse relationship between the amount of rhTNFa present in the experiment and the degree of inhibition (see Figure 3). Thus, the inhibitory activity is overcome competitively by increasing the amount of rhTNFa.

Example 11

Inhibition of TKFa-enhanced PGE1 formation by skin fibroblasts

Human skin fibroblasts were seeded at a concentration of 2.0 x 10 cells per well and grown for 48 hours. As a blank, cells were then stimulated with DMEM buffer to which 10% FCS had been added. Cells were also stimulated with 20 rhTNFa at concentrations of 0.5 to 5 mg / ml, and the effect of the TNFaR of Example 5 was studied in that buffer in three dilutions (1:20, 1:50 and 1:80) . After incubation for 72 hours, the formation in the supernatants was measured by radioimmunoassay using a serum against PGE (see J. M. Dayer et al. in 25 J. Clin. Invest. 67 (1979) 1386).

The results reported in Table 4 below show that the ability of rhTNFa to stimulate PGE 1 formation by dermal fibroblasts was inhibited by TNFaR in all three dilutions. At the 1:80 dilution, the inhibition by TNFaR was partially overcome by increasing the concentration of rhTNFa.

35 $ 900779.

- 21 -

Table 4

Concentration of PGE Formation by Human Fibro-RhTNF on Human Blasts (ng / ml) - Fibroblasts. , (pg / ml) Dilution of the TNFR_ 5 _not_1: 80_1 ^ 50_1: 20 0 50.6 ± 7.4 88.8 ± 5.6 103.0 ± 8.9 111.0+ 9.4 500 160, 0 ± 14.1 126.0 ± 9.3 115.9 ± 6.6 113.9 ± 7.1 2000 331.7 ± 28.4 217.2 ± 10.7 156.7 + 10.7 115, 3 + 21.3 5000 381.7 ± 19.6 257.2 ± 13.7 253.1 ± 21.2 221.6 ± 16.0 10

Three different experiments were performed with the same fibroblast strain. Buffer or buffer containing TNFaR were incubated with different concentrations of rhTNFa. The FGE1 formation by the cultured skin fibroblasts was measured after 3 days. The figures given are the means of three cultures ± SFGem (n = 3).

Example 12

Determination of the degree of inhibition of rhTNFa binding

Recombinant-human TNFa was iodinated by the iodogen method of Fraker and Speck jr., Biochem. Biophys. Res. Comm.

80 (1978) 849. The specific activity of the -TNFa

was 2.2 x 10 rpm / ng and gave electrophoresis on SDS-PAGE

a single band with a molecular weight of 17 kDa. The human 6 macrophage cell line U937 was incubated in 10 cell aliquots for 2 hours at 4 ° C in 200 µl medium containing RPMI 1640 (from Gibco, Paisley, Scotland) supplemented with 100 µg / ml streptomycin (100 U / ml penicillin), 1.0% glutamine and 10% fetal calf 125 serum, and additionally containing 0.04% sodium azide and 0.5 ng of I-TNFa. Inhibition of binding was done by adding TNFaR in various dilutions (1:20, 1: 200 and 1: 2000).

Nonspecific binding was measured in the presence of a 100-fold excess of unnoticed rhTNFa, and the free radioactivity was separated from the bound Ι-TNFa by centrifugation through an oil mixture, as described by Robb et al. In J. Exp. Med. 154 (1981) 1455. Cell-bound I / TNFa 35 was measured in a γ counter (from the LKB in Bromma, Sweden) and the percent binding inhibition was calculated according to formula II.

6900779.

- 22 -

Percentage of binding inhibition = "rpm with TNFaR - rpm by non-specific 100 x 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - By - binding

Example 13 * 125

Effect of TNFaR on binding of I-TNFa to U937 cells U937 cells were pre-incubated at 20 ° C for 1 hour in the culture medium of Example 12, either with I-TNFa 125 alone or with I TNFa and a 100- fold excess not marked rhTNFa. The U937 cells were then centrifuged at 4 ° C and washed three times with 50 ml of phosphate-buffered saline. The cells incubated alone with I-TNFa were divided into 15 four aliquots and either alone with buffer or with three solutions. incubated several dilutions (1:20, 1: 200 and 1: 2000) of the TNFaR of Example 5.

With these three dilutions, the specific binding 125 of I-TNFa to U937 cells at 4 ° C was suppressed by 100%, 80% and 35%, respectively. The blank portion, which did not contain TNFaR at all, showed no inhibition. The binding inhibition by the two weaker solutions was found to be increased to 125% and 60%, respectively, when I-TNFa was preincubated in dilutions of 1: 200 and 1: 2000 with the TNFaR before cells were added.

This experiment was repeated with cells pre-incubated with I-TNFa and a 100-fold excess of unlabeled TNFa, so that the percent binding inhibition could be corrected for the nonspecific binding.

30 Example 14

Dissociation of a preformed TNFa complex with U937 125 U937 cells were pre-incubated with I-TNFa for 1 hour as described in Example 12. The cells were centrifuged and washed and incubated either at 4 ° or at 37 ° C, with or without the TNFaR of Example 5. The 125-cell-bound I-TNFa appeared to dissociate faster in the presence of TNFaR than in the absence and this was found to occur in a time and temperature dependent manner (see Figure 6).

8900773 - 23 - *

Example 15 TNFaR is not proteolytic for TNFa 125 Ι-TNFa was incubated with TNFaR for 1 hour at 20 ° with three different dilutions (1:20, 1: 200 and 1: 2000), and also with buffer only. Electrophoresis on SDS-PAGE and auto-radiography revealed the I-TNFct to run as a single band, both in the absence and in the presence of TNFaR, showing that the inhibitor does not cause proteolysis.

Example 16 Effect of the TNFaR on the binding of IL-1 to its receptor

The influence of the TNFaR of Example 5 was investigated on the binding of IL-1 to LAF (lymphocyte activating factor), with induction by both IL-Ια and IL-18. (This test is described by P. Seckinger et al in J. Immunol. 139 (1987) 151541 for a protein that inhibits IL-1.) Both with IL-Ια and IL-18 3, one dose-dependent incorporation of H-thymidine (meaning multiplication of thymocytes) in concentrations up to 200 µg / ml. Addition of TNFaR at levels that inhibit rhTNFa had no significant effect on the IL-1 mediated thymocyte proliferation, demonstrating that the inhibition is specific for TNFa.

The results obtained are illustrated by the accompanying drawings, of which Figure 1 shows the efficacy profile of the gel filtrate over Sephacryl S-200 of crude TNFaR from urine. 1 ml fractions were tested at a dilution of 1:10 in the presence of 1.0 µg / ml actinomycin D for the influence on cytotoxicity of 1.0 ng / µl rhTNFa (o-o). The line (-) represents the absorbance of the fractions at 280 nm. The blocks on the left represent the lysis of the cells, measured by dye uptake in response to actinomycin D (0) and actinomycin D plus hrTNFa (E2) without urine. The molecular weight marks are dextran blue (DB), bovine serum albumin (BSA), ovalbumin (OA), α-chymotrypsino-35 none (aCT), ribunuclease A (RNase) and phenol red (J-red).

Figure 2 shows the activity profile of the chromatographic focusing on a Mono-P column of crude TNFaR from urine. 1 ml fractions were tested at a 1:10 dilution to influence the cytotoxicity of 0.2 ng / ml rhTNFa in 8900778.

- 24 - presence of 1.0 µg / ml actinomycin D (o-o). The line (-—) represents the absorbance at 280 nm of the fractions and (—-—-) represents their pH. The bars are as shown for Figure 1.

Figure 3 shows the reversibility of the TNFaR action. The line (o-o) shows the extinctions measured in the presence of actinomycin D, rhTNFa and TNFaR at 570 nm, the line (· - ·) shows the only in the presence of actinomycin and

rhTNFa measured extinctions at 570 nm again; the bar (Θ) adjusts the absorbance at 570 nm in the presence of actinomycin D alone

(1.0 µg / ml) for.

Figure 4 shows the efficacy profile of a gel filtrate over Sephacryl S-200 of purified TNFaR of Example 5. 9 ml fractions were sterilized and assayed at a 1:50 dilution against 1.0 mg / ml rhTNFa, in the presence of 1.0 µg / ml actinomycin D, the cytotoxicity at L929 being the criterion (oo) . The line (-) represents the absorbance of the fractions at 280 nm. The bars are as defined for Figure 1.

Figure 5 shows the SDS-PAGE electrophoresis of purified TNFaR of Example 7. This chromatography was performed as described by U. Laemmli et al. (Loc. Cit.). Samples were applied to a 15% polyacrylamide gel with a 3% gel in the head, and these gels were stained with silver as described by C. Merril et al. In Proc, Natl. Acad. Sci. USA 76 (1979) 4335.

Samples run under non-reducing conditions were tested for biological activity by cutting 2 mm slices from the gel and eluting the proteins overnight with 200 µl 10 mM Tris.HCl buffer of pH 7.4 containing 2 mM EDTA. contained. The 30 fractions were assayed on L929 cells in a 1:10 dilution in the presence of 0.15 ng / ml rhTNFa.

Figure 6 shows the effect of TNFaR on the binding of 125 I-TNFa to U937 cells. The TNFaR was incubated with the U937-35 cells in three different dilutions in the presence of I-TNFa, as described in Example 13. The open squares (D - □) represent the incubation in the presence of TNFaR and the open triangles (Δ-Δ) represent the blanks.

Closed symbols represent a 30 minute pre-incubation of the 125 TNFaR at 20 ° with I-TNFa before the cells were added.

8900779.

- 25 -

Figure 7 represents the dissociation of a preformed TNFα complex with U937. The U937 cells were pre-incubated 125 with Ι-TNFa, washed and then with TNFaR as described in Example 14, either at 4 ° (Q-0) or at 37 ° C (I-). At the indicated times, the hg cellular radioactivity was measured and the percentages of specific binding were determined. In the graph, the value obtained for the blank without inhibitor is subtracted from the results obtained at the two temperatures; 100% therefore corresponds to the 10 result without TNFaR added.

8900779.

Claims (23)

1. A protein that inhibits the actions exerted by tumor necrosis factor α (TNFa) but does not inhibit other proteins that share some but not all biological actions with TNFa. 2. Protein which selectively inhibits the activity of tumor necrosis factor α and which exhibits one or more of the following characteristics: (a) a molecular weight between 40 and 60 kDa, determined by chromatography on a molecular sieve, (b) an isoelectric point between 5.5 and 6.1, determined by chromatographic focusing, (c) inhibition of the standard TNF assay of the differential cytotoxicity for mouse cells L929 treated with actinomycin D, (d) inhibition of the TNF-mediated release of PGE 1 from human fibroblasts and synovial cells, (e) the inhibition of binding of TNFa to Ü937 cells, as evidenced by suppression of the incorporation of radioactive 125 labeled TNFa (I-TNFa), (f) it on a temperature-dependent promoting dissociation of a complex previously formed from TNFa and U937 cells, (g) not breaking down TNF by proteolytic cleavage, (h) not inhibiting the binding of IL-1 and its receptor, 125 b similar to the uptake of radioactive IL-1 (I-IL-Ια) by the mouse thymoma subline EL4-6.1. The protein of claim 2, however, having a molecular weight of about 33 kDa, as determined by electrophoresis on SDS-PAGE. Protein according to claim 2 or 3, with at least the characteristics (a) and (b) and with one or more of the characteristics (c) to (h). 5 Asp-Ser-Val-Cys-Pro-Gln-Gly-Lys-Tyr-Ile-His-Pro-Gln-Cys-Asn-Ser-Ile-Asn-Ser-Thr-Lys. Protein according to claim 2 or 3, with all characteristics (a) to (h). Protein according to any one of claims 1 to 5, which corresponds to a naturally occurring TNFa inhibitor. Protein according to any one of claims 1 to 6 with the amino end sequence at 8900779.-amino amino acid sequence: Asp-Ser-Val-Cys-Pro-Gln-Lys-Tyr-Ile-His-Pro- Gln-Cys-Asn-Ser-Ile. Protein according to claim 7 with the following amino acid sequence at the amino end: Protein according to any one of claims 1 to 8 with one or more omissions, substitutions, insertions, inversions or additions of allele or other origin in the series of amino acids, which however is at least 80% homologous with the mother protein and in essentially the same biological properties as that mother protein. The protein of claim 9 which is at least 90% homologous to the mother protein. Protein according to any of the preceding claims in a substantially homogeneous form. Protein according to any of the preceding claims, which is a recombinant protein. Protein according to any one of claims 1 to 12, which is a glycosylated protein. Protein according to any one of claims 1 to 12, which is in a substantially non-glycolysed state. Exogenous DNA containing a nucleotide sequence encoding a protein according to any one of claims 1 to 11. A cDNA containing a nucleotide sequence encoding a protein according to any one of claims 1 to 11. A recombinant expression vector containing DNA according to claim 15 or 16. 18. A host cell transformed with an expression vector according to claim 17. A method of preparing a TNFa inhibitor, for which one cultivates a cell according to claim 18 and isolates the TNFaR protein. 20. Recombinant protein prepared by the method of claim 19. 21. A method of preparing a TNFaR protein, comprising: (a) concentrating urine from patients with fever, (b) precipitating with ammonium sulfate, (c) anion exchange chromatography, 8900779. -58 - (d) cation exchange chromatography, (e) gel filtration, (f) affinity chromatography and (g) reverse phase liquid chromatography. Protein characterized in that it is substantially identical to the protein obtained by the method of claim 21. A pharmaceutical composition containing a TNFa inhibitor according to any one of claims 1 to 14 or 22 or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier therefor. -ooooo- 8300779. -23 "Improvement of errata in the description associated with patent application no. 89.00779 Dutch proposed by applicant dated May 1, 1989 .________________________ In the third line of claim 7 (second line of that page) insert between "Gin" and "Lys" in "-Gly-" This improvement is based on page 18 of page 4 and line 8 of page 9. JKr / JvdB 8900779?