DE3913101A1 - DNA encoding TNF binding protein and TNF- receptor - Google Patents

Abstract

DNA (I) coding for a tumour necrosis factor (TNF) receptor or fragment with a specified sequence in the figure is new. Also new are (1) DNA (II) encoding TNF binding proteins; (2) recombinant DNA molecules containing (I) or (II); (3) host organisms transformed with the recombinant mols.; and (4) polypeptides, (A1) and (A2), encoded by (I) or (II) respectively. (II) has a choice of 2 N-terminal sequences. Preferably the recombinant mols. are plasmids ADTNF-BP, pADTNF-R, pADBTNF-BP or pADBTNF-R. A1 has the sequence in the figure.

Description

The invention relates to DNA, in particular to recombinant DNA for the production of proteins with the ability to bind TNF- α .

Tumor necrosis factor (TNF- α ) was first found in the serum of mice and rabbits infected with Bacillus Calmette-Guerin and injected with endotoxin and recognized for its cytotoxic and antitumor properties (1). It is mainly produced by activated macrophages and monocytes. Numerous cell types that are targets for TNF have surface receptors with high affinity for this polypeptide (2); Lymphotoxin (TNF- b ) binds to the same receptor (3, 4). TNF- α is identical to a factor called cachectin (5), which suppresses lipoprotein lipase and leads to hypertriglyceridemia in chronic inflammatory and malignant diseases (6, 7). TNF- α may be involved in the regulation of growth as well as in the differentiation and function of cells involved in inflammation, immune processes and hematopoiesis.

TNF can exert a beneficial effect on the host organism by stimulating neutrophils (8, 9) and monocytes, as well as by inhibiting the replication of viruses (10, 11). In addition, TNF- a activates the immune system against parasites and acts directly and / or indirectly as a mediator in immune reactions, inflammatory processes and other processes in the organism, the mechanisms of action in many cases are still unclear.

However, the administration of TNF- α (12) may also be accompanied by deleterious phenomena (13) such as shock and tissue damage that can be abrogated by antibodies to TNF- α (14). A number of observations suggest a role of endogenously released TNF- α in various pathological conditions. Thus, TNF- α seems to be a mediator of cachexia, which is used in chronic invasive, eg. B. parasitic diseases can occur. TNF- α also appears to play an essential role in the pathogenesis of gram-negative bacterial shock (endotoxin shock); it may be involved in some, if not all, of the effects of lipopolysaccharides (22). Similarly, TNF has been implicated in tissue damage associated with inflammatory processes in joints and other tissues, as well as in lethality and morbidity of graft-versus-host reaction (GHVR) (15). There was also a correlation between TNF levels in serum and the fatal outcome of meningococcal disease (16).

Furthermore, it has been observed that the administration of TNF- α over a prolonged period causes a condition of anorexia and wasting that has similar symptoms to cachexia associated with neoplastic and chronic infectious diseases (23).

It has been reported to have TNF inhibitory activity Protein from the urine of fever patients reported, from the effect of which is suspected that they rely on a competitive mechanism  Receptor level itself (similar to the effect of the Interleukin-1 inhibitor (17)) (24).

In preliminary experiments of the present invention, a protein which inhibits the biological effects of TNF- α by preventing it from binding to its cell surface receptor by interacting with TNF- α could be identified from dialysis obese uremia patients.

It was found that the TNF- α binding protein also has affinity for TNF- β , this affinity was determined to be about 1/50 of the affinity for TNF- α .

This protein, called TNF- α binding protein or TNF-BP in the following, was further purified to homogeneity. It has a molecular weight of about 30,000, determined by SDS-PAGE, the N-terminal amino acid sequence was compared with Asp-Ser-Vol-X-Pro-Gln-Gly-Lys-Tyr-Ile-His-Pro-Gln- certainly. (In addition, the following N-terminal sequence was detected in traces: Leu- (Val) - (Pro) - (His) -Leu-Gly-X-Arg-Glu-.)

The native TNF-BP was called glycoprotein demonstrated.

It has the following amino acid composition, indicated in amino acid residues per molecule and determined as the mean of a 24- and 48-hour Hydrolysis, determined:  

A content of glucosamine was using Amino acid analysis detected.

TNF-BP forms a complex with TNF- α ; the molar ratio of TNF- α to TNF-BP was determined to be 1: 1. TNF-BP could be detected in the serum, but not in the urine of healthy persons. An elevated titer was determined in the serum and urine of dialysis patients with uremia. The starting material for the purification of TNF- a binding protein is thus the urine of uremic patients under continuous dialysis treatment (hereinafter referred to as dialysis or urinary dialysis).

The binding experiments showed a dose-dependent inhibition of TNF binding to the cell by concentrated dialysis. The possibility of interpreting that the observed decrease in binding could be caused by TNF- α itself or TNF- β , which may compete for binding, in the urine was confirmed by the finding that the reduction in binding by application of TNF- α - and TNF- β antibodies could not be reversed, excluded. It has also been shown that the effect of the inhibitor is not due to its attachment to the cell; Rather, the formation of a complex between TNF- α and TNF-BP could be detected directly. TNF-BP thus differs in its mode of action from the interleukin-1 inhibitor, which competes with interleukin-1 for binding to the cell surface receptor (17). The interleukin-1 inhibitor does not interfere with the action of TNF- α . It has also been demonstrated that the TNF- α binding protein has affinity for TNF- β .

The purification of the TNF binding protein can - optionally after concentration of the dialysis hair in several steps according to usual methods of Protein chemistry be performed, for. B. by means fractionated ammonium sulphate precipitation, Ion exchange and gel permeation chromatography, Adsorption, z. B. on hydroxyapatite or hydrophobic Adsorption, affinity chromatography, reverse Phase chromatography, taking these steps basically in any order, optionally with intervening dialysis or gel filtration, can be performed.  

Pre-purification in two steps by ion exchange chromatography over a DEAE column followed by gel chromatography (Sephadex G-75) resulted in a 62-fold enrichment. The further purification is preferably carried out by means of affinity chromatography by TNF- α bound to solid support material (eg sepharose); finally, if necessary, purified to homogeneity by further chromatographic steps, such as reverse phase chromatography.

The homogenous protein was obtained in highly purified form by concentrating urine from dialysis patients by ultrafiltration, dialyzing the concentrated urine, and first enriching in a first purification step four times by DEAE-Sephacel chromatography. Further enrichment was carried out by means of affinity chromatography by TNF- α bound to Sepharose. The final purification was carried out by reverse phase chromatography (FPLC).

The highly purified protein became the N-terminal Amino acid sequence elucidated.

The occurrence of elevated titers of TNF-binding Protein in both the serum and the urine of Uremia patients can be explained by the fact that Proteins of this size, usually the subjected to glomerular filtration and by Tubulus cells are catabolized at Illnesses with loss of nephron (eg Glomerulonephritis) with diminution of Excretion and endogenous catabolism Accumulate, be accumulated in the blood as well as in the urine (18).  

TNF-BP obviously has the function of a Regulators of TNF activity with the ability to the concentration changes of free, biological to buffer active TNF. TNF-BP is also likely to Influence excretion of TNF by the kidney, because the complex formed with TNF, the one Molecular weight of about 75,000, determined by means Gel permeation chromatography on Sephadex G 75 has, unlike TNF not by the Glomerulus is restrained.

TNF-BP represents one of three main urinary protein components of dialysis patients who have affinity for TNF and elute together with TNF-BP from the affinity chromatography column. However, the other two proteins apparently bind in a manner that does not interfere with the binding of TNF- α to its cell surface receptor.

Based on the results obtained for Biological effect of TNF-BP could be this protein around the soluble part of the Act TNF receptor.

Due to its ability to inhibit the biological activity of TNF- α and TNF- β , the TNF-binding protein is suitable for use in indications where a decrease in TNF activity in the organism is indicated.

TNF-BP can be used for the prophylactic and therapeutic treatment of the human or animal body in indications where a damaging effect of TNF- α occurs. Such diseases are in particular inflammatory as well as infectious and parasitic diseases or shock states in which endogenous TNF- α is released. It also includes pathological conditions that can occur as side effects in the therapy with TNF- α , especially at high doses, eg. Severe hypotension, disorders of the central nervous system. As pharmaceuticals, in particular pharmaceutical preparations for parenteral use into consideration, for. B. in the form of lyophilisates or solutions, optionally together with physiologically acceptable additives, such as stabilizers. Due to its TNF- α binding properties, TNF-BP is also suitable as a diagnostic agent for the determination of TNF- α , eg, as one of the components in radioimmunoassays or enzyme immunoassays, optionally together with antibodies against TNF- α .

Because of its properties, this protein is a pharmacologically valuable ingredient that is made natural sources are not in sufficient quantity can be represented by protein-chemical methods.

There was therefore a need to produce this protein (or related proteins with the ability to bind TNF- α ) by recombinant means, in order to make it available for therapeutic use in sufficient quantity.

It is an object of the present invention to provide DNAs coding for proteins capable of binding TNF- α , in order on their basis to enable the production of recombinant DNA molecules with which suitable host organisms can be transformed To produce proteins with TNF-BP activity.

The object has been achieved according to the invention, in that based on the N-terminal amino acid sequence and amino acid sequences of tryptic peptides, obtained from the highly purified TNF-BP, Hybridization probes were prepared with and Help these probes from a suitable cDNA library a cDNA that is part of the for TNF-BP encoding DNA.

Such a DNA hybridizes with DNAs (or RNAs) coding for TNF-BP or related proteins with the ability to bind TNF- α , or for proteins whose processing gives TNF-BP or TNF-BP related proteins ( Hereinafter, these DNAs (or RNAs) are referred to simply as "TNF-BP DNAs" and "TNF-BP RNAs", respectively). Processing is understood to mean the in vivo cleavage of partial sequences of a protein. In this case, N-terminal cleavage of a signal and / or other sequences and optionally additionally, if it is about the protein to the extracellular soluble part of a receptor, which protrudes with the N-terminal portion of the cell membrane, C- terminal the cleavage of the transmembrane and cytoplasmic region of the receptor-forming part of the protein take place.

Among TNF-BP DNAs (or TNF-BP RNAs) are also cDNAs derived from mRNAs generated by alternative  Splicing (or these mRNAs themselves), incorporated. Under "alternative" splicing is the Removal of introns understood at the of same mRNA precursor different Splice acceptor and / or splice donor sites be used. The resulting mRNAs differ from each other by the whole or partial presence or absence of certain exon sequences, where appropriate can come to a shift of the reading frame.

The subject of the invention is thus to claim 1 defined DNA including all Variants of which are suitable with TNF-BP DNAs or TNF-BP RNAs to hybridize.

The variants mentioned include z. B. those DNA molecules generated by PCR amplification obtained from primers whose nucleotide sequence not exactly with the searched sequence agrees, for example, due to too Cloning provided Restriction interfaces or due to at the amino acid sequence analysis, for example, not unique determined amino acids.

The DNA molecules of the invention can be used in order to convert cDNA libraries from cDNA libraries, containing TNF-BP DNAs. Furthermore you can the DNAs of the invention as Hybridization probes for mRNA preparations can be used to isolate TNF-BP RNAs and out of it z. B. enriched cDNA libraries produce a much simplified and enable more efficient screening. Another one  Field of application is the isolation of the desired DNAs from genomic DNA libraries using the DNAs according to the invention as hybridization probes.

The invention furthermore relates to TNF-BP-DNAs which hybridize with the abovementioned DNA molecules, including their degenerate variants, and modified TNF-BP-DNAs coding for proteins with the ability to bind TNF- α .

The invention further relates to recombinant DNA molecules containing such DNA sequences.

Such recombinant DNA molecules are useful for cloning DNA encoding proteins capable of binding TNF- α as well as for their expression in corresponding host organisms.

The invention furthermore relates to polypeptides, which are encoded by such DNAs.

Because of their ability to bind TNF- α , these polypeptides are useful in the prophylactic and therapeutic treatment of the human or animal body in indications where a deleterious effect of TNF- α occurs.

Since TNF-BP has also been shown to inhibit TNF- β , it may (or the related polypeptides) in appropriate dosage, optionally modified in view of increased affinity for TNF- β , also for inhibiting the action of TNF- β can be used in the organism.

The invention therefore further relates to pharmaceutical preparations containing an effective inhibiting amount of TNF-BP or a related polypeptide with the ability to bind TNF the biological activity of TNF- α and / or TNF- β .

By "modified DNAs" are meant those DNAs which are e.g. By mutation, transposition or addition and by truncation of TNF-BP DNAs, provided that these modified sequences encode proteins capable of binding TNF- α .

In the context of the present invention, the ability to bind TNF- α means the property of a protein to bind to TNF- α in such a way that the binding of TNF- α to the functional part of the receptor is prevented and the effect of TNF - α is inhibited or abolished in the human or animal organism This definition also includes the ability of a protein to bind to other proteins, such as TNF- β , and to inhibit their action.

As well as any modifications of the DNA sequence the selection is made for the expression suitable host organisms, in particular with regard to to this biological property; Furthermore go in the production of recombinant Proteins usual criteria such as compatibility with the chosen vector, processability, Isolation of the protein, Expression characteristics, safety and  Cost aspects in the decision on the Host organism. The choice of a suitable one Vector results from that for the transformation provided host. Basically, all vectors are suitable, the TNF-BP DNAs of the invention replicate and express.

In the case of TNF-BP-DNA, the possible relevance of the two criteria found for the natural protein, glycosylation (see preliminary experiment 12) and high proportion of cysteine residues (see preliminary experiment 9) for the characteristic TNF- α to take account. Therefore, eukaryotes, in particular suitable expression systems of higher eukaryotes, are expediently used for the expression.

Searching cDNA Libraries Using Hybridization probes derived from amino acid sequences short peptides are derived, due to the Degeneration of the genetic code sometimes on bigger difficulties. In addition, it is made more difficult this procedure, when used by a protein, such as As the TNF-BP, is not known in which Tissues it is synthesized. In this case can in case of failure of this method under circumstances can not be determined with certainty whether it is on the choice of an inappropriate cDNA library or the lack of specificity of Hybridization probes is due.

To solve this problem, the procedure was therefore as follows according to the invention: The cDNA library used was a library of the fibrosarcoma cell line HS 913 T, which had been induced with TNF- α and was present in lambda gt11.

To use this library lambda DNA with TNF-BP Sequences became the big one Sensitivity of polymerase chain reaction (PCR) exploited. With the help of this method can from a entire cDNA library an unknown DNA sequence flanked by Oligonucleotides based on known Amino acid part sequences designed and described as Hybridization probes were used. On such longer DNA fragment can be referred to as Hybridization probe, e.g. B. for the isolation of cDNA clones, especially of the original one cDNA clones are used.

The "Polymerase Chain Reaction" ("Polymerase Chain Reaction ", PCR) is a method to DNA enzymatically amplified in vitro. The method based on the repetition of a cycle of the three Stages: heat denaturation of the DNA, binding of Oligodeoxynucleotide primers complementary thereto Sequences and extension of primers with DNA Polymerase, successively under controlled temperature conditions several times is repeated. For PCR amplification of DNA become two oligodeoxynucleotide primers used, which is the DNA segment to be amplified flank. These oligodeoxynucleotides are so designed to be at the opposite Bind DNA strands and are oriented so that the DNA synthesis by the polymerase over between the primer region takes place. This will the amount of this DNA segment in each cycle doubled, because the extended products also complementary to one of the primers and capable of binding it. this leads to  to an exponential accumulation of the specific, DNA fragments flanked by the primers. The use of a thermostable DNA polymerase the bacterium Thermus aquaticus allows the Automation of PCR. Because this enzyme is his Activity even after prolonged incubation at high Temperature (95 ° C), which prevents the denaturation of DNA is necessary, a single addition of the Enzyme for performing many PCR cycles (31).

In particular, the task was as follows solved:

From the highly purified TNF- α binding protein, the N-terminal amino acid sequence as well as the amino acid sequences of peptides obtained by tryptic digestion of the protein were determined.

With regard to their use in the PCR for the production of oligonucleotides, these sequences were selected on the one hand from the N-terminus and on the other hand from a tryptic peptide such that the complexity of mixed oligonucleotides for the hybridization with cDNA is as low as possible. On the basis of these two areas, a set of mixed oligonucleotides was produced in each case, the nucleus derived from the N-terminal region being synthesized corresponding to the mRNA and the tryptic peptide derived reverse complementary to the mRNA. To facilitate subsequent cloning of a PCR amplified segment, the tryptic peptide-derived set of oligonucleotides was provided with a BamHI restriction site. Lambda DNA was then isolated from the TNF- α- induced fibrosarcoma cDNA library and from this a TNF-BP sequence was amplified by PCR. The resulting fragment was cloned and sequenced; it has 158 nucleotides and contains the sequences coding for the tryptic peptide 20 between the two sequences derived from the primer oligonucleotides.

This DNA fragment may subsequently become radioactive marked and as a probe, z. B. for the isolation of cDNA clones from the fibrosarcoma library, be used. This can be done in this way that plaques first hybridize with the probe, Phages of hybridizing plaques isolated and Lambda DNA can be obtained from this.

Thereupon single-stranded DNA can be analyzed by PCR prepared and sequenced directly. (These Method can be used to quickly Sequence information of inserts recombinant To obtain lambda clones without the usual way of Subcloning of DNA fragments in M13 or having to follow similar vectors.)

The invention will be apparent from the following Preliminary tests and examples explained in more detail:

Preliminary test 1 competition binding assay

The presence of TNF-BP was checked by competition with the binding of radioactively labeled recombinant TNF- α (125 I-TNF- α ) to HL-60 cells. For radioactive labeling with ¹²⁵I-TNF- α , the two-phase method of Tejedor and Ballesta was used (19). For this purpose, 50 μl borate buffer (50 mM, pH 8.4), 10 μl KI (0.125 mM in free borate buffer) and 1 mCi 125 I (Amersham) with 1 μg recombinant TNF- α (manufactured by Genentech containing 36 × 10⁶ E / mg, corresponding to 646 U / pmole) containing 0.05% Tween 10 mixed. Based on the assumption that labeled TNF- α does not differ from unlabelled with regard to the binding properties, the specific activity was determined by displacement analysis.

To carry out the competition binding assay became a subclone of HL-60 cells (HL-60-10) (20, 21).

The cells were cultured in suspension culture in RPMI-1640 medium containing 10% fetal bovine serum (FBS). Logarithmic growth phase (7.5 x 10⁶) cells were washed with binding buffer (RPMI-1640, 10% FBS, 20 mM Hepes pH 7.4) and perfused with 50 mM 125 I-TNF- α for 2 hours at 4 ° C Rotation in 1.5 ml Eppendorf centrifuge tubes in a total volume of 300 ul incubated. It was then centrifuged for 10 seconds at 8000 xg, the precipitate taken up with ice-cold binding buffer and washed twice to separate free from membrane-bound125 I-TNF.

The radioactivity was measured with a gamma counter (LKB 1272 Clinigamma). The specific binding was defined as the difference between total binding and nonspecific binding that occurred with a 1000-fold excess of unlabelled TNF- α (the non-specific binding was 5-20% of total binding). Subsequently, the influence of dialyzed urine on the binding of 125 I-TNF- α was measured.

The binding inhibition caused by different volumes of a concentrated dialysis fiber (the starting urine used was conserved with EDTA (10 g / l), TRIS (6 g / l), NaN₃ (1 g / l), benzamidine hydrochloride (1 g / l), versus 0 , 15M NaCl, 5mM Hepes, pH 7.4, and 1mM benzamidine hydrochloride dialyzed and stored at -20 ° C) was used to construct a standard curve with the urine volume against the ratio of bound 125 I-TNF- α to maximum bound ¹²⁵I-TNF- α (B / Bmax) was applied. One unit of TNF binding protein was defined as that amount of TNF-BP which reduces the ratio B / Bmax to 0.5.

In order to prevent a reduction of the binding of 125 I-TNF- α to the cells by TNF- α or TNF- β possibly present in the sample, these had to be removed or blocked.

For this purpose, antisera were used which had been prepared by immunization of rabbits with TNF- α or TNF- β . The immunoglobulin of 1 μl of TNF- α- antiserum was adsorbed on 25 mg of protein A-Sepharose (Pharmacia) in 200 μl of binding buffer at 37 ° C for one hour. In order to remove any TNF- α contained in the biological samples, they were incubated with the anti-TNF- α bound to protein A-Sepharose for 12 hours at 4 ° C. in a volume of 750 μl with rotation. After subsequent centrifugation at 10,000 xg for 5 minutes, the supernatant was used for the competition binding assay.

In a comparative experiment it was possible to show that with the aid of this procedure the competition is completely abolished by 10 nM exogenous recombinant TNF- α .

To do this, various concentrations of TNF- α (1-100 nM) in 7 μl of binding buffer were incubated with 1 μl of anti-TNF- α adsorbed on 25 mg of protein A-Sepharose for 12 hours at 4 ° C with rotation. After centrifugation, the supernatants were subjected to the competition binding assay. The results of this comparative experiment are shown in FIG. 2a: The values obtained after preincubation with anti-TNF- α (⚫ - - - ⚫) were compared with the corresponding values for TNF- α , which had not been pre-incubated (○ -). - - ○). The results show that the decrease in the binding of125I-TNF- α can be eliminated by 10 nM TNF- α when TNF- α is preincubated with antibodies.

Since antibodies to TNF- β did not bind to 125 I-TNF- α , they could be added directly to the biological sample. 10 μl of antiserum were mixed with the sample, incubated for one hour and then the competition assay was carried out.

In a comparative experiment, it was shown that the addition of anti-TNF-β by the exogenous 100 nM recombinant (produced by Genentech, containing 230 × 10⁶ U / mg according β caused 3,96 E / pmol TNF inhibition of binding is released For this purpose, different amounts of anti-TNF- β (1.25-20 μl) were added to the control samples with constant TNF- β concentration After incubation for 60 minutes, the mixtures were subjected to the competition binding assay Fig. 2b shows that 10 μl of anti-TNF β completely abolishes the binding inhibition caused by TNF- β 100 nM.

The results of the competition binding experiments are shown in Fig. 1: The binding of 50 pM ¹²⁵I-TNF- α at 4 ° C in the presence of 20x concentrated dialysis is represented by the curve of the curve ⚫ - - - ((the bars show the Standard error of the arithmetic mean).

The fact that the measured reduction in the binding of125I-TNF- α to the cell is not due to the presence of TNF- α or TNF- β in the sample is shown by the identical curve in samples with prior treatment with TNF- α antibodies bound on protein A-Sepharose (○ - - - ○), or with the addition of 10 μl of TNF- β- antiserum (- - -).

Preliminary test 2

This experiment was performed to check whether TNF-BP has affinity for TNF- β . The procedure was as in the first experiment with the difference that the cells were incubated with 50 pM ¹²⁵I-TNF- β . It has been found that TNF-BP has an affinity to TNF- β which is about 1/50 of its affinity for TNF- a ; ie, 50 units of TNF-BP are needed to inhibit the binding of TNF- β to the cell by 50%.

Preliminary test 3 Detecting the formation of a complex of TNF-a TNF-BP from biological fluids

These experiments were performed with a mixture of 125 I-TNF- α with dialyzed urine or serum by gel chromatography from Sepahcryl. For this, mixtures with and without a 250-fold molar excess of unlabelled TNF- α were prepared.

500 μl sample was mixed for 10 minutes at 23 ° C. with 125 I-TNF- α at a final concentration of 200 pM and on a Sephacryl 200 superfine column (1.5 × 60 cm, Pharmacia) containing 0.15 M NaCl, 5 mM Hepes pH 7.4 had been applied. The column was eluted with 0.15 M NaCl, 5 mM Hepes pH 7.4 at a flow rate of 25 ml / h. Before gel chromatography, a portion of the samples were additionally incubated with a 250-fold molar excess of unlabeled TNF- α (50 nM).

Figure 3A shows the chromatogram of 125 I-TNF- α alone (- - -) and a mixture of 200 pM 125 I-TNF- α with dialyzed urine from a healthy subject (⚫ - - - ⚫).

Figure 3B shows the elution profiles of mixtures of 200 pM 125I-TNF- α with dialyzed urine from a uremic patient in the absence (⚫ - - - ⚫) and presence (○ - - - ○) of a 250-fold excess of unlabelled TNF- α ).

Fig. 3C shows the elution profiles of mixtures of 200 pM ¹²⁵I-TNF- α with serum of a healthy person in the absence (⚫ - - - ⚫) and presence (○ - - - ○) of a 250-fold excess of unlabelled TNF- α .

Figure 3D shows the elution profiles of mixtures of 200 pM ¹²⁵I-TNF- α with serum from a uremic patient in the absence (⚫ - - - ⚫) and presence (○ - - - ○) of a 250-fold excess of unlabelled TNF- α . Arrows indicate the dead volume V ₀ and the elution volumes of bovine serum albumin (I; molecular weight 67,000), ovalbumin (II; 43,000), chymotrypsinogen (III; 25,000) and ribonuclease (IV; 13,700).

Using dialyzed urine from a healthy subject, ¹²⁵I eluted-TNF- α as a single peak corresponding to a molecular weight of approximately 35,000 ( Figure 3A); the use of dialysis of a uremia patient, serum of a healthy person and serum of a uraemia patient resulted in two major peaks, corresponding to molecular weights of about 35,000 (free ¹²⁵I-TNF- α ) and about 75,000 (complex between ¹²⁵I-TNF- α and TNF -BP).

Preliminary test 4 Partial purification of TNF-BP

TNF-BP was made from several samples of Dialyseharn Uremia patients partially cleaned.

a) concentration

For this purpose, each 300 ml of urine were through Pressure ultrafiltration (Diaflo cells, equipped with UM-10 membranes (Amicon Inc.) and subsequent dialysis against 10 mM Tris HCl, pH Concentrated 8.0, 0.3% sodium azide.

b) ion exchange chromatography

To perform the ion exchange chromatography, a DEAE-Sephacel column (1.5 x 60 cm, Pharmacia) was charged with the sample (15 ml of concentrated dialysis sera containing 192 U / ml TNF-BP). Elution was with 150 ml of NaCl / 10 mM Tris / HCl, pH 8.0 gradient, the NaCl concentration being 0 to 0.4M. The steep gradient used at the end of the elution was used to check whether additional TNF-BP can be eluted at high ionic strength. The fractions containing the TNF binding protein were pooled, lyophilized, resuspended and dialyzed against 0.15 M NaCl, 5 mM Hepes pH 7.4. The ion exchange chromatogram is shown in Fig. 4A (to obtain the absorbance values, multiply the values shown by 0.3).

c) Gel Permeation Chromatography

The protein solution obtained from step b) (5 ml, containing 375 U / ml TNF-BP) was applied to a Sephadex G-75 superfine column (2.6 × 90 cm, Pharmacia) equilibrated with 0.15 M NaCl, 5 mM Hepes, pH 7.4, applied. The column was eluted at a flow rate of 7 ml / hour. The chromatogram is shown in Fig. 4B; the arrows indicate the dead volume ( V ₀), the elution volumes of bovine serum albumin (I, molecular weight 67,000), ovalbumin (II, 43,000) and chymotrypsinogen (III, 25,000) (to obtain the values for absorption are multiply by 0.03).

The fractions containing TNF-BP were united, lyophilized, resumed, against 0.12 M NaCl, 5 mM Hepes pH 7.4 and dialysed through Filtration sterilized. The determination of Protein concentration was carried out using the Biorad Protein microassay and with albumin as standard.

The partial cleaning brought a 62-fold Enrichment of the TNF binding protein, the Molecular weight was determined to be about 50,000.  

Preliminary test 5 Inhibition of biological activity of recombinant TNF by TNF-BP

The blocking of the growth inhibitory effect of TNF- α by TNF-BP was measured on HL-60-10 cells in agar culture. 2000 cells were layered in 1 ml of 0.3% agar on 0.5% agar, each in growth medium containing 10% fetal bovine serum, in 35 mm tissue culture dishes; the top agar layer of the culture dishes contained various concentrations of TNF- α (0-4 pM, starting from stock solutions containing 80 pM or 320 pM TNF- α in growth medium) and a uniform concentration of TNF-BP (13 U / ml, starting from a Stock solution containing 100 U / ml in 0.15 M NaCl, 5 mM Hepes, pH 7.4). The colonies were counted after 10 days.

Fig. 5A shows the blocking of growth inhibition of recombinant TNF- α by TNF-BP in agar culture: ⚫ - - - ⚫ TNF- α alone; ○ - - - ○ TNF- α with addition of 13 U / ml TNF-BP. TNF-BP alone does not affect cell growth.

The results of experiments with different TNF-BP concentrations (0.4-27 U / ml, stock solutions 1000, 250, 62 and 16 U / ml) at a constant concentration of TNF- α (2 pM, stock solution 80 pM) are in Fig. 5B (bars each indicate the standard deviation of the arithmetic mean). The results of these experiments show the dose-dependent effect of TNF-BP on the biological effect of TNF- α .

Preliminary test 6 Binding behavior of cells after pretreatment with TNF-BP

7.5 x 10⁶ HL-60-10 cells in 300 ml RPMI, 10% FBS, 20 mM Hepes pH 7.4 were incubated for 20 min at 37 ° C with TNF-BP (14 U / ml), then twice with washed ice-cold binding buffer. Thereafter, the competition binding test was carried out with ¹²⁵I-TNF- α as indicated in Example 1; the control experiments were carried out with untreated cells.

An average B / Bmax ratio of 0.95 was obtained. This result shows that the effect of TNF-BP is not due to its eventual binding to the cells.

Preliminary test 7 Preparation of highly purified TNF-BP a) Concentration of the urine

200 l of dialysis sutures from uremia patients in bottles containing EDTA (10 g / l), tris (6 g / l), NaN3 (1 g / l) and benzamidine hydrochloride (1 g / l) and stored cool, were by ultrafiltration by means of a highly permeable hemocapillary filter with an asymmetric hollow-fiber emulsion (FH 88H, Gambro) to 4.2 l with a protein content of 567 g  concentrated. The concentrated urine was against 10 mM / l Tris HCl, pH 8 dialyzed. During this The process became as in the following steps (except in reverse phase chromatography), 1 m / M / l benzamidine hydrochloride added to counteract proteolytic digestion. All subsequent cleaning steps were, if not otherwise stated, carried out at 4 ° C.

b) ion exchange chromatography

This step became, as in the preliminary experiment 1 described, performed. In addition were DEAE Sephacel columns (2.5 × 40 cm) with samples containing concentrated and dialysed urine each about 75 g of protein, fed. Was eluted with 800 ml of a NaCl / 10 mM Tris / HCl pH 8 gradient, wherein the NaCl concentration was 0 to 0.4M. The TNF-BP containing fractions of seven Columns with a total protein content of 114 g were stored at -20 ° C.

c) Affinity chromatography

To prepare the TNF Sepharose column, rTNF-a (15 mg) in 0.1 M NaHCO₃, 1 M NaCl, pH 9 (Coupling buffer) to 1.5 g cyanogen bromide activated Sepharose 4B (Pharmacia) coupled. The Sepharose was swollen in 1 mM HCl and coupled with buffer washed. After addition of rTNF-a, the Suspension for 2 hours at room temperature to rotate. The excess of CNBr groups was made by one-half hour rotation with 1M Ethanolamine, pH 8 blocked. The TNF Sepharose  was alternated several times in 1 M NaCl, 0.1 M Sodium acetate pH 8 and 1 M NaCl, 0.1 M boric acid pH 4 washed and then in phosphate buffered Saline with 1mM benzamidine hydrochloride stored. The fractions obtained from step b) were adjusted to a concentration of 0.2 M NaCl, 10 mM Tris / HCl, pH 8 set. The TNF Sepharose was packed in a column and with 0.2 M NaCl, 10 mM Tris HCl, pH 8, and containing the TNF-BP Fractions, corresponding to about 30 g of protein, at applied at a flow rate of 10 ml / h and extensively with 0.2 M NaCl, 10 mM Tris HCl, pH 8 washed until in the eluate at 280 nm no absorption was more detectable. Subsequently, TNF-BP was used 0.2 M glycine / HCl, pH 2.5 eluted.

The course of the affinity chromatography is shown in FIG. 6: ○ - - - ○ absorbance at 280 nm; ⚫ - - - ⚫ TNF-BP, measured in the competition binding test.

TNF-BP containing fractions from 4 separations were combined and after addition of Polyethylene glycol (MG 6000) - up to one Final concentration of 10 mg / ml - lyophilized. The lyophilized sample was in distilled water dissolved and dialyzed against distilled water. (The dialyzed sample (4 ml) was taken in stored frozen).

This purification step brought over the previous one further enrichment by about 9000 times. (The degree of purification in this step could not be accurately determined because a certain proportion of TNF- α leaked out of the column, possibly masking some of the TNF-BP, and such masking inevitably has an inaccuracy in quantitating the activity of the protein result.) SDS-PAGE (performed as described in Preliminary Example 8) of the fractions containing TNF-BP showed the elution of three major components with molecular weights of 28,000, 30,000, and 50,000 ( Figure 8, lane A).

d) reverse phase chromatography

An aliquot (1 ml) of the fractions obtained from step c) with an addition of 0.1% trifluoroacetic acid was applied to a ProRPC HR 5/10 column (Pharmacia) attached to a FPLC system (Pharmacia). The column was equilibrated with 0.1% trifluoroacetic acid and charged at room temperature with a linear 15 ml gradient from 10% to 50% by volume acetonitrile containing 0.1% trifluoroacetic acid; the flow rate was 0.3 ml / min. Fractions of 0.5 ml were collected and the absorbance at 280 nm as well as the activity of the TNF- α binding protein were determined by the competition binding assay as indicated in Example 1, using 0.01 μl sample each. TNF-BP eluted as a single peak of activity corresponding to a sharp UV absorption peak. The results of these studies are shown in FIG .

This last cleaning step brought one Increase in specific activity by about 29 times, the overall increase in activity over the Starting material (concentrated dialysis hair) that was about 1.1 × 10⁶ times.  

SDS-PAGE of the reduced and non-reduced sample, performed as indicated in Prerequisite 8, demonstrated the presence of a single polypeptide of molecular weight about 30,000 ( Figure 8, lanes B, C).

Preliminary test 8 SDS polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was prepared according to the method of Laemmli (25) on 18 cm long, 16 cm wide and 1.5 mm thick Flat gels with 10 pockets by means of a LKB 2001 Electrophoresis unit performed. The Protein content of the samples from the Purification steps c) and d) (pre-experiment 7) was determined by Bio-Rad Protein Assay or from the Absorbance at 280 nm calculated using one Absorption of 1.0 a content of 1 mg TNF-BP / ml 000 000dnet was.

The samples containing approximately 25 μg of protein (from preliminary experiment 7c) or approximately 5 μg (from 7d) in reduced ( β- mercaptoethanol) and non-reduced form were placed on a 3% collecting gel and a 5- to 20% strength applied linear polyacrylamide gradient gel. The electrophoresis was run at 25 mA / gel without cooling. As molecular weight markers (Pharmacia), phosphorylase B (MW 94,000), bovine serum albumin (MW 67,000), ovalbumin (MW 43,000), carbonic anhydrase (MW 30,000), soybean trypsin inhibitor (MW20,100) and α-lactalbumin (MW14,000) were used 400). The gels were stained with Coomassie Blue in 7% acetic acid / 40% ethanol and decolorized in 7% acetic acid / 25% ethanol.

The result of the SDS-PAGE is shown in FIG. 8: Lane A (affinity chromatography): 3 proteins with molecular weights of about 28,000, about 30,000 and about 50,000 were detected.

Lanes B and C (reverse phase chromatography): Electropherograms of the reduced (B) and the unreduced (C) highly purified protein.

The result of SDS-PAGE shows that TNF-BP is off a single polypeptide chain with a Molecular weight of about 30 000 consists. (The Difference between this molecular weight and the gel by means of gel permeation chromatography on Sephadex G-75 determined molecular weight of approx. 50,000 could be explained by the fact that in Gel permeation chromatography based on Molecular shape of the protein a higher Molecular weight is simulated. Another Possibility of explaining this discrepancy could not undermine the formation of TNF-BP dimers denaturing conditions.)

Preliminary test 9 amino acid analysis

The amino acid analysis was done with the Beckman High Performance Analyzer System 6300 after Standard procedure performed.

It gave the following amino acid composition, expressed in moles of amino acid per mole of protein and in Mol% amino acid, determined as the mean of a 24- and a 48-hour hydrolysis:  

Furthermore, glucosamine was detected in the samples.

Preliminary test 10 a) Sample preparation

15 μg of the purified after preliminary experiment 7d) Protein was desalted via reverse phase HPLC and further cleaned. This was a Bakerbond WP C18 column (Baker; 4.6 x 250 mm) and 0.1% Trifluoroacetic acid in water (eluent A) or in Acetonitrile (eluent B) used as the mobile phase.  The gradient increase was 20 to 68% eluent B in 24 min. The detection was carried out in parallel at 214 nm and at 280 nm. The TNF-BP containing Fraction was collected, dried and in 75 μl Dissolved 70% formic acid and directly for the Amino acid sequence analysis used.

b) amino acid sequence analysis

Automatic amino acid sequence analysis was performed on an Applied Biosystem 477A Liquid Phase Sequenator by on-line determination of the released phenylthiohydantoin derivatives using Applied Biosystems Analyzer Model 120 A PTH. It gave the following N-terminal sequence as the main sequence: (about 80% of the protein amount):
Asp-Ser-Val-X-Pro-Gln-Gly-Lys-Tyr-Ile-His-Pro-Gln.
In addition, the following secondary sequence had to be demonstrated:
Leu (Val) - (Pro) - (His) Leu-Gly-X-Arg-Glu.
(The parenthesized amino acids could not be clearly identified.)

Preliminary test 11 SDS-PAGE

The sample preparation was carried out as in the preliminary experiment 10, with the difference that the sample amount was 10 μg. The sample was taken up in 50 μl of water and divided into 4 portions. One of the four aliquots was reduced for purity by SDS-PAGE by the method of Laemmli (25) with DTT (dithiothreitol) and separated into minigels (Höfer, 55 x 80 x 0.75, 15%); as the molecular weight marker was used in the preliminary test 8 indicated. The staining was done by the method of Oakley (30). The electropherogram is shown in FIG . It shows a single band at a molecular weight of about 30,000.

Preliminary test 12 Examination for sugar content by means of affinoblotting

These were two of the remaining portions from the Sample preparation for preliminary test 11 gelelektrophoretisch (36) separated and on Nitrocellulose blotted.

The transfer to nitrocellulose (NC, pore size 0.45 mm) was carried out with a semi-dry apparatus Sartorius SM 17 556) during 2 h at a Amperage of 1.1 mA / cm² gel area. Subsequently at the blot, the NC was in for 1 h Blocking buffer (1% BSA in PBS, pH 7.2) at Room temperature with shaking incubated (PBS: 137 mM NaCl, 2.7 mM KCl, 4.2 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.2).

The staining of the glycoproteins was carried out with two different lectins that are different from each other To distinguish specificity for the sugar residues: Concanavalin A (ConA) reacts with glucose and Mannose, and wheat germ lectin (WGA) reacts with N-acetylglucosamine and N-acetylneuraminic acid. WGA was using glutaraldehyde with Horseradish peroxidase coupled.  

The controls were for each lectin Control proteins with known glycan structure with applied (Con A: ovalbumin, WGA: fetuin).

The development with ConA was carried out as follows:

• - Rinse several times with TBS (500 mM NaCl, 20 mM TRIS, pH 7.4),
• Incubation with ConA (25 mg ConA / ml) in TBSK (TBS with 1 mM MnCl₂ and 1 mM CaCl₂),
• - Rinse 4 × 15 min in TBSK with 0.1% TWEEN 20,
• Incubation with peroxidase (50 mg / ml in TBSK)
• - Rinse 4 × 15 min in TBSK with 0.1% TWEEN 20,
• Incubation in TBS for 15 minutes,
• - Incubation in the staining medium (60 mg 4-chloro-1-naphthol, 15 ml methanol, 85 ml TBS, 60 ml H₂O₂) until blue bands visible become.
• - The staining was done by rinsing with water completed.

Development with WGA / peroxidase became as follows run:

• - rinse with TBS several times,
• Incubation with lectin conjugate (20 ml / ml in PBS) for 2 h,
• - rinse with TBS several times,
• Incubation in TBS for 15 minutes,
• Incubation with staining medium (see above) until blue bands become visible.

The result of the affinoblot is shown in FIG. 10:
Lanes 1 (control) and 2 show the reaction with ConA, lanes 3 (control) and 4 show the reaction with WGA.

The result of the affinoblot shows that it is TNF-BP is a glycoprotein.

Preliminary test 13 Influence of TNF-BP on the cytotoxic effect of TNF- α

The determination of the cytotoxic effect of TNF-a on WEHI 164 clone 13 cells was performed as described in (29). Target cells were plated on microtiter plates at a concentration of 20,000 to 40,000 cells per well in 100 μl of RPMI tissue culture medium containing 10% FBS. TNF- α was added in two different concentrations (100 pM / l, 1000 pM / l) along with various concentrations of highly purified TNF-BP. After 20 hours, the proportion of dead cells was determined as described in (29). The titration curves of this experiment are shown in FIG. 11 (⚫ - - - ⚫: 100 pM TNF- α ; ○ - - - ○: 1000 pM TNF- α ).

example 1 a) Tryptic Peptide Mapping

About 60 μg of the purified after preliminary experiment 7d) Protein was desalted via reverse phase HPLC and thus further purified. This was a Bakerbond WP C18 column (Baker, 4.6 × 250 mm) and 0.1% trifluoroacetic acid in water (eluent A) or in acetonitrile (eluent B) as a mobile phase used. The gradient increase was 20 to 68% eluent B in 24 min. The detection took place parallel at 214 nm and at 280 nm. The TNF-BP  containing fraction (retention time about 13.0 min) was collected, dried and in 60 ul 1% Ammonium bicarbonate dissolved.

This solution was 1% w / w, corresponding to 0.6 μg Trypsin (Boehringer Mannheim) was added and the Reaction mixture for 6 hours at 37 ° C incubated. Thereafter, another 1% w / w of trypsin was added and the incubation continued overnight.

To reduce the disulfide bridges, the Reaction mixture then with 60 .mu.l 6 M Urea and with 12 ul of 0.5 M dithiothreitol and 2 hours at room temperature ditched.

The separation of the resulting tryptic Cleavage peptides were by reverse phase HPLC, wherein a Delta Pak C18 column (Waters, 3.9 x 150 mm, 5 @m particle diameter, 100 A Pore diameter) at 30 ° C and 0.1% Trifluoroacetic acid in water (eluent A) or in Acetonitrile (eluent B) used as the mobile phase were. The gradient increase was 0 to 55% Eluent B in 55 min, then 55% B for 15 min maintained. The flow rate was 1 ml / min, the Detection was carried out in parallel at 214 nm (0.5 AUFS) and at 280 nm (0.05 AUFS).

b) Sequence analysis of tryptic peptides

Some of the tryptic won after a) Colloid peptides of TNF-BP were the automatic Subjected to amino acid sequence analysis. In addition were the corresponding fractions from the reverse phase Collected by HPLC, dried and 70% in 75 ul  Formic acid dissolved. These solutions became direct for sequencing in an Applied Biosystems 477 A Pulsed Liquid Phase Sequenator used. Table 1 contains the results of the sequence analysis of tryptic peptides, those in parentheses mentioned amino acids not with certainty could be detected. The specification "X" means that at this point the amino acid is not could be identified. In fraction 8, the amino acid could be in position 6 can not be identified. The sequence -X-N-S- for heading 6-8 suggests that the Amino acid 6 is present in glycosylated form.

Striking is the extensive identity of the Sequence of occurring only in small quantities Fraction 12 with that determined in preliminary experiment 10 Subsequence of the N-terminus. Because the proteins of the Main and secondary sequence on an analytical Reverse phase HPLC column (preliminary experiment 10a) not could be with the protein the minor sequence extended by one at the N-terminus Form of TNF-BP act by processing for the most part in the protein with the main sequence is transferred.

fraction amino acid sequence 1 D - S - V - C - P - Q - G - K 2 X - X - L - S - (C) - S - K 5 E - N - E - (C) - V - S - (C) - (S) - N - (C) - K - (K) 8th Y - I - H - P - Q - X - N - S - I - X - X - X - K 11 E - C - E - S - G - S - F - T - A - S - E - N - (N) - (K) 12 L - V - P - H - L - G - D - R 14 / I G - T - Y - L - Y - N - D - C - P - G - P - G - Q - 14 / II (E) - M - G - Q - V - (E) - (I) - (S) - X - X - X - (V) - (D) - 20 G - T - Y - L - Y - N - D - C - P - G - P - G - Q - D - T - X - X - R 26 Q - N - T - V - (C) - T - X - (H) - A - G - F - (F) - L - (R) 27 S - L - E - (C) - T - K - L - (C) - L - P - Q - I - E - N -

(To the description of the examples below simplify, often become recurrent methods or descriptions briefly described.)

"Cutting" or "digesting" DNA refers on the catalytic cleavage of the DNA by means of Restrictionendonucleases (restriction enzymes) for these specific jobs (Restriction sites). restriction endonucleases are available for purchase and are among those of conditions recommended by the manufacturers (buffers, Bovine serum albumin (BSA) as carrier protein, Dithiothreitol (DTT) as oxidation protection) used. Restriction endonucleases are included  a capital letter, mostly followed by Lowercase letters and usually a Roman Numeral designated. The letters depend on that Microorganism from which the relevant Restriction endonuclease was isolated (e.g., Sma I: Serratia marcencens). Usually about 1 μg DNA with one or more units of the Enzyme cut in about 20 ul buffer solution. Normally, an incubation period of 1 Hour at 37 ° C, but may be loud Use instructions of the manufacturer vary become. After cutting, sometimes the 5'-phosphate group by incubation with alkaline Phosphatase removed from calf intestine (CIP). This serves to prevent an unwanted reaction the specific place in a subsequent one Ligase reaction (eg circularization of a linearized plasmid without insertion of a second DNA fragment). If not otherwise stated, DNA fragments are after cutting usually not with restriction endonucleases dephosphorylated. Reaction conditions for the Incubation with alkaline phosphatase are z. B. the M13 cloning and sequencing manual (Cloning and Sequencing Handbook, Fa Amersham, PI / 129/83/12) remove. After incubation, protein will pass through Extraction with phenol and chloroform removed and the DNA from the aqueous phase by addition of Ethanol precipitated.

"Isolation" of a particular DNA fragment means the by the restriction digestion obtained DNA fragments separation of z. B. on a 1% agarose gel. After electrophoresis and visualizing the DNA in UV light Staining with ethidium bromide (EtBr) will be the desired fragment based on  Molecular weight markers localized and through further electrophoresis on DE 81 paper (Schleicher and Schuell). The DNA is flushed with Low salt buffer (200 mM NaCl, 20 mM Tris pH = 7.5, 1 mM EDTA) and then with a High salt buffer (1 M NaCl, 20 mM Tris pH = 7.5, 1 mM EDTA) eluted. The DNA is made by adding Ethanol precipitated.

"Transformation" means introducing DNA into an organism so that the DNA replicable there is, either extrachromosomally or chromosomally integrated. Transformation of E. coli follows in the M13 Cloning and Sequencing Manual (Cloning and Sequencing Handbook, Fa. Amersham, PI / 129/83/12) specified method.

"Sequencing" a DNA means the determination the nucleotide sequence. For this purpose, the first to sequencing DNA with different Cut restriction enzymes, and the fragments are in appropriately cut M13 mp 8, mp 9, mp 18 or mp 19 double-stranded DNA introduced, or the DNA is fragmented by ultrasound, fixed the ends and, the size-selected ones Fragments cut into Sma I, dephosphorylated M13 mp 8 DNA introduced (shotgun method). After Transformation of E. coli JM 101 becomes single-stranded DNA from recombinant M13 phages according to the M13 cloning and sequencing manual (Cloning and Sequencing Handbook, Fa. Amersham, PI / 129/83/12) isolated and according to the dideoxy method (35) sequenced. (As an alternative to using the Klenow fragment of E. coli DNA polymerase I offers the T7 DNA polymerase ("Sequenase, United States Biochemical Corporation). The Sequence reactions will be according to the manual  "Sequenase: Step-by-Step Protocols for DNA Sequencing With Sequenase "performed.)

Another sequencing method is cloning the DNA to be sequenced into a vector, the including an origin of replication of a DNA single stranded phage (M13, f1) carries (e.g. Bluescribe or Bluescript M13 by Stratagene). After transformation of E. coli JM101 with the Recombinant molecule can be transformed with the transformants a helper phage, z. M13K07 or R408 from Progema, become infected. As a result you get a mixture of helper phages and packed, single-stranded recombinant vector. The Processing of the sequencing template is analogous to the M13 method. The evaluation of the sequences is carried out by means of originally developed by R. Staden (32) and Ch. Pieler (33) modified computer programs.

"Ligate" refers to the process of formation of phosphodiester bonds between two ends of Double-stranded DNA fragments. Usually between 0.02 and 0.2 μg of DNA fragments in 10 μl with about 5 units of T4 DNA ligase ("ligase") in one suitable buffer solution is ligated (33) (T. Maniatis et al., Molecular cloning, 1982, p. 474).

"Preparation" of DNA from transformants means the isolation of plasmid DNA from bacteria using the alkaline SDS method, modified according to Birnboim and Doly (T. Maniatis et al., Molecular Cloning, 1982, pp. 368-369) under Omission of the lysozyme. This will be the bacteria used from 1.5 to 50 ml of culture.

"Oligonucleotides" are short polydeoxynucleotides, which are chemically synthesized. This was the  Applied Biosystems Synthesizer Model 381A used. The oligonucleotides are correspondingly Model 381A User Manual (Applied Biosystems) worked up. Sequence primers will be without further Cleaning used directly. Other oligonucleotides be up to a chain length of 70 by the "OPC" method purified (OPC = oligonucleotide purification column, Applied Biosystems, Product Bulletin, January 1988). Longer oligonucleotides are synthesized by polyacrylamide gel electrophoresis (6% Acrylamide, 0.15% bisacrylamide, 6 M urea, TBE buffer) and after elution from the Gel desalted over a Gel-25 Sepharose column.

Example 2 Production of TNF-binding protein-specific hybridization probes

The selection of oligonucleotides was made with respect to their use for amplification of cDNA by PCR:
a) From the N-terminal amino acid sequence of the TNF-binding protein (obtained from preliminary experiment 10 and example 1, fraction 1)

Asp-Ser-Val-Cys-Pro-Gln-Gly-Lys-Tyr-Ile His-Pro-Gln

a heptapeptide region was chosen which allows the lowest complexity of a mixed oligonucleotide to hybridize to cDNA:
These are amino acids 6 through 12. In order to reduce the complexity of the mixed oligonucleotide, four mixed oligonucleotides with a complexity of 48 were prepared. The oligonucleotides were prepared in the direction of the mRNA, they are thus oriented to the 3 'end of the sequence:

b) From the amino acid sequence of a tryptic peptide (Fraction 11 of tryptic digestion) of amino acid sequence

Glu-Cys-Glu-Ser-Gly-Ser-Phe-Thr-Ala-Ser- (Glu / Cys) - Asn-Asn-Lys (see Example 1)

one peptide region was selected and another Synthesis of mixed oligonucleotides:

The oligonucleotides were complementary to the mRNA synthesized and thus are at the 5'end of the sequence oriented. To the amplified DNA fragment in Be able to efficiently clone the connection to the PCR, also became a BamHI linker at the 5'end of the Oligonucleotides provided. Are z. B. the Oligonucleotides TNF-BP # 4 / 5-8 together with TNF-BP # 3 / 1-4 for the PCR on the entire lambda DNA one Used as a library, can be approximately resulting DNA fragment can be recut with BamHI. The Partner oligonucleotides give a straight end at 5'Terminus, the fragment can thus in the SmaI-BamHI sites of a suitable vector cloned become.

Each mixed oligonucleotide TNF-BP # 4/5 to 8 is one Mix of 48 single nucleotides and considered some codins do not, namely:

Thr ACG Ala GCG and GCT Ser TCG and TCC Asn AAT

GCT considers the possibility of that the triplet CGG complementary to GCC (Ala) can be effective by forming a G-T bridge, for TCG (Ser) and AAT (Asn), the same applies with respect to AGT or TTG.

ACG, GCG and TCG are extremely rare codons (CG rule) and were therefore not considered.

Example 3 Amplification of a coding for TNF-BP Partial sequence from a cDNA library a) Isolation of lambda DNA from a cDNA library

The preparation of the cDNA library was carried out according to the method described in EP-A1-02 93 567 for the human placental cDNA library with the difference that 10⁹ fibrosarcoma cells of the cell line HS 913 T, which under stimulation with human TNF- α (10 ng / ml) were used. Lambda gt10 was used instead of lambda gt10 (cDNA synthesis: Amersham RPN 1256, EcoRI digested lambda gt 11 arms: Promega Biotech, in vitro packaging of the ligated DNA: Gigapack Plus, Stratagene).

5 ml of the phage supernatant of the amplified cDNA Library of human fibrosarcoma cell line HS913T in lambda gt11 were treated with 0.5 μg RNase A and 0.5 μg DNase I is added and left for one hour at 37 ° C incubated. The mixture was stirred at 5000 xg for 10 min centrifuged, the supernatant by extraction with Free phenol and chloroform from protein and the  DNA from the aqueous phase by addition of ethanol precipitated. The lambda DNA was digested in TE buffer (10 mM Tris pH = 7.5; 1 mM DETA).

b) PCR amplification of a TNF-BP sequence from a cDNA library

For the application of the PCR on DNA of the HS913T cDNA Library, 16 individual reactions were performed, in which in each case one of the 4 mixed oligonucleotides EBI-1639, EBI-1540, EBI-1641, EBI-1642 first Primer and one of the four mixed oligonucleotides EBI-1653, EBI-1654, EBI-1657, EBI-1658 second Primer was used. Each of these Mischoligonukleotide contains 48 different Oligonucleotides of equal length. Amplification by PCR was in 50 μl Reaction volume containing 250 ng of lambda DNA cDNA library, 50mM KCl, 10mM Tris pH = 8.3, 1.5mM MgCl₂, 0.01% gelatin, 0.2mM each of the 4 desoxy-nucleotide triphosphates (dATP, dGTP, dCTP, dTTP), 200 pmoles first and second primer, 1.25 Units tag polymerase [Perkin-Elmer Cetus] instead. To prevent the evaporation, the solution became with a few drops of mineral oil (0.1 ml) overlayed. The PCR was in a DNA Thermal Cycler (Perkon Elmer Cetus) as follows The samples were held for 5 minutes at 94 ° C heated to denature the DNA, and then subjected to 40 amplification cycles. One cycle consisted of 40 seconds of incubation 94 ° C, 2 minutes incubation at 55 ° C and 3 minutes Incubation at 72 ° C. At the end of the last cycle the samples were held at 72 ° C for an additional 7 minutes incubated to make sure the last one Primer extension runs completely. To Cooling to room temperature, the samples were with  Free phenol and chloroform from protein and the DNA precipitated with ethanol.

5 μl of each of the 16 PCR samples were placed on a Agarose gel applied and the length of the after amplified DNA fragments determined by electrophoretic separation. The strongest DNA band, a fragment of 0.16 kb in length, was seen in the PCR samples with the Oligonucleotide EBI-1653 as the first primer and a Oligonucleotides EBI-1639, EBI-1640, EBI-1641 or EBI-1642 as a second primer were. As with the primer pair EBI-1653 and EBI-1642 amplified sample the largest amount contained this 0.16 kb DNA fragment, this was Sample selected for further work-up.

Example 4 Cloning and sequencing one by PCR Amplification of recovered DNA fragments

The resulting PCR product of primers EBI-1642 and EBI-1653 was cut with BamHI and subsequently electrophoretically in an agarose gel (1.5% NuSieve GTG agarose plus 1% Seakem GTG agarose, FMC Corporation) separated by size. The Main band, a DNA fragment of 0.16 kb in length, was eluted from the gel and ethanol precipitated. This DNA fragment was with BamHI / SmaI cut plasmid pUC18 (Pharmacia) ligated and E. coli JM101 with the ligation mixture transformed. The after the minipreparation method were prepared by cutting with the restriction enzymes PvuII and EcoRI-BamHI and subsequent electrophoresis in agarose gels characterized. The plasmid pUC18 contains two Interfaces for PvuII, which are in a 0.32 kb  DNA fragment flanking the polycloning site. Very short DNA inserts in the polycloning site of the Plasmids can be more easily imitated after cutting with PvuII Agarose gel be made visible as the Length increased by 0.32 kb. By cutting with EcoRI and BamHI can do this in terms with BamHI and SmaI cut plasmid vector ligated DNA fragment including some base pairs of the polylinker sequence to be obtained. A clone with the desired insert was designated pTNF-BP3B. The entire DNA insert of this clone was subcloned of an EcoRI-BamHI fragment in M13mp18 (Pharmacia) using the modified dideoxy method Sequenase (United States Biochemical Corporation) sequenced.

Analysis of PCR amplified DNA revealed following sequence (only the non-coding strand is shown, about the derived Amino acid sequence):

The first 20 and the last 29 nucleotides (in Italics) correspond to the sequences of the Primer oligonucleotides EBI-1642 and the complement, respectively from EBI-1653. The amino acids 38 to 43 confirm the remaining sequence of the tryptic peptide 11.

Furthermore contains the generated by PCR DNA fragment the sequence of the peptide of the fraction 20 of the tryptic digest (amino acids 20 to 34, underlined). This proved that the clone pTNF-BP3B was derived from a cDNA coding for Can encode TNF binding protein. pTNF-BP3B thus provides a probe, z. B. for Browse cDNA libraries for TNF binding protein encoding cDNAs, is.

literature

1. Carswell, EA, LJ Old, RL Kassel, S. Green, N. Fiore, and B. Williamson. 1975. An endotoxin-induced serum factor that causes necrosis of tumors.
Proc. Natl. Acad. Sci. UNITED STATES. 25: 3666-3670.
2. Old, LJ 1987. Tumor necrosis factor. Polypeptides mediator network.
Nature (London). 326: 330-331.
3. Aggarwal, BB, TE Eessalu, PE Haas. 1985. Characterization of receptors for human tumor necrosis factor 4 and their regulation by gamma interferon.
Nature 1985, 318: 665-667.
4. Gullberg, U., U., M. Lantz, E. Nilsson, C. Peetre, G. Adolf, and I. Olsson. 1987. Characterization of a Relationship Between the T-lymphocyte derived differentiation inducing factor (DIF) and lymphotoxin: A common receptor system for DIF, lymphotoxin and tumor necrosis factor downregulated by phorbol diesters.
Eur. J. Haematol. 39: 241-251.
5. Beutler, B., D. Greenwald, JD Hulmes, M. Chang, Y.-CE Pan, J. Mathison, R. Ulevitch, and A. Cerami. 1985. Identity of tumor necrosis factor and the macrophage-secreted factor cachectin.
Nature 316: 552-554.
6. Torti, FM, B. Dieckmann, B. Beutler, A. Cerami, and GM Ringold. 1985. A macrophage factor inhibits adipocyte gene expression: An in vitro model for cachexia.
Nature (London). 229: 867-869.
7. Mahoney Jr., JR, BA Beutler, NL Trang, W. Vine, Y. Ikeda, M. Kawakami, and A. Cerami. 1985. Lipopolysaccharide-treated raw 264.7 cells produce a mediator that inhibits lipoprotein lipase in 3T3-L1 cells.
J. Immunol. 134: 1673-1675.
8. Shalaby, MR, BB Aggarwal. E. Rinderknecht, LP Svedersky, BS Finkle, and MA Palladino, Jr. 1985. Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factor.
J. Immunol. 135: 2069-2073.
9. Klebanoff, SJ, MA Vadas, JM Harlan, LH Sparks, JR Gamble, JM Agosti, and AM Waltersdorp. 1986. Stimulation of neutrophilis by tumor necrosis factor.
J. Immunol. 136: 4220-4225.
10. Mestan, J., W. Digel, S. Mittnacht, H. Hillen, D. Blohm, A. Moller, H. Jacobson, and H. Kirchner. 1986. Antiviral effects of recombinant tumor necrosis factor in vitro.
Nature (London). 323: 816-819.
11. Wong, GHW, and DV Goeddel. 1986. Tumor necrosis factors a and β inhibit virus replication and synergize with interferons.
Nature (London). 323: 819-822.
12. Cerami, A., and B. Beutler. 1988. The role of cachetine in endotoxic shock and cachexia. Immunol. Today. 9: 28-31.
13. Tracey, KJ, B. Beutler, SF Lowry, J. Merryweather, S. Wolfe, IW Milsark, RJ Hariri, TJ Fahey III, A. Zentella, JD Albert, GT Shires, and A. Cerami. 1986. Shock and tissue injury induced by recombinant human recombinant human cachectin.
Science (Wash. DC). 234: 470-474.
14. Tracey, KJ, Y. Fong, DG Hesse, KR Manogue, AT Lee, GC Kuo, SF Lowry, and C. Cerami. 1987. Anti-cachectin / TNF monoclonal antibodies prevent septic shock during lethal bacteraemia.
Nature (London). 330: 662-666.
15. Piguet, PF, G., Gray, B., Allet, and P., Vassalli. 1987. Tumor necrosis factor (TNF) is an important mediator of the mortality and morbidity induced by the graft-versus-host reaction (GHVR).
Immunobiol. 175: 27
16. Libra, A., A. Halstensen, and T. Espevik, 1987. Association between tumor necrosis factor in serum and fatal outcome in patients with meningococcal disease.
Lancet. ii: 355-357.
17. Seckinger, P., JW Lowenthal, K. Williamson, J.-M. Dayer and HR MacDonald. 1987. A urine inhibitor of interleukin 1 activity that blocks ligand binding.
J. Immunol. 139: 1546-1549.
18. Strober, W., and TA Waldmann. 1974. The role of the kidney in the metabolism of plasma proteins.
Nephron. 13: 35-66.
19. Tejedor, F., and JPG Ballesta. 1982. Iodination of biological samples without loss of functional activity. Analyte. Biochem. 127: 143-149.
20. Gullberg, U., E. Nilsson, MG Sarngadharan, and I. Olsson. 1986. T Lymphocyte-Derived Differentation-Inducing Factor Inhibits Proliferation of Leukemic and Normal Hemopoietic Cells.
Blood 6, 1986: 1333-1338.
21. Peetre, C., U., Gullberg, E. Nilsson, and I. Olsson. 1986. Effects of Recombinant Tumor Necrosis Factor on Proliferation and Differentiation of Leukemic and Normal Hemopoietic Cells in Vitro.
J. Clin. Invest. 78: 1694-1700.
22. Beutler B., Cerami A., tumor necrosis, cachexia, shock, and inflammation:
A common mediator. Ann. Rev. Biochem. 57: 505-18, 1988.
23. Oliff A., Defeo-Jones D., Boyer M. et al. Tumors secreting human TNF / cachectin induce cachexia in mice. Cell 1987: 555-63.
24. Seckinger P. Isaaz S., Dayer JM A human inhibitor for tumor necrosis factor a. J. Exp. Med. 1988: 1511-6.
25. Laemmli UK Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 1970; 227: 680-4.
26. Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ A gas-liquid solid phase peptide and protein sequenator. J. Biol, Chem. 1981; 256: 7990-7.
27. Liao Z., Grimshaw RS, Rosenstreich DL Identification of a specific interleukin I inhibitor in the urine of febrile patients. J. Exp. Med. 1984; 159: 126-36.
28. Seckinger P., Williamson K., Balavoine JF et al. A urine inhibitor of interleukin I activity effects both interleukin Ia and I beta but not tumor necrosis factor a. J. Immunol. 1987: 139: 1541-5.
29. Espevik T., Nissen-Meyer J. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor / tumor necrosis factor from human monocytes. J. Immunol. Meth. 1986; 95: 99-105.
30. Oakley, BR, Cherry DR, Morris R. Analyt. Biochem. 1986; 105: 361-363.
31. Saiki, RK, Science 239 (1988): 487-491.
32. Staden, R., Nucleic Acid Res. 10 (1982): 4731-4751.
33. Pieler Ch., 1987, Dissertation, University of Vienna.
34. Maniatis, T., et al., Molecular Cloning, 1982: p. 474th
35. Sanger et al., Proc. Natl. Acad. Sci. 74 1977: 5463-5467.

Claims (23)

1. DNA, characterized in that it has the formula or a variant of a DNA of this formula which hybridizes with DNA selected from the group

• a) DNA encoding TNF- α binding protein,
• b) DNA encoding a protein whose processing gives TNF-BP,
• c) DNA encoding a TNF-BP related protein having the ability to bind TNF- α , such as cDNA derived from mRNA obtained by alternative splicing,
• d) DNA encoding a protein whose processing yields a TNF-BP related protein capable of binding TNF- α , such as cDNA derived from mRNA obtained by alternative splicing.

2. DNA, according to claim 1, characterized in that it has a nucleotide sequence of the formula where R1 and R2 are optionally provided restriction sites and / or the nucleotide sequence of the formula is optionally modified, and that it hybridizes with DNA encoding TNF- α binding protein or for a protein whose processing results in TNF- α binding protein. 3. DNA, characterized in that it is in accordance with DNA Claim 1 hybridized and is selected from the Group defined in claim 1 a) to d) DNAs, including their degenerate variants. 4. DNA, characterized in that it hybridizes with DNA according to claim 2 and for TNF- α binding protein or for a protein whose processing results in the TNF- α binding protein encodes, including their degenerate variants. 5. Recombinant DNA molecule containing an in Claim 3 defined DNA sequence. 6. Recombinant DNA molecule according to claim 5 to Cloning of a DNA defined in claim 3. 7. Recombinant DNA molecule according to claim 5, replicable in prokaryotic or eukaryotic Host organisms, for the expression of a claim 3 defined DNA, said DNA functional with Expression control sequences is connected. 8. Recombinant DNA molecule containing an in Claim 4 defined DNA sequence.   9. Recombinant DNA molecule according to claim 8 for Cloning of a DNA defined in claim 4. 10. Recombinant DNA molecule according to claim 8, replicable in prokaryotic or eukaryotic Host organisms, for expression of a claim 4 defined DNA, said DNA functional with Expression control sequences is connected. 11. Host organism transformed with at least one Recombinant DNA molecule according to one of the claims 7 or 10. 12. Polypeptide, characterized in that it is from a DNA according to claim 3 is encoded. 13. polypeptide, characterized in that it is from a DNA according to claim 4 is encoded. 14. Polypeptide according to claim 13, characterized in that it contains the N-terminal amino acid sequence of the formula having. 15. A method for producing a polypeptide according to Claims 12-14, characterized in that a suitable host organism with recombinant DNA according to claim 3 or 4 transformed and is grown and the expressed protein is isolated becomes.   16. A polypeptide according to any one of claims 12-14 for the prophylactic or therapeutic treatment of the human or animal body in indications in which a damaging effect of TNF- α occurs. 17. Polypeptide according to any one of claims 12-14 to Treatment of inflammatory diseases. 18. Polypeptide according to any one of claims 12-14 to Treatment of infectious diseases. 19. Polypeptide according to any one of claims 12-14 to Treatment of parasitic diseases. 20. Polypeptide according to any one of claims 12-14 to Treatment of shock states. 21. Polypeptide according to any one of claims 12-14 for the treatment of pathological conditions that occur as side effects in the therapy with TNF- α . 22. Pharmaceutical compositions containing an effective inhibiting the biological activity of TNF- α amount of one or more polypeptides according to any one of claims 12-14. 23. A pharmaceutical preparation containing an effective biological inhibiting activity of TNF- β amount of one or more polypeptides according to any one of claims 12-14.