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2017-09-07 PTAB case IPR2017-02066 filed (Not Instituted - Merits) litigation https://portal.unifiedpatents.com/ptab/case/IPR2017-02066 Petitioner: Institution date: 2018-03-09 Termination date: 2018-03-09 "Unified Patents PTAB Data" by Unified Patents is licensed under a Creative Commons Attribution 4.0 International License.
This is a division of application Ser. No. 08/095,640, filed Jul. 21, 1993; now U.S. Pat. No. 5,610,279, which is a continuation application of Ser. No. 07/580,013, filed Sep. 10, 1990, now abandoned. This application claims priority under 35 U.S.C. §119 to application Serial Numbers 3319/89, 746/90 and 1347/90, filed on Sep. 12, 1989, Mar. 8, 1990 and Apr. 20, 1990, respectively, all in Switzerland. This application also claims priority under 35 U.S.C. §119 to European Patent Application Number 90116707.2—(now Patent Number EP 0417563), filed Aug. 31, 1990.
Tumor necrosis factor α (TNFα, also cachectin), discovered as a result of its hemorragic-necrotizing activity on certain tumors, and lymphotoxin (TNFβ) are two closely related peptide factors  from the class of lymphokines/cytokines which are both referred to herein-after as TNF [see references 2 and 3]. TNF possesses a broad cellular spectrum of activity. For example, TNF has inhibitory or cytotoxic activity on a series of tumor cell lines [2,3], stimulates the proliferation of fibroblasts and the phagocytic/cytotoxic activity of myeloic cells [4, 5, 6], induces adhesion molecules in endothelial cells or exerts an inhibitory activity on the endothelium [7, 8, 9, 10], inhibits the synthesis of specific enzymes in adipocytes  and induces the expression of histo-compatibility antigens . Many of these TNF activities are produced via induction of other factors or by synergistic effects with other factors such as interferons or interleukins [13-16].
This invention % further comprises DNA sequences encoding the proteins: described above, proteins encoded by these sequences, and antibodies to any of these proteins.
This invention comprises. DNA sequences which combine two partial DNA sequences, one sequence encoding soluble fragments of TNF binding proteins and the other partial sequence encoding all domains except the first domain of the constant region of the heavy chain of human immunoglobulin IgG, IgA, IgM, or IgE, and the recombinant proteins encoded by these sequences.
FIG. 1. Nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) for cDNA clone derived from 55 kD TNF-BP. The 19 amino acid transmembrane region is underlined. Hypothetical glycosylation sites are identified by asterisks.
FIG. 3. Sandwich assays of cells transfected with plasmid pK19. Culture supernatants of cells transfected 30 with pK19 were incubated with anti-55 kD TNF-BP antibody followed by 125I-TNFα. Columns 1, 5, and 8 are controls. Columns 2, 3, 4, 5, and 6 are five parallel transfections.
FIG. 4. Nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID NO: 4) for cDNA clones derived from 75/6510 TNF-BP.
TNF binding proteins may be isolated from tissues and purified to homogeneity, or isolated from cells which contain membrane-bound TNF binding protein, and purified to homogeneity. One possible method for growing cells and isolating cell extract is described in Example 2, however, other cells types and other growth and isolation methods are well known in the art. Purification of TNF-binding proteins from cell extracts may be performed using the methods described in Examples 4, 5, and 6 in combination with the assay described in Example 1. TNF-binding proteins isolated and purified by these methods were sequenced by well-known methods, as described in Example 7. From these amino acid sequences, DNA probes were produced and used to obtain mRNA encoding TNF binding proteins from which cDNA was made, all by known methods described in Examples 8 and 11. Other well-known methods for producing cDNA are known in the art and may effectively be used. In general, any TNF-binding protein can be isolated from any cell or tissue expressing such proteins using a cDNA probe such as the probe described above, isolating mRNA and transcribing the mRNA into cDNA. Thereafter, the protein can be produced by inserting the cDNA into an expression vector as described in Example 9, such as a virus, plasmid, cosmid, or other vector, inserting the expression vector into a cell, such as the COS cell-described in Example 9 or the insect cell described in Example 10, proliferating the resulting cells, and isolating the expressed TNF-binding protein from the medium or from cell extract as described above. Alternatively, TNF-binding proteins may be chemically synthesized using the sequence described above and an amino acid synthesizer, or manual synthesis using chemical conditions well known to form peptide bonds between selected amino acids. Analogues and fragments of TNF-binding proteins may be produced by the above methods. In the case of analogues, the proteins may be chemically modified, or modified by genetic engineering as described above. These fragments and analogues may then be tested for TNF-binding activity using methods such as the assay of Example 1.
A process for the isolation of the TNF-BP in accordance with the invention is also an object of the present invention. This process comprises carrying out essentially the following purification steps in sequence: production of a cell or tissue extract, immune affinity chromatography and/or single or multiple ligand affinity chromatography, high resolution liquid chromatography (HPLC) and preparative SDS-polyacrylamide gel electro phoresis (SDS-PAGE). The combination of the individual purification steps, which are known from the state of the art, is essential to the success of the process in accordance with the invention, whereby individual steps have been modified and improved having regard to the problem to be solved. Thus, for example, the original combined immune affinity chromatography/TNFα-ligand affinity chromatography step originally used for the enrichment of TNF-BP from human placenta  has been altered by using a BSA-Sepharose 4B pre-column. For the application of the cell or membrane extract, this pre-column was connected in series with the immune affinity column followed by the ligand affinity column. After the application of the extract the two afore-mentioned columns were coupled, each eluted and the TNF-BP-active fractions were purified again via a ligand affinity column. The use of a detergent-containing solvent mixture for the performance of the reversed-phase HPLC step is essential to the invention.
(c) DNA sequences which, because of the degeneracy of the genetic code, do not hybridize with sequences as defined under (a) and (b), but which code for polypeptides, having exactly the same amino acid sequence.
That is to say, the present invention embraces not only allelic variants, but also those DNA sequences which result from deletions, substitutions and additions from one or more nucleotides of the sequences given in FIG. 1 or FIG. 4, whereby in the case of the proteins coded thereby there come into consideration, just as before, TNF-BP. One sequence which results from such a deletion is described, for example, in Science 248, 1019-1023, (1990).
There are preferred first of all those DNA sequences which code for such a protein having an apparent molecular weight of about 55 kD, whereby the sequence given in FIG. 1 is especially preferred, and sequences which code for non-soluble as well as soluble fragments of such proteins. A DNA sequence which codes, for example, for such a non-soluble protein fragment extends from nucleotide −185 to 1122 of the sequence given in FIG. 1. DNA sequences which code for soluble protein fragments are, for example, those which extend from nucleotide −185 to 633 or from nucleotide −14 to 633 of the sequence given in FIG. 1. There are also preferred DNA sequences which code for a protein of about 75/65 kD, whereby those which contain the partial cDNA sequences shown in FIG. 4 are preferred. Especially preferred DNA sequences in this case are the sequences of the open reading frame of nucleotide 2 to 1,177. The peptides IIA, IIC, IIE, IIF, IIG and IIH are coded by the partial cDNA sequence in FIG. 4, whereby the insignificant deviations in the experimentally determined amino acid sequences are based on the cDNA-derived sequence with highest probability from the limited resolution of the gas phase sequencing. DNA sequences which code for insoluble (deposited on Oct. 17, 2006 with the American Type Culture Collection under Accession No. PTA 7942) as well as soluble fractions of TNF-binding proteins having an apparent molecular weight of 65 kD/75 kD are also preferred. DNA sequences for such soluble fragments can be determined on the basis of the amino acid sequences derived from the nucleic acid sequences coding for such non-soluble TNF-BP.
The present invention is also concerned with the recombinant proteins coded by any of DNA sequences described above. Of course, there are thereby also included such proteins in whose amino acid sequences amino acids have been exchanged, for example by planned mutagenesis, so that the activity of the TNF-BP or fragments thereof, namely the binding of TNF or the interaction with other membrane components participating in the signal transfer, have been altered or maintained in a desirable manner. Amino acid exchanges in proteins and peptides which do not generally alter the activity of such molecules are known in the state of the art and are described, for example, by H. Neurath and R. L. Hill in “The'Proteins” (Academic Press, New York, 1979, see especially FIG. 6, page 14). The most commonly occurring exchanges are: Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly as well as these in reverse. The present invention is also concerned with vectors which contain any of the DNA sequences described above in accordance with the invention and which are suitable for the transformation of suitable pro- and eukaryotic host systems, whereby there are preferred those vectors whose use leads to the expression of the proteins which are coded by any of the DNA sequences described above in accordance with the invention. Finally, the present invention is also concerned with pro- and eukaryotic host systems transformed with such vectors, as well as a process for the production of recombinant compounds in accordance with the invention by cultivating such host systems and subsequently isolating these compounds from the host systems themselves or their culture supernatants.
Starting materials for the TNF-BP in accordance with the invention are quite generally cells which contain such TNF-BP [in membrane-bound form] and which are generally accessible without restrictions to a person skilled in the art, such as, for example, HL60 [ATCC No. CCL 240], U 937 [ATCC No. CRL 1593], SW 480 [ATCC No. CCL 228] and HEp2 cells [ATCC No. CCL 23]. These cells can be cultivated according to known methods of the state of the art  or, in order to produce high cell densities, according to the procedure already described generally and described in detail in. Example 2 for HL60 cells. TNF-BP can then be extracted from the cells, which are centrifuged-off from the medium and washed, according to known methods of the state of the art using suitable detergents, for example Triton X-114, 1-0-n-octyl-β-D-glucopyranoside (octylglucoside) or 3-[(3-cholylamido-propyl)-dimethylammonio]-1-propane sulphonate (CHAPS), especially using Triton X-100. For the detection of such TNF-BP there can be Used the usually used detection methods for TNF-BP, for example a polyethylene glycol-1-induced precipitation of the 125I-TNF/TNF-BP complex , especially filter-binding tests with radioactively labelled TNF according to Example 1. In order to produce the TNF-BP in accordance with the invention, the general methods of the state of the art used for the purification of proteins, especially of membrane proteins, such as, for example, ion exchange chromatography, gel filtration, affinity chromatography, HPLC and SDS-PAGE can be used. Especially preferred methods for the production of TNF-BP in accordance with the invention are affinity chromatography, especially with TNF-a as the ligand bound to the solid phase, and immune-affinity chromatography, HPLC and SDS-PAGE. The elution of TNF-BP bands which are separated using SDS-PAGE can be effected according to known methods of protein chemistry, for example using electroelution according to Hunkapiller et al. , whereby according to present knowledge the electro-dialysis times given there generally have to be doubled. Thereafter, traces of SDS which still remain can then be removed in accordance with Bosserhoff et al. .
The thus-purified TNF-BP can be characterized by methods of peptide chemistry which are known in the state of the art, such as, for example, N-terminal amino acid sequencing of enzymatic well as chemical peptide cleavage. Fragments obtained by enzymatic or chemical cleavage can be separated according to usual methods such as, for example, HPLC and can themselves be subjected to further N-terminal sequencing. Such fragments which themselves bind TNF can be identified using the afore-mentioned detection methods for TNF-BP and are likewise objects of the present invention.
Starting from the thus-obtained amino acid sequence information or the DNA and amino acid sequences given in FIG. 1 as well as in FIG. 4 there can be produced, taking into consideration the degeneracy of the genetic code, according to methods known in the state of the art suitable oligonucleotides . By means of these, again according to known methods of molecular biology [42,43], cDNA or genomic DNA banks can be searched for clones which contain nucleic acid sequences coding for TNF-BP. More-over, using the polymerase chain reaction (PCR)  cDNA fragments can be cloned by completely degenerating the amino acid sequence of two spaced apart relatively short segments while taking into consideration the genetic code and introducing into their complementarity suitable oligo-nucleotides as a “primer”, whereby the fragment lying between these two sequences can be amplified and identified. The determination of the nucleotide sequence of a such a fragment permits an independent determination of the amino acid sequence of the protein fragment for which it codes. The cDNA fragments obtainable by PCR can also, as already described for the oligonucleotides themselves, be used according to known methods to search for clones containing nucleic acid sequences coding for TNF-BP from cDNA or genomic DNA banks. Such nucleic acid sequences can then be sequenced according to known methods . On the basis of the thus-determined sequences and of the already known sequences for certain receptors, those partial sequences which code for soluble TNF-BP fragments can be determined and cut out from the complete sequence using known methods .
Suitable expression vectors include, for example, vectors such as pBC12MI [ATCC 67 109], pSV2dhfr [ATCC 37 146], pSVL [Pharmacia, Uppsala, Sweden], pRSVcat [ATCC 37 152] and pMSG [Pharmacia, Uppsala, Sweden]. The vectors “pK19” and “pN123” used in Example 9 are especially preferred vectors. These can be isolated according to known methods from E. coli strains HB101(pK19) and HB101(pN123) transformed with them . These E. coli strains have been deposited on the 26 Jan. 1990 at the Deutschen Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM) in Braunschweig, FRG, under DSM 5761 for HB101(pK19) and DMS 5764 for HB101(pN123). For the expression of proteins which consist of a soluble fragment of non-soluble TNF-BP and an immunoglobulin fragment, i.e. all domains except the first of the constant region of the heavy chain, there are especially suitable pSV2-derived vectors as described, for example, by German, C. in “DNA Cloning” [Vol. II., edt. by Glover, D. M., IRL Press, Oxford, 1985]. The vectors pCD4-Hp (DSM 5315, deposited on 21 Apr. 1989), pDC4-Hγ1 (DSM 5314, deposited on 21 Apr. 1989) and pCD4-Hy3 (DSM 5523, deposited on 14 Sep. 1989) which have been deposited at the Deutschen Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM) in Braunschweig, FRG, and which are described in detail in European Patent Application No. 90107393.2 are especially preferred vectors. This European Patent Specification and the equivalent Applications referred to in Example 11 also contain data with respect to the further use of these vectors for the expression of chimeric proteins (see also Example 11) and for the construction of vectors for the expression of such chimeric proteins with other immunoglobulin fragments.
The manner in which these cells are transfected depends on the chosen expression system and vector system. An overview of these methods is to be found e.g. in Pollard et al., “DNA Transformation of Mammalian Cells” in “Methods in Molecular Biology” [Nucleic Acids Vol. 2, 1984, Walker, J. M., ed, Humana, Clifton, N.J. ]. Further methods are to be found in Chen and Okayama [“High-Efficiency Transformation of Mammalian Cells by Plasmid DNA”, Molecular and Cell Biology 7, 2745-2752, 1987] and in Feigner [Feigner et al., “Lipofectin: A highly efficient, lipid-mediated. DNA-transfection procedure”, Proc. Nat. Acad. Sci. USA 84, 7413-7417, 1987].
The baculovirus expression system, which has already been used successfully for the expression of a series of proteins (for an overview see Luckow and Summers, Bio/Technology 6, 47-55, 1988), can be used for the expression in insect cells. Recombinant proteins can be produced in authentic form or as fusion proteins. The thus-produced proteins can also be modified such as, for example, glycosylated (Smith et al., Proc. Nat. Acad. Sci. USA 82, 8404-8408, 1987). For the production of a recombinant baculovirus which expresses the desired protein there is used a so-called “transfer vector”. Under this there is to be understood a plasmid which contains the heterologous DNA sequence under the control of a strong promoter, e.g. that of the polyhedron gene, whereby this is surrounded on both sides by viral sequences. The vectors “pN113”, “pN119” and “pN124” used in Example 10 are especially preferred vectors. These can be isolated according to known methods from E. coli strains HB101(pN113), HB101(pN119) and HB101(pN124) transformed with them. These E. coli strains have been deposited on the 26 Jan. 1990 at the Deutschen Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM) in Braunschweig, FRG, under DSM 5762 for HB101(pN113), DSM 5763 for HB101(pN119) and DSM 5765 for HB101(pN124). The transfer vector is then transfected into the insect cells together with DNA of the wild type baculovirus. The recombinant viruses which result in the cells by homologous recombination can then be identified and isolated according to known methods. An overview of the baculovirus expression system and the methods used therein is to be found in Luckow and Summers .
The TNF-BP obtained in accordance with the invention can also be used as antigens to produce polyclonal and monoclonal antibodies according to known-techniques [44, 45] or according to the procedure described in Example 3. Such antibodies, especially monoclonal antibodies against the 75 kD TNF-BP species, are also an object of the present invention. Those antibodies which are directed against the 75 kD TNF-BP can be used for the isolation of TNF-BP by modifications of the purification procedure described in detail in Examples 4-6 which are familiar to a person skilled in the art.
On the basis of the high binding affinity of TNF-BP in accordance with the invention for TNF (Kd value in the order of 10−9-10−10 M), these or fragments thereof can be used as diagnostics for the detection of TNF in serum or other body fluids according to methods known in the state of the art, for example in solid phase binding tests or in combination with anti-TNF-BP antibodies in so-called “sandwich” tests.
The TNF-BP in accordance with the invention as well as their physiologically compatible salts, which can be manufactured according to methods which are known in the state of the art, can also be used for the production of pharmaceutical preparations, primarily those for the treatment of illnesses in which TNF is involved in their course. For this purpose, one or more of the said compounds, where desired or required in combination with other pharmaceutically active substances, can be processed in a known manner with the usually used solid or liquid carrier. materials. The dosage of such preparations can be effected having regard to the usual criteria in analogy to already used preparations of similar activity and structure.
The TNF-BP were detected in a filter test with human radioiodinated 125I-TNF. TNF (46, 47) was radioactively labelled with Na125I (IMS40, Amersham, Amersham, England) and iodo gene (#28600, Pierce Eurochemie, Oud-Beijerland, Netherlands) according to Fraker and Speck . For the detection of the TNF-BP, isolated membranes of the cells or their solubilized, enriched and purified fractions were applied to moist nitrocellulose filter (0.45μ, BioRad, Richmond, Calif., USA). The filters were then blocked in buffer solution with 1% skimmed milk powder and subsequently incubated with 5·105 cpm/ml of 125I-TNFα (0.3-1.0·108 cpm/μg) in two batches with and without the addition of 5 μg/ml of non-labelled TNFα, washed and dried in the air. The bound radio-activity was detected semiquantitatively by autoradiography or counted in a gamma-counter. The specific 125I-TNF-α binding was determined after correction for unspecific binding in the presence of unlabelled TNF-α in excess. The specific TNF-binding in the filter test was measured at various TNF concentrations and analyzed according to Scatchard, whereby a Kd value of ·10−9-10−10 M was determined.
HL60 cells [ATCC No. CCL 240] were cultivated on an experimental laboratory scale in a RPMI 1640 medium [GIBCO catalogue No. 074-01800], which contained 2 g/l NaHCO3 and 5% foetal calf serum, in a 5% CO2 atmosphere, and subsequently centrifuged.
The following procedure was used to produce high cell densities on an industrial scale. The cultivation was carried out in a 75 l Airlift fermenter (Fa. Chemap, Switzerland) with a working volume of 58 l. For this there was used the cassette membrane system “PROSTAK” (Millipore, Switzerland) with a membrane surface of 0.32 m2(1 cassette) integrated into the external circulation circuit. The culture medium (see Table 1) was pumped around with a Watson-Marlow pump, Type 603U, with 5 l/min. After a steam sterilization of the installation, whereby the “PROSTAK” system was sterilized separately in autoclaves, the fermentation was started with growing HL-60 cells from a 20 l Airlift fermenter (Chemap). The cell cultivation in the inoculation fermenter was effected in a conventional batch process in the medium according to Table 1 and an initial cell titre of 2×105 cells/ml. After 4 days the HL60 batch was transferred with a titre of 4.9×106 cells/ml into the 75 l fermenter. The pH value was held at 7.1 and the pO2 value was held at 25% saturation, whereby the oxygen introduction was effected through a microporous frit. After initial batch fermentation, on the 2nd day the perfusion at a cell titre of 4×106 cells/ml was started with 30 l of medium exchange per day. On the filtrate side of the medium the conditioned medium was removed and replaced by the addition of fresh medium. The added medium was fortified as follows: Primatone from 0.25% to 0.35%, glutamine from 5 mM to 6 mM and glucose from 4 g/l to 6 g/l. The perfusion rate was then increased on the 3rd and 4th day to 72 l of medium/day and on the 5th day to 100 l of medium/day. The fermentation had finished after 120 hours of continuous cultivation. Exponential cell growth up to 40×106 cells/ml took place under the given fermentation conditions. The duplication time of the cell population was 20-22 hours to 10×106 cells/ml and then increased to 30-36 hours with increasing cell density. The proportion of living cells lay at 90-95% during the entire fermentation period. The HL-60 batch was then cooled down in the fermenter to about 12° C. and the cells were harvested by centrifugation (Beckman centrifuge [Model J-6B, Rotor JS], 3000 rpm, 10 min., 4° C.).
The centrifugate was washed with isotonic phosphate buffer (PBS; 0.2 g/l KCl, 0.2 g/l KH2PO4, 8.0 g/1 NaCl, 2.16 g/l Na2HPO4.7H20), which had been treated with 5% dimethylformamide, 10 mM benzamidine, 100 U/ml aprotinin, 10 μM leupeptin, 1 μM pepstatin, 1 mM o-phenanthroline, 5 mM iodoacetamide, 1 mM phenyl-methylsulphonyl fluoride (referred to hereinafter as PBS-M). The washed cells were extracted at a density of 2.5·108 cells/ml in PBS-M with Triton X-100 (final concentration 1.0%). The cell extract was clarified by centrifugation (15,000×g, 1 hour; 100,000×g, 1 hour).
A centrifugation supernatant from the cultivation of HL60 cells on an experimental laboratory scale, obtained according to Example 2, was diluted with PBS in the ratio 1:10. The diluted supernatant was applied at 4° C. (flow rate: 0.2 ml/min.) to a column which contained 2 ml of Affigel 10 (Bio Rad Catalogue No. 153-6099) to which had been coupled 20 mg of recombinant human TNF-α [Pennica, D. et al. (1984) Nature 312, 724; Shirai, T. et al. (1985) Nature 313, 803; Wang, A. M. et al. (1985) Science 228, 149] according to the recommendations of the manufacturer. The column was washed at 4° C. and a throughflow rate of 1 ml/min firstly with 20 ml of PBS which contained 0.1% Triton X 114 and thereafter with 20 ml of PBS. Thus-1-enriched TNF-BP was eluted at 22° C. and a flow rate of 2 ml/min with 4 ml of 100 mM glycine, pH 2.8, 0.1% decyl-maltoside. The eluate was concentrated to 10 μl in a Centricon 30 unit [Amicon].
10 μl of this eluate were mixed with 20 μl of complete Freund's adjuvant to give an emulsion. 10 μl of the emulsion were injected according to the procedure described by Holmdahl, R. et al. [(1985), J. Immunol. Methods 32, 379] on each of days 0, 7 and 12 into a hind paw of a narcotized Balb/c mouse.
The immunized mice were sacrificed on day 14, the popliteal lymph nodes were removed, minced and suspended by repeated pipetting in Iscove's medium (IMEM, GIBCO Catalogue No. 074-2200) which contained 2 g/l NaHCO3. According to a modified procedure of De St. Groth and Scheidegger [J. Immunol. Methods (1980), 35, 1] 5×107 cells of the lymph nodes were fused with 5×107PAI mouse myeloma cells (J. W. Stocker et al., Research Disclosure, 217, May 1982, 155-157) which were in logarithmic growth. The cells were mixed, collected by centrifugation and resuspended in 2 ml of 50% (v/v) polyethylene glycol in IMEM at room temperature by slight shaking and diluted by the slow addition of 10 ml of IMEM during careful shaking for 10 minutes. The cells were collected by centrifugation and resuspended in 200 ml of complete medium [IMEM+20% foetal calf serum, glutamine (2.0 mM), 2-mercaptoethanol (100 μl), 100 μM hypoxanthine, 0.4 μM aminopterine and 16 μM thymidine (HAT)]. The suspension was distributed on 10 tissue 10 culture dishes each containing 96 wells and incubated at 37° C. for 11 days without changing the medium in an atmosphere of 5% CO2 and a relative humidity of 98%.
The antibodies are distinguished by their inhibitory action on the binding of TNF to HL60 cells or by their binding to antigens in the filter test according to Example 1. The following procedure was used to detect the biological activity of anti(TNF-BP) antibodies: 5×106 HL60 or U937 cells were incubated in complete RPMI 1640 medium together with affinity-purified monoclonal anti-(TNF-BP) antibodies or control antibodies (i.e. those which are not directed against TNF-BP) in a concentration range of 1 ng/ml to 10 μg/ml. After incubation at 37° C. for one hour the cells were collected by centrifugition and washed with 4.5 ml of PBS at 0° C. They were resuspended in 1 ml of complete RPMI 1640 medium (Example 2) which additionally contained 0.1% sodium azide and 125I-TNFα (106 cpm/ml) with or without the addition of unlabelled TNFα (see above). The specific radioactivity of the 125I-TNFα amounted to 700 Ci/mmol. The cells were incubated at 4° C. for 2 hours, collected and washed 4 times at 0° C. with 4.5 ml of PBS which contained 1% BSA and 0.001% Triton X 100 (Fluka). The radioactivity bound to the cells was measured in a γ-scintillation counter. The cell-bound radioactivity of cells which had not been treated with anti-(TNF-BP) antibodies was determined in a comparative experiment (approximately 10 000 cpm/5×106 cells).
For the further purification, a monoclonal anti-(55 kD TNF-BP) antibody (2.8 mg/ml gel), obtained according to Example 3, TNFα (3.9 mg/ml gel) and bovine serum albumin (BSA, 8.5 mg/ml gel) were each covalently coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) according to the directions of the manufacturer. The cell extract obtained according to Example 2 was passed through the thus-prepared columns which were connected in series in the following sequence: BSA-Sepharose pre-column, immune affinity column (anti-(55 kD-TNF-BP) antibody), TNFα-ligand affinity column. After completion of the application the two last-mentioned columns were separated and washed individually with in each case 100 ml of the following buffer solutions: (1) PBS, 1,0% Triton X-100, 10 mM benzamidine, 100 U/ml aprotinin; (2) PBS, 0.1% Triton X-100, 0.5M NaCl, 10 mM ATP, 10 mM benzamidine, 100 U/ml aprotinin; and (3) PBS, 0.1% Triton X-100, 10 mM benzamidine, 100 U/ml aprotinin. Not only the immune affinity column, but also the TNFα-ligand affinity column were then each eluted with 100 mM glycine pH 2.5, 100 mM NaCl, 0.2% decylmaltoside, 10 mM benzamidine, 100 U/ml aprotinin. The fractions of each column which were active in the filter test according to Example 1 were thereafter combined and neutralized with 1M Tris pH 8.0.
The thus-combined TNF-BP active fractions of the immune affinity chromatography on the one hand and of the TNFα-ligand affinity chromatography on the other hand were, for further purification, again applied to in each case one small TNFa-ligand affinity column. Thereafter, these two columns were washed with in each case 40 ml of (1) PBS, 1.0% Triton X-100, 10 mM benzamidine, 100 U/ml aprotinin, (2) PBS, 0.1% Triton X-100, 0.5M NaCl, 10 mM ATP, 10 mM benzamidine, 100 U/ml aprotinin, (3) PBS, 0.1% Triton X-100, (4) 50 mM Tris pH 7.5, 150 mM NaCl, 1.0% NP-40, 1.0% desoxycholate, 0.1% SDS, (5) PBS, 0.2% decyl-maltoside. Subsequently, the columns were eluted with 100 mM glycine pH 2.5, 100 mM NaCl, 0.2% decylmaltoside. Fractions of 0.5 ml from each column were collected and the fractions from each column which were active according to the filter test (Example 1) were combined and concentrated in a Centricon unit (Amicon, molecular weight exclusion 10,000).
The active fractions obtained according to Example 4 were each applied according to their different source (immune or ligand affinity chromatography) to C1/C8 reversed phase HPLC columns (ProRPC, Pharmacia, 5×20 mm) which had been equilibrated with 0.1% trifluoroacetic acid, 0.1% octylglucoside. The columns were then eluted with a linear acetonitrile gradient (0-80%) in the same buffer at a flow of 0.5 ml/min. Fractions of 1.0 ml were collected from each column and the active fractions from each column were combined (detection according to Example 1).
Under the mentioned conditions there were obtained for samples which has been obtained according to Example 4 by TNF-α-ligand affinity chromatography of immune affinity chromatography eluates and which had been further separated by HPLC according to Example 5 two bands of 55 kD and 51 kD as well as three weaker bands of 38 kD, 36 kD and 34 kD. These bands were transferred electro-phoretically during 1 hour at 100 V in 25 mM Tris, 192 mM glycine, 20% methanol on to a PVDF membrane (Immobilon, Millipore, Bedford, Mass. USA) in a Mini Trans Blot System (BioRad, Richmond, Calif., USA). Thereafter, the PVDF membrane was either protein-stained with 0.15% Serva-Blue (Serva, Heidelberg, FRG) in methanol/water/glacial acetic acid (50/40/10 parts by volume) or blocked with skimmed milk powder and subsequently incubated with 125I-TNFα according to the filter test conditions described in Example 1 in order to detect bands having TNF-BP activity. This showed that all bands produced in the protein staining bonded TNFα specifically. In the Western blot according to Towbin et al.  all of these bands also bonded the monoclonal anti-55 kD-TNF-BP antibody produced according to Example 3. In this case, a procedure according to that described in Example 1 with Na125I radioactively-labelled, affinity-purified (mouse immuno-globulin-Sepharose-4B affinity column) rabbit-anti-mouse-immunoglobulin antibody was used for the autoradiographic detection of this antibody.
Gly-Ser-Gln-Gly-Pro-Glu-Gln-Gln-X-X-Leu-Ile-X-Ala-Pro (SEQ ID NO: 14), in which X stands for an amino acid residue which could not be determined.
Starting from the amino acid sequence according to formula IA there were synthesized having regard to the genetic code for the amino acid residues 2-7 and 17-23 corresponding completely degenerated oligonucleotides in suitable complementarity (“sense” and “antisense” oligonucleotides). Total cellular RNA was isolated from HL60 cells [42,43] and the first cDNA strand was synthesized by oligo-dT priming or by priming with the “antisense” oligonucleotide using a cDNA synthesis kit (RPN 1256, Amersham, Amersham; England) according to the instructions of the manufacturer. This cDNA strand and the two synthesized degenerate “sense” and “anti-sense” oligonucleotides were used in a polymerase chain reaction (PCR, Perkin Elmer Cetus, Norwalk, Conn., USA according to the instructions of the manufacturer) to synthesize as a cDNA fragment the base sequence coding for the amino acid residues 8-16 (formula IA). The base sequence of this cDNA fragment accorded to: 5′-AGGGAGAAGAGAGATAGTGTGTGTCCC-3′ (SEQ ID NO: 16). This cDNA fragment was used as a probe in order to identify according to a known procedure a cDNA clone coding for the 55 kD TNF-BP in a Xgt11-cDNA gene bank from human placenta (42, 43). This clone was then cut according to usual methods from the X-vector and cloned in the plasmids pUC18 (Pharmacia, Uppsala, Sweden) and pUC19 (Pharmacia, Uppsala, Sweden) and in the M13 mp 18/M13 mp 19 bacteriophage (Pharmacia, Uppsala, Sweden) (42, 43). The nucleotide sequence of this cDNA clone was determined using a Sequenase kit (U.S. Biochemical, Cleveland, Ohio, USA) according to the details of the manufacturer. The nucleotide sequence and the amino acid sequence derived therefrom for the 55 kD TNF-BP and its signal peptide (amino acid “−28” to amino acid “0”) is given in FIG. 1 using the abbreviations for bases such as amino acids usual in the state of the art. From sequence comparisons with other already known receptor protein sequences there can be determined a N-terminal domain containing approximately 180 amino acids and a C-terminal domain containing 220 amino acids which are separated from one another by a transmembrane region of 19 amino acids (underlined in FIG. 1) which is typical according to the sequence comparisons. Hypothetical glycosylation sites are characterized in FIG. 1 by asterisks above the corresponding amino acid.
Essentially analogous techniques were used to identify 75/65 kD TNF-BP-coding partial cDNA sequences, whereby however, in this case genomic human DNA and completely degenerated 14-mer and 15-mer “sense” and “antisense” oligonucleotides derived from peptide IIA were used in order to produce a primary 26 by cDNA probe in a polymerase chain reaction. This cDNA probe was then used in a HL-60 cDNA library to identify cDNA clones of different lengths. This cDNA library was produced using isolated HL60 RNA and a cDNA cloning kit (Amersham) according to the details of the manufacturer. The sequence of such a cDNA clone is given in FIG. 4, whereby repeated sequencing lead to the following correction. A threonine coded by “ACC” not “TCC”, has to be at position 3 instead of the serine.
Vectors starting from the plasmid “pN11” were constructed for the expression in COS cells. The plasmid “pN11” contains the efficient promoter and enhancer of the “major immediate-early” gene of human cytomegalovirus (“HCMV”; Boshart et al., Cell 41, 521-530, 1985). After the promoter there is situated a short DNA sequence which contains several restriction cleavage sites, which are present only once in the plasmid (“polylinker”), inter alia the cleavage sites for HindIII, BalI, BamHI and PvuII (see sequence).
After these cleavage sites there are situated three translation stop codons in all three reading frames. After the polylinker sequence there is situated the 2nd intron and the polyadenylation signal of the preproinsulin gene of the rat (Lomedico et al., Cell 18, 545-558, 1979). The plasmid also contains the replication origin of the SV40 virus and a fragment from pBR322 which confers E. coli-bacteria ampicillin resistance and permits the replication of the plasmid in E. coli.
For the construction of the expression vector “pN123”, this plasmid “pN11” was cleaved the restriction endo-nuclease PvuII and subsequently treated with alkaline phosphatase. The dephosphorylated vector was thereafter isolated from an agarose gel (V1). The 5′-projecting nucleotides of the EcoRI-cleaved 1.3 kb fragment of the 55 kD TNF-BP-cDNA (see Example 8) were filled in using Klenow enzyme. Subsequently, this fragment was isolated from an agarose gel (F1). Thereafter, V1 and F1 were joined together using T4-ligase. E. coli HB101 cells were then transformed with this ligation batch according to known methods . By means of restriction analyses and DNA sequencing according to known methods  there were identified transformants which had been transformed with a plasmid and which contained the 1.3 kb EcoRI fragment of the 55 kD TNF-BP-cDNA in the correct orientation for expression via the HCMV-promoter. This vector received the designation “pN123”.
with the pN11 vector (columns # 1, 5) and of a control with HL60 cell lysate (column # 8) are given in FIG. 3.
E. coli HB101 was transformed with the ligation batch and transformants containing a plasmid in which the oligonucleotide had been incorporated correctly were identified by restriction analysis and DNA sequencing according to known methods (see above); this plasmid was named “pNR704”. For the construction of the transfer vector “pN113”, this plasmid “pNR704” was cleaved with EcoRI, treated with alkaline phosphatase and the thus-1-produced vector trunk (V2) was subsequently isolated from an agarose gel. The 1.3 kb fragment of the 55 kD TNF-BP-cDNA cleaved with EcoRI as above was ligated with fragment V2. Transformants obtained with this ligation batch, which contained a plasmid containing the cDNA insert in the correct orientation for the expression via the polyhedron promoter, were identified (see above). The vector isolated therefrom received the designation “pN113”.
Two stop codons of the translation after amino acid 182 and a cleavage site for the restriction endo-nuclease Asp718 are incorporated with the above adaptor. After carrying out ligation the batch was digested with EcoRI and Asp718 and the partial 55 kD TNF-BP fragment (F3) was isolated. Furthermore, the plasmid “pNR704”, likewise cleaved with Asp718 and EcoRI, was ligated with F3 and the ligation batch was transformed into E. coli HB101. The identification of the transformants which contained a plasmid in which the partial 55 kD TNF-BP cDNA had been correctly integrated for the expression was effected as already described. The plasmid isolated from these transformants received the name “pN119”.
The following procedure was used for the construction of the transfer vector “pN124”. The cDNA fragment coding for the extracellular part of the 55 kD TNF-BP, described in Example 9, was amplified with the specified oligo-nucleotides with the aid of PCR technology as described in Example 9. This fragment was cleaved with BamHI and Asp718 and isolated from an agarose gel (F4). The plasmid “pNR704” was also cleaved with BamHI and Asp718 and the vector trunk (V4) was isolated (see above). The fragments V4 and F4 were ligated, E. coli HB101 was transformed therewith and the recombinant transfer vector “pN124” was identified and isolated as described.
The following procedure was used for the transfection 10 of the insect cells. 3 μg of the transfer vector “pN113” were transfected with 1 μg of DNA of the Autographa californica nuclear polyhedrosisvirus (AcMNPV) (EP 127839) in Sf9 cells (ATCC CRL 1711). Polyhedron-negative viruses were identified and purified from “plaques” . Sf9 cells were again infected with these recombinant viruses as described in . After 3 days in the culture the infected cells were investigated for TNF-binding using 125I-TNFα. For this purpose, the transfected cells were washed from the cell culture dish with a Pasteur pipette and resuspended at a cell density of 5×106 cells/ml of culture medium  which contained 10 ng/ml of 125I-TNF-α, not only in the presence of, but also in the absence of 5 μg/ml of non-labelled TNF-α and incubated on ice for 2 hours. Thereafter, the cells were washed with pure culture medium and the cell-bound radio-activity was counted in a γ-counter (see Table 2).
This cDNA fragment was ligated in the pCD4-Hy3 vector [DSM 5523; European Patent Application No. 90107393.2; Japanese Patent Application No. 108967/90; U.S. Pat. No. 51,077,390] from which the CD4-cDNA had been removed via the SstI restriction cleavage sites. SstI cleavage sites are situated in vector pCD4-Hγ3 not only in front of, but also behind the CD4-partial sequence fragment. The construction was transfixed in J558 myeloma cells (ATCC No. TIB6) by means of protoplast fusion according to Oi et al. (Procd. Natl. Acad. Sci. USA 80, 825-829, 1983). Transfectants were selected by adding 5 μg/ml of mycophenolic acid and 250 g/ml of xanthin (Traunecker et al., Eur. J. Immunol. 16, 851-854 ) in basic medium (Dulbecco's modified Eagle's Medium, 10% foetal calf serum, 5×10−5M 2-mercaptoethanol). The expression product secreted by the transfixed cells could be purified using usual methods of protein chemistry, e.g. TNF-BP-antibody affinity chromatography. Unless not already specifically indicated, standard procedures as described e.g. by Freshney, R. I. in “Culture of Animal Cells”, Alan R. Liss, Inc., New York (1983) were used for the cultivation of the cell lines employed, for the cloning, for the selection or for the expansion of the cloned cells.
wherein said protein specifically binds human TNF.
2. The protein of claim 1, wherein the soluble fragment comprises the peptides LCAP (SEQ ID NO: 12) and VFCT (SEQ ID NO: 8).
3. The protein of claim 2, wherein the soluble fragment further comprises the peptide LPAQVAFXPYAPEPGSTC (SEQ ID NO: 10).
4. The protein of claim 1, wherein said human immunoglobulin IgG heavy chain is IgG1.
5. The protein of claim 4, wherein said domains of the constant region of the human immunoglobulin heavy chain consist essentially of the immunoglobulin amino acid sequence encoded by pCD4-Hγ1 vector (deposited at Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM 5314).
6. A pharmaceutical composition comprising the protein of claim 4 and a pharmaceutically acceptable carrier material.
7. The protein of claim 1, wherein the protein is purified.
8. The protein of claim 1, wherein the protein is produced by CHO cells.
9. The protein of claim 1, wherein the protein consists of (a) the soluble fragment of the receptor and (b) all of the domains of the constant region of the human immunoglobulin IgG heavy chain other than the first domain of the constant region.
10. The protein of claim 1, wherein said domains of the constant region of the human immunoglobulin heavy chain consist essentially of the immunoglobulin amino acid sequence encoded by pCD4-Hγ1 vector (deposited at Deutschen Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM 5314) or by pCD4-Hγ3 vector (deposited at Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM 5523).
11. The protein of claim 1, wherein the protein consists essentially of the extracellular region of the insoluble human TNF receptor and all the domains of the constant region of a human IgG1 immunoglobulin heavy chain other than the first domain of the constant region.
12. A pharmaceutical composition comprising the protein of claim 11 and a pharmaceutically acceptable carrier material.
14. The protein of claim 13, wherein the protein is purified.
15. The protein of claim 13, wherein the protein is produced by CHO cells.
16. The protein of claim 13, wherein the protein consists of (a) the soluble fragment of the receptor and (b) all of the domains of the constant region of the human IgG1 heavy chain other than the first domain of the constant region.
17. The protein of claim 13, wherein the soluble fragment further comprises the peptide LPAQVAFXPYAPEPGSTC (SEQ ID NO: 10).
19. The protein of claim 18, wherein the soluble fragment comprises the peptides LCAP (SEQ ID NO: 12) and VFCT (SEQ ID NO: 8).
20. The protein of any one of claim 18 or 19, wherein said human immunoglobulin heavy chain is IgG1.
21. The protein of claim 20, wherein said domains of the constant region of the human immunoglobulin heavy chain consist essentially of the immunoglobulin amino acid sequence encoded by the DNA insert of pCD4-Hγ1 vector (deposited at Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM 5314).
22. The protein of claim 18, wherein the protein is purified.
23. The protein of claim 18, wherein the protein is produced by CHO cells.
24. The protein of claim 18, wherein the protein consists of (a) the soluble fragment of the receptor and (b) all of the domains of the constant region of the human IgG1 heavy chain other than the first domain of the constant region.
25. The protein of claim 18, wherein said domains of the constant region of the human immunoglobulin heavy chain consist essentially of the immunoglobulin amino acid sequence encoded by pCD4-Hγ1 vector (deposited at Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM 5314) or by pCD4-Hγ3 vector (deposited at Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM 5523).
wherein the protein is produced by CHO cells.
27. The protein of claim 26, wherein the soluble fragment comprises the peptide LPAQVAFXPYAPEPGSTC (SEQ ID NO: 10).
28. The protein of claim 26, wherein the protein is purified.
29. A pharmaceutical composition comprising the protein of any of claim 1, 18, 26, or 27 and a pharmaceutically acceptable carrier material.
31. The protein of claim 30, consisting of the soluble fragment and all the domains of the constant region of the human immunoglobulin IgG heavy chain other than the first domain of said constant region.
32. The protein of claim 30, wherein the protein is expressed by a mammalian host cell.
33. The protein of claim 32, wherein the mammalian host cell is a CHO cell.
34. The protein of claim 32, consisting of the soluble fragment and all the domains of the constant region of the human immunoglobulin IgG heavy chain other than the first domain of said constant region.
35. The protein of claim 30, wherein the protein consists essentially of the extracellular region of the human tumor necrosis factor (TNF) receptor amino acid sequence encoded by the cDNA insert, and all the domains of the constant region of a human IgG1 immunoglobulin heavy chain other than the first domain of the constant region.
36. A pharmaceutical composition comprising the protein of claim 35 and a pharmaceutically acceptable carrier material.
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CA2266439C (en) 2009-06-16 Neutrokine .alpha.

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