WO1998002557A2 - Fnt-alpha convertase mammalienne - Google Patents

Fnt-alpha convertase mammalienne Download PDF

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Publication number
WO1998002557A2
WO1998002557A2 PCT/US1997/011637 US9711637W WO9802557A2 WO 1998002557 A2 WO1998002557 A2 WO 1998002557A2 US 9711637 W US9711637 W US 9711637W WO 9802557 A2 WO9802557 A2 WO 9802557A2
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Prior art keywords
tnf
convertase
leu
substrate
nucleic acid
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PCT/US1997/011637
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WO1998002557A3 (fr
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Barbara Dalie
Xuedong Fan
Daniel Lundell
Charles A. Lunn
Jimmy C. Tan
Paul J. Zavodny
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Schering Corporation
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Priority to JP10506072A priority Critical patent/JP2000500660A/ja
Priority to EP97931547A priority patent/EP0912746A2/fr
Priority to AU35149/97A priority patent/AU3514997A/en
Publication of WO1998002557A2 publication Critical patent/WO1998002557A2/fr
Publication of WO1998002557A3 publication Critical patent/WO1998002557A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)

Definitions

  • the present invention relates to mammalian tumor necrosis factor- ⁇ (TNF- ⁇ ) convertase enzymes. More particularly, it relates to bovine, human and other TNF- ⁇ convertases, isolated nucleic acids and recombinant vectors encoding the enzymes, methods for making the enzymes, fragments or fusion proteins thereof using recombinant DNA methodology or chemical synthesis, and to methods for using the enzymes in screening systems to identify TNF- ⁇ convertase inhibitors for the treatment of various diseases, and nucleic acids encoding a TNF- ⁇ convertase.
  • This invention further relates to antibodies, both polyclonal and monoclonal, which specifically bind to the TNF- ⁇ convertases, and to fragments and fusion proteins of the TNF- ⁇ convertases of the invention.
  • TN F- ⁇ also known as cachectin, is a 17 kDa (kilodalton) protein produced by cells of the monocyte/macrophage lineage, and by other cells.
  • TNF- ⁇ is beneficial, e.g., in that it is believed to be a part of host anti-tumor defenses. It also produces detrimental effects, however, including, e.g.
  • TNF- ⁇ is a key mediator of inflammation (including inflammatory diseases such as arthritis) and mammalian responses to injury, invasion by pathogens, and neoplasia.
  • TNF- ⁇ The biosynthesis of human TNF- ⁇ proceeds by way of a membrane-bound precursor containing 233 amino acid residues [Wang et al, Science 225: 149- 154 (1985); Muller et al., Nature 335: 265 -267 ( 1987)], which is processed during cellular activation by cleavage of a 76-residue peptide to produce the mature, secreted form of TNF- ⁇ .
  • the enzyme(s) responsible for this cleavage, called TNF- ⁇ convertase has until the present invention been elusive for most mammalian species.
  • TNF- ⁇ convertase A putative TNF- ⁇ convertase, called PR-3, has been isolated and cloned from human neutrophils, and it has been suggested that this enzyme can be used in screens to identify TNF- ⁇ convertase inhibitors. See International Patent Applications Publication Numbers WO 94/00555 and WO 95/24501. This enzyme, however, is not believed to be the physiologically relevant human TNF- ⁇ convertase because it is a serine protease, whereas the relevant enzyme is believed to be a metalloproteinase.
  • the source of the serine protease, neutrophils, is not believed to be important in the production of TNF- ⁇ , and the serine protease does not cleave the precursor form of TNF- ⁇ ( proTNF- ⁇ ) at the point expected for the physiologically relevant human enzyme.
  • TNF- ⁇ In view of the important role of TNF- ⁇ in many disease processes, there is a need for agents that can selectively block the biosynthesis of mature, secreted TNF- ⁇ .
  • the search for such agents would be greatly facilitated by the availability of substantially pure mammalian TNF- ⁇ convertases.
  • the present invention fills the foregoing need by providing materials and methods for identifying specific inhibitors of TNF- ⁇ convertase. More particularly, this invention provides substantially pure mammalian TNF- ⁇ convertases capable of converting proTNF- ⁇ to the mature, secreted form. This invention further provides isolated or recombinant nucleic acids encoding mammalian TNF- ⁇ convertases, and recombinant vectors and host cells comprising such nucleic acids.
  • This invention further provides a method for making a mammalian TNF- ⁇ convertase, comprising culturing a host cell comprising a nucleic acid encoding a mammalian TNF- ⁇ convertase under conditions in which the nucleic acid is expressed.
  • the method further comprises isolation of the TNF- ⁇ convertase from the culture.
  • This invention also provides polypeptides comprising a fragment of a TNF- ⁇ convertase having an amino acid sequence corresponding to the sequence of at least about 8 contiguous residues of the complete enzyme sequence.
  • the polypeptides comprise at least about 12, more preferably at least about 20, and most preferably at least about 30 such residues.
  • this invention provides fusion proteins comprising a
  • TNF- ⁇ convertase or a polypeptide thereof covalently linked to a fusion partner .
  • the present invention also provides antibodies, both polyclonal and monoclonal, that specifically bind to one or more of the TNF- ⁇ convertases or to a polypeptide thereof. Also provided are anti- idiotypic antibodies, both monoclonal and polyclonal, which specifically bind to the foregoing antibodies.
  • This invention still further provides a method of treatment comprising administering to a mammal afflicted with a medical condition caused or mediated by TNF- ⁇ , an effective amount of an antibody, or an antigen-binding fragment thereof, that specifically binds to a mammalian TNF- ⁇ convertase, and pharmaceutical compositions comprising such antibodies or fragments and pharmaceutically acceptable carriers.
  • the present invention also provides a method for identifying an inhibitor of a mammalian TNF- ⁇ convertase, comprising:
  • the contacting of the convertase with the sample in the presence of substrate occurs on the surface of a mammalian host cell comprising one or more nucleic acids encoding a mammalian TNF- ⁇ convertase and a substrate of the convertase.
  • Fig. 1 is an elution profile from an HPLC column showing DNP- proTNF- ⁇ cleavage products
  • Fig. 2 is a graphical representation of results from an assay in which human proTNF- ⁇ was cleaved by membrane-type matrix metalloproteases MT-MMP1 and MT-MMP3; and
  • Fig. 3 is a graphical representation of results from an assay in which cleavage of human proTNF- ⁇ by MT-MMP1 was inhibited by varying amounts of an MMP inhibitor.
  • the mammalian TNF- ⁇ convertases of the present invention are functionally characterized by an ability to process, through proteolytic cleavage, the conversion of the membrane-bound form of a precursor form of TNF- ⁇ , referred to herein as "proTNF- ⁇ ", to the soluble, mature form.
  • This processing entails cleavage of the first 76 amino-terminal residues of the human precursor protein, the entire sequence of which is defined in the Sequence Listing by SEQ ID NO: 1 .
  • This sequence taken from Human Cytokines, B. Aggarwal and J. Gutterman, Eds., 1992, Blackwell Scientific Publications, Oxford, pp. 276-277, is in agreement with the Swiss-Prot sequence, Accession Code: Swiss-Prot P01375.
  • GenBank sequence [Accession No. M 10988; Wang et al, Science 228: 149 ( 1985)], which has a serine residue at position - 14, instead of the phenylalanine residue shown at that position in SEQ ID NO: 1.
  • cleavage of proTNF- ⁇ by the TNF- ⁇ convertases of this invention occurs at an Ala- Val peptide bond, resulting in mature human TNF- ⁇ having a valine residue at the amino terminus (i.e., beginning with the Val residue at position 1 of SEQ ID NO: 1 ).
  • the foregoing cleavage point is different from that observed for the serine protease PR-3 mentioned above. Robache-Gallea et al. [J. Biol. Chem.
  • TNF- ⁇ convertases of the present invention are further characterized by their presence in cells that make TNF- ⁇ . They may be present in other cells as well, however, and may even be ubiquitously expressed. Control could be exerted at the level of transcription of the proTNF- ⁇ message, or specific controllers of the T N F- ⁇ convertases could be present in different cell types.
  • TNF- ⁇ convertases cleave and process only pro TNF- ⁇ ; they could have other substrates as well. It also may be that different TNF- ⁇ convertases process proTNF- ⁇ in different cell types. Thus, one convertase might carry out processing in T and NK cells, while a different enzyme might function in macrophages. It therefore may not be necessary that a given TNF- ⁇ convertase be present in all cell types that make TNF- ⁇ .
  • TNF- ⁇ convertase of this invention be present in at least one type of cell that makes TNF- ⁇ , whether the other possibilities discussed in the foregoing paragraph are correct or not is not essential to the invention.
  • This bovine enzyme is further characterized by behavior observed in various chromatographic systems during purification, as is described in detail in the Example below, and by an apparent molecular weight in SDS-PAGE under reducing conditions of about 65 kDa.
  • the present invention also encompasses another bovine TNF- ⁇ convertase and enzymes from other mammalian species, including human TNF- ⁇ convertases.
  • TNF- ⁇ convertases as defined in this invention are as follows:
  • the proteins of the present invention are useful in rational drug discovery screens for the identification of compounds that selectively block the conversion of proTNF- ⁇ to soluble, mature TNF- ⁇ . They have this utility because when introduced into cells used in the screens, e.g., by transfection of nucleic acids encoding the proteins, they produce TNF- ⁇ convertase activity which can act on an appropriate substrate as described herein. Inhibitors can be identified by measuring inhibition of this activity.
  • bovine TNF- ⁇ convertase in one embodiment means an enzyme having the above-mentioned subsequence and purification characteristics, or a significant fragment of such a protein which substantially retains the proteolytic activity and specificity disclosed herein.
  • "bovine TNF- ⁇ convertase” means bovine ADAM 10 [GenBank Accession No. Z21961 ; Wolfsberg et al, J. Cell Biol. 131 :215 ( 1995)], which the present inventors have surprisingly found is a TNF- ⁇ convertase.
  • bovine-derived enzyme exhibiting similar enzymatic activity which specifically binds to an antibody elicited against either of the bovine TNF- ⁇ convertases, or to a proteolytically active fragment from one of those enzymes.
  • Such antibodies typically bind to a bovine or other TNF- ⁇ convertase with high affinity, e.g., with an affinity constant of at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably better than about 3 nM.
  • bovine ADAM 10 is a TNF- ⁇ convertase and a comparison of its amino acid sequence with available sequence information on human ADAM 10 shows the two proteins to be 96% homologous
  • human ADAM 10 is also a TNF- ⁇ convertase as defined herein.
  • the present inventors have also discovered that the human membrane-type metalloproteases MT-MMP1 [Sato et al, Nature 370:61 (1994)], MT-MMP2 [Will et al, Eur. J. Biochem. 231 :602 (1995)] and MT-MMP3 [Takino et al, J. Biol. Chem. 270:23013 ( 1995)] are also TNF- ⁇ convertases as defined herein.
  • the present inventors have further cloned a cDNA encoding a novel human protein.
  • this protein produces such processing.
  • the sequence of this DNA, together with the predicted amino acid sequence, is substantially as defined in the Sequence Listing by SEQ ID NO: 21 .
  • polypeptide means a fragment or segment, e.g., of a TNF- ⁇ convertase which comprises a subsequence of the complete amino acid sequence of the enzyme containing at least about 8, preferably at least about 12, more preferably at least about 20, and most preferably at least about 30 or more contiguous amino acid residues, up to and including the total number of residues in the complete enzyme.
  • polypeptides of the invention can comprise any part of the complete sequence of a TNF- ⁇ convertase.
  • they could be produced by proteolytic cleavage of an intact enzyme, they can also be made by chemical synthesis or by the application of recombinant DNA technology and are not limited to polypeptides delineated by proteolytic cleavage sites.
  • analog(s) means a TNF- ⁇ convertase which has been modified by deletion, addition, modification or substitution of one or more amino acid residues in the wild-type enzyme. It encompasses allelic and polymorphic variants, and also muteins and fusion proteins which comprise all or a significant part of a TNF- ⁇ convertase, e.g., covalently linked via a side-chain group or terminal residue to a different protein, polypeptide or moiety (fusion partner).
  • amino acid substitutions are preferably "conservative", with residues replaced with physicochemically similar residues, such as Gly/Ala, Asp/Glu, Val/Ile/Leu, Lys/Arg, Asn/Gln and Phe/Trp/Tyr. Analogs having such conservative substitutions typically retain substantial proteolytic acti vity. Other analogs, which have non- conservative substitutions such as Asn/Glu, Val/Tyr and His/Glu, may substantially lack proteolytic activity. Nevertheless, such analogs are useful because they can be used as antigens to elicit production of antibodies in an immunologically competent host.
  • the antibodies can be used, e.g., for the immunopurification or immunoassay of the wild-type enzymes .
  • Whether a particular analog exhibits convertase activity can be determined by routine experimentation as described herein. Some analogs are truncated variants in which residues have been successively deleted from the amino- and/or carboxyl-termini, while substantially retaining the characteristic proteolytic activity.
  • Modifications of amino acid residues may include but are not limited to aliphatic esters or amides of the carboxyl terminus or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino- terminal amino acid or amino-group containing residues, e.g., lysine or arginine.
  • This invention also encompasses physical variants having substantial amino acid sequence homology with the amino acid sequences of the TNF- ⁇ convertases or polypeptides.
  • amino acid sequence homology, or sequence identity is determined by optimizing residue matches and, if necessary, by introducing gaps as required.
  • Homologous amino acid sequences are typically intended to include natural allelic, polymorphic and interspecies variations in each respective sequence.
  • Typical homologous proteins or peptides will have from 25- 100% homology (if gaps can be introduced) to 50- 100% homology (if conservative substitutions are included), with the amino acid sequence of the TNF- ⁇ convertases. Primate species convertases are of particluar interest.
  • Observed homologies will typically be at least about 35%, preferably at least about 50%, more preferably at least about 75%, and most preferably at least about 85% or more. See Needleham et al, J.
  • Glycosylation variants include, e.g., analogs made by modifying glycosylation patterns during synthesis and processing in various alternative eukaryotic host expression systems, or during further processing steps.
  • Particularly preferred methods for producing glycosylation modifications include exposing the TNF- ⁇ convertases to glycosylating enzymes derived from cells which normally carry out such processing, such as mammalian glycosylation enzymes .
  • deglycosylation enzymes can be used to remove carbohydrates attached during production in eukaryotic expression systems .
  • TNF- ⁇ convertases containing modifications, such as incorporation of unnatural amino acid residues, or phosphorylated amino acid residues such as phosphotyrosine, phosphoserine or phosphothreonine residues.
  • modifications include sulfonation, biotinylation, or the addition of other moieties, particularly those which have molecular shapes similar to phosphate groups.
  • Analogs of TNF- ⁇ convertases can be prepared by chemical synthesis or by using site-directed mutagenesis [Gillman et al, G e n e 5:81 (1979); Roberts et al, Nature 328:131 (1987) or Innis (Ed.), 1990, PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, NY] or the polymerase chain reaction method [PCR; Saiki e t al., Science 239:481 ( 1988)], as exemplified by Daugherty et al. [Nucleic
  • TNF- ⁇ convertases typically entails retention of at least about 50%, preferably at least about 75%, more preferably at least about 80%, and most preferably at least about 90% of the proTNF- ⁇ processing activity and/or specificity of the corresponding wild-type enzyme.
  • isolated nucleic acid means a nucleic acid such as an RNA or DNA molecule, or a mixed polymer, which is substantially separated from other components that are normally found in cells or in recombinant DNA expression systems. These components include but are not limited to ribosomes, polymerases, serum components, and flanking genomic sequences.
  • the term thus embraces a nucleic acid which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
  • a substantially pure molecule includes isolated forms of the molecule.
  • An isolated nucleic acid will generally be a homogeneous composition of molecules but may, in some embodiments, contain minor heterogeneity. Such heterogeneity is typically found at the ends of nucleic acid coding sequences or in regions not critical to a desired biological function or activity.
  • a "recombinant nucleic acid” is defined either by its method of production or structure. Some recombinant nucleic acids are thus made by the use of recombinant DNA techniques which involve human intervention, either in manipulation or selection. Others are made by fusing two fragments not naturally contiguous to each other. Engineered vectors are encompassed, as well as nucleic acids comprising sequences derived using any synthetic oligonucleotide process .
  • a wild-type codon may be replaced with a redundant codon encoding the same amino acid residue or a conservative substitution, while at the same time introducing or removing a nucleic acid sequence recognition site.
  • nucleic acid segments encoding desired functions may be fused to generate a single genetic entity encoding a desired combination of functions not found together in nature.
  • restriction enzyme recognition sites are often the target of such artificial manipulations, other site- specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design. Sequences encoding epitope tags for detection or purification as described above may also be incorporated.
  • a nucleic acid "fragment” is defined herein as a nucleotide sequence comprising at least about 17, generally at least about 25, preferably at least about 35, more preferably at least about 45, and most preferably at least about 55 or more contiguous nucleotides.
  • This invention further encompasses recombinant DNA molecules and fragments having sequences that are identical or highly homologous to those described herein.
  • the nucleic acids of the invention may be operably linked to DNA segments which control transcription, translation, and DNA replication.
  • “Homologous nucleic acid sequences” are those which when aligned and compared exhibit significant similarities. Standards for homology in nucleic acids are either measures for homology generally used in the art by sequence comparison or based upon hybridization conditions, which are described in greater detail below.
  • Substantial nucleotide sequence homology is observed when there is identity in nucleotide residues in two sequences (or in their complementary strands) when optimally aligned to account for nucleotide insertions or deletions, in at least about 50%, preferably in at least about 75%, more preferably in at least about 90%, and most preferably in at least about 95% of the aligned nucleotides.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 30 nucleotides, preferably at least about 65% over a stretch of at least about 25 nucleotides, more preferably at least about 75%, and most preferably at least about 90% over about 20 nucleotides. See, e.g., Kanehisa,
  • the lengths of such homology comparisons may encompass longer stretches and in certain embodiments may cover a sequence of at least about 17, preferably at least about 25, more preferably at least about 50, and most preferably at least about 75 nucleotide residues.
  • Stringency of conditions employed in hybridizations to establish homology are dependent upon factors such as salt concentration, temperature, the presence of organic solvents, and other parameters.
  • Stringent temperature conditions usually include temperatures in excess of about 30°C, often in excess of about 37°C, typically in excess of about 45°C, preferably in excess of about 55°C, more preferably in excess of about 65°C, and most preferably in excess of about 70°C.
  • Stringent salt conditions will ordinarily be less than about 1000 mM, usually less than about 500 mM, more usually less than about 400 mM, preferably less than about 300 mM, more preferably less than about 200 mM, and most preferably less than about 150 mM.
  • salt concentrations of 100, 50 and 20 mM are used. The combination of the foregoing parameters, however, is more important than the measure of any single parameter. See, e.g., Wetmur et al, J. Mol. Biol. 31 :349 ( 1968).
  • substantially pure is defined herein to mean a TNF- ⁇ convertase or other material that is free from other contaminating proteins, nucleic acids, and other biologicals derived from an original source organism or recombinant DNA expression system. Purity may be assayed by standard methods and will typically exceed at least about 50%, preferably at least about 75%, more preferably at least about 90%, and most preferably at least about 95% purity. Purity evaluation may be made on a mass or molar basis.
  • Antigenic (i.e., immunogenic) fragments of the TNF- ⁇ convertases of this invention may similarly be produced. Regardless of whether they cleave proTNF- ⁇ , such fragments, like the complete TNF- ⁇ convertases, are useful as antigens for preparing antibodies, using standard methods, that can bind to the complete enzymes. Shorter fragments can be concatenated or attached to a carrier. Because it is well known in the art that epitopes generally contain at least about five, preferably at least about 8, amino acid residues [Ohno et al, Proc. Natl. Acad. Sci. USA 52 : 2945
  • fragments used for the production of antibodies will generally be at least that size. Preferably, they will contain even more residues, as described above. Whether a given fragment is immunogenic can readily be determined by routine experimentation. Although it is generally not necessary when complete TNF- ⁇ convertases are used as antigens to elicit antibody production in an immunologically competent host, smaller antigenic fragments are preferably first rendered more immunogenic by cross-linking or concatenation, or by coupling to an immunogenic carrier molecule (i.e., a macromolecule having the property of independently eliciting an immunological response in a host animal).
  • an immunogenic carrier molecule i.e., a macromolecule having the property of independently eliciting an immunological response in a host animal.
  • Cross-linking or conjugation to a carrier molecule may be required because small polypeptide fragments sometimes act as haptens (molecules which are capable of specifically binding to an antibody but incapable of eliciting antibody production, i.e., they are not immunogenic). Conjugation of such fragments to an immunogenic carrier molecule renders them more immunogenic through what is commonly known as the "carrier effect" .
  • Suitable carrier molecules include, e.g. , proteins and natural or synthetic polymeric compounds such as polypeptides, polysaccharides, lipopolysaccharides etc.
  • Protein carrier molecules are especially preferred, including but not limited to keyhole limpet hemocyanin and mammalian serum proteins such as human or bovine gammaglobulin, human, bovine or rabbit serum albumin, or methylated or other derivatives of such proteins.
  • keyhole limpet hemocyanin and mammalian serum proteins such as human or bovine gammaglobulin, human, bovine or rabbit serum albumin, or methylated or other derivatives of such proteins.
  • Other protein carriers will be apparent to those skilled in the art.
  • the protein carrier will be foreign to the host animal in which antibodies against the fragments are to be elicited.
  • Covalent coupling to the carrier molecule can be achieved using methods well known in the art, the exact choice of which will be dictated by the nature of the carrier molecule used.
  • the immunogenic carrier molecule is a protein
  • the fragments of the invention can be coupled, e. g. , using water soluble carbodiimides such as dicyclohexylcarbodiimide or glutaraldehyde.
  • Coupling agents such as these can also be used to cross-link the fragments to themselves without the use of a separate carrier molecule. Such cross-linking into aggregates can also increase immunogenicity. Immunogenicity can also be increased by the use of known adjuvants, alone or in combination with coupling or aggregation.
  • Suitable adjuvants for the vaccination of animals include but are not limited to Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate); Freund's complete or incomplete adjuvant; mineral gels such as aluminum hydroxide, aluminum phosphate and alum ; surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N , N - di oc tadec y l - N ' , N ' - bi s ( 2- h ydrox yme th y l ) propanedi ami ne , methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran, dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides such as muramyl dipeptide, di
  • Serum produced from animals immunized using standard methods can be used directly, or the IgG fraction can be separated from the serum using standard methods such as plasmaphoresis or adsorption chromatography with IgG-specific adsorbents such as immobilized Protein A.
  • monoclonal antibodies can be prepared .
  • Hybridomas producing monoclonal antibodies against the TNF- ⁇ convertases of the invention or antigenic fragments thereof are produced by well-known techniques. Usually, the process involves the fusion of an immortalizing cell line with a B-lymphocyte that produces the desired antibody.
  • non-fusion techniques for generating immortal antibody-producing cell lines can be used, e.g., virally-induced transformation [Casali et al, Science 234 :416 ( 1986)] .
  • Immortalizing cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Most frequently, rat or mouse myeloma cell lines are employed as a matter of convenience and availability.
  • peripheral blood lymphocytes are used if cells of human origin are employed, or spleen or lymph node cells are used from non-human mammalian sources.
  • a host animal is injected with repeated dosages of the purified antigen (human cells are sensitized in vitro), and the animal is permitted to generate the desired antibody-producing cells before they are harvested for fusion with the immortalizing cell line.
  • Techniques for fusion are also well known in the art, and in general involve mixing the cells with a fusing agent, such as polyethylene glycol.
  • Hybridomas are selected by standard procedures, such as HAT (hypoxanthine-aminopterin-thymidine) selection. Those secreting the desired antibody are selected using standard immunoassays, such as Western blotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), or the like. Antibodies are recovered from the medium using standard protein purification techniques [Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)] .
  • Monoclonal antibodies can also be produced using well known phage library systems. See, e.g., Huse, e t al, Science 246: 1215 ( 1989); Ward, et al, Nature 341 :544 ( 1989). Antibodies thus produced, whether polyclonal or monoclonal, can be used, e.g., in an immobilized form bound to a solid support by well known methods, to purify the TNF- ⁇ convertases by immunoaffinity chromatography.
  • Antibodies against the antigenic fragments can also be used, unlabeled or labeled by standard methods, as the basis for immunoassays of the TNF- ⁇ convertases.
  • the particular label used will depend upon the type of immunoassay. Examples of labels that can be used include but are not limited to radiolabels such as 32 P, 1 25 I, 3 H and , 4 C; fluorescent labels such as fluorescein and its derivatives, rhodamine and i ts derivati ves , dansyl and umbel liferone ; chemiluminescers such as luciferia and 2,3-dihydro-phthalazinediones; and enzymes such as horseradish peroxidase, alkaline phosphatase, lysozyme and glucose-6-phosphate dehydrogenase.
  • the antibodies can be tagged with such labels by known methods.
  • coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bisdiazotized benzadine and the like may be used to tag the antibodies with fluorescent, chemiluminescent or enzyme labels.
  • the general methods involved are well known in the art and are described, e.g., in Immunoassay: A Practical Guide, 1987, Chan (Ed.), Academic Press, Inc.,
  • the antibodies of the present invention can also be used to identify particular cDNA clones expressing the TNF- ⁇ convertases, in expression cloning systems.
  • Neutralizing antibodies that bind to the catalytic site of a TNF- ⁇ convertase may also be used as inhibitors to block substrate binding, and hence catalytic activity. This can be done using complete antibody molecules, or well known antigen binding fragments such as Fab, Fc, F(ab)2 _ and Fv fragments.
  • the antibodies and antigen-binding fragments thereof can be used therapeutically to block the activity of a TNF- ⁇ convertase, and thereby to treat any medical condition caused or mediated by TNF- ⁇ .
  • Such antibodies and fragments are preferably chimeric or humanized, to reduce antigenicity and human anti-mouse antibody (HAMA) reactions.
  • HAMA human anti-mouse antibody
  • the dosage regimen involved in a therapeutic application will be determined by the attending physician, considering various factors which may modify the action of the antibodies or binding fragments, e.g., the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration, and other clinical factors.
  • compositions of this invention is typically parenteral, by intraperitoneal, intravenous , subcutaneous , or intramuscular injection, or by infusion or by any other acceptable systemic method.
  • treatment dosages are titrated upward from a low level to optimize safety and efficacy.
  • daily antibody dosages will fall within a range of about 0.01 to 20 mg protein per kilogram of body weight.
  • the dosage range will be from about 0.1 to 5 mg protein per kilogram of body weight.
  • Dosages of antigen binding fragments from the antibodies will be adjusted to account for the smaller molecular sizes and possibly decreased half-lives (clearance times) following administration.
  • Various modifications or derivatives of the antibodies or fragments such as addition of polyethylene glycol chains (PEGylation), may be made to influence their pharmacokinetic and/or pharmacodynamic properties.
  • TNF- ⁇ convertase inhibitors of the invention are not limited to neutralizing antibodies or binding fragments thereof.
  • This invention also encompasses other types of inhibitors, including small organic molecules and inhibitory substrate analogs.
  • GI 129471 a metalloprotease inhibitor designated GI 129471 , which has been shown to block TNF- ⁇ secretion, both in vitro and in vivo [McGeehan et al, Nature 370 :558 ( 1994)] .
  • Another such example is N- ⁇ D,L- [2-(hydroxyaminocarbonyl)methyl]-4- methylpentanoyl ⁇ L- 3-(2'naphthyl )-alanyl-L alanine , 2-aminoethyl amide [Mohler et al, Nature 570:218 ( 1994)], which has been shown to protect mice against a lethal dose of endotoxin.
  • SCH 43534 is a compound, designated SCH 43534, which is mentioned in an example below.
  • This compound is a peptide-based hydroxamate inhibitor of collagenase structurally similar to the foregoing compounds, the inhibitory activity of which validates use of an assay of the invention to identify a T ⁇ F- ⁇ convertase inhibitor.
  • the foregoing small organic molecules are not specific inhibitors of a T ⁇ F- ⁇ convertase but inhibit other metalloproteases as well.
  • specific inhibitors of a T ⁇ F- ⁇ convertase which do not inhibit other metalloproteases can also be identified using the methods of this invention if desired.
  • An "effective amount" of a composition of the invention is an amount that will ameliorate one or more of the well known parameters that characterize medical conditions caused or mediated by TNF- ⁇ .
  • compositions of this invention could be administered in simple solution, they are more typically used in combination with other materials such as carriers, preferably pharmaceutical carriers.
  • Useful pharmaceutical carriers can be any compatible, non-toxic substance suitable for delivering the compositions of the invention to a patient. Sterile water, alcohol, fats, waxes, and inert solids may be included in a carrier. Pharmaceutically acceptable adjuvants (buffering agents, dispersing agents) may also be incorporated into the pharmaceutical composition.
  • compositions useful for parenteral administration of such drugs are well known; e.g. Remington 's Pharmaceutical Science, 17th Ed. (Mack Publishing Company, Easton, PA, 1990).
  • compositions of the invention may be introduced into a patient's body by implantable drug delivery systems [Urquhart et al, Ann. Rev. Pharmacol. Toxicol. 24 : 199 ( 1984)] .
  • Therapeutic formulations may be administered in many conventional dosage formulation.
  • Formulations typically comprise at least one acti ve ingredient, together with one or more pharmaceutically acceptable carriers.
  • Formulations may include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman et al. (eds.) ( 1990), The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and R e m in g ton ' s Pharmaceutical Sciences, supra, Easton, Penn.; Avis et al.
  • the present invention also encompasses anti-idiotypic antibodies, both polyclonal and monoclonal, which are produced using the above- described antibodies as antigens. These antibodies are useful because they may mimic the structures of the proteases.
  • the proteins, polypeptides and antigenic fragments of this invention can be purified by standard methods, including but not limited to salt or alcohol precipitation, preparative disc-gel elec trophoresi s, i soe lectric focusing , high pres sure liquid chromatography (HPLC), reversed-phase HPLC, gel filtration, cation and anion exchange and partition chromatography, and countercurrent distribution.
  • HPLC high pres sure liquid chromatography
  • HPLC high pres sure liquid chromatography
  • reversed-phase HPLC gel filtration, cation and anion exchange and partition chromatography
  • countercurrent distribution e.g., in Guide to Protein Purification, Methods in Enzymology, Vol. 182, M. Lieber, Ed., 1990, Academic Press, New York, NY. More specific methods applicable to purification of the bovine TNF- ⁇ convertases are described below.
  • Purification steps can be followed by carrying out assays for TNF- ⁇ convertase activity as described below.
  • a convertase is being isolated from a cellular or tissue source
  • inhibitors include, e.g., 4-(2- aminoethyl )-benzenesulfonyl fluoride hydrochloride ( AEB S F) , peps tati n , leupeptin , and me thoxysu cc i nyl -A l a- Al a- Pro- V al- chloromethylketone (AAPV).
  • Nucleic acids encoding the TNF- ⁇ convertases or fragments thereof can be prepared by standard methods.
  • DNA can be chemically synthesized using, e.g., the phosphoramidite solid support method of Matteucci et al. [J. Am. Chem. Soc. 7 05 : 3 1 85
  • nucleic acids encoding the TNF- ⁇ convertases can readily be modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. Such modifications result in novel DNA sequences which encode antigens having immunogenic or antigenic activity in common with the wild- type enzymes. These modified sequences can be used to produce wild- type or mutant enzymes, or to enhance expression in a recombinant DNA system.
  • Insertion of the DNAs encoding the TNF- ⁇ convertases into a vector is easily accomplished when the termini of both the DNAs and the vector comprise compatible restriction sites. If this cannot be done, it may be necessary to modify the termini of the DNAs and/or vector by digesting back single-stranded DNA overhangs generated by restriction endonuclease cleavage to produce blunt ends, or to achieve the same result by filling in the single-stranded termini with an appropriate DNA polymerase.
  • desired sites may be produced, e.g., by ligating nucleotide sequences (linkers) onto the termini. Such linkers may comprise specific oligonucleotide sequences that define desired restriction sites.
  • Restriction sites can also be generated by the use of the polymerase chain reaction (PCR). See, e.g., Saiki et al, Sc ien ce 259 :487 ( 1988).
  • PCR polymerase chain reaction
  • the cleaved vector and the DNA fragments may also be modified if required by homopolymeric tailing.
  • TNF- ⁇ convertases of this invention can be carried out by conventional methods in either prokaryotic or eukaryotic cells. Although strains of E. coli are employed most frequently in prokaryotic systems, many other bacteria such as various strains of Pseudomonas and Bacillus are know in the art and can be used as well.
  • Prokaryotic expression control sequences typically used include promoters, including those derived from the ⁇ -lactamase and lactose promoter systems [Chang et al, Nature 795: 1056 ( 1977)] , the tryptophan (trp) promoter system [Goeddel et al, Nucleic Acids Res. 5 :4057 ( 1980)], the lambda PL promoter system [Shimatake et al, Nature 292: 128 ( 1981 )] and the tac promoter [De Boer et al, Proc. Natl. Acad. Sci. USA 292 : 128 ( 1983)] . Numerous expression vectors containing such control sequences are known in the art and available commercially.
  • Eukaryotic expression systems typically insect, mammalian or yeast host cells, for which many expression vectors are known in the art and commercially available.
  • the enzymes are employed in basic screening systems. Essentially, these systems provide methods for bringing together a mammalian TNF- ⁇ convertase, an appropriate substrate for the enzyme, and a sample to be tested for the presence of an inhibitor of the enzyme. If the sample contains such an inhibitor, substantially reduced cleavage of the substrate will be observed, compared to what would be observed in the absence of an inhibitor, e.g., using a "control" sample containing only buffer.
  • a basic screening method comprises:
  • an inhibitor of the TNF- ⁇ convertase in the sample is identified by measuring substantially reduced cleavage of the substrate, compared to what would be measured in the absence of such inhibitor.
  • sample is defined herein to mean any solution, whether aqueous, organic of some combination of the two, that may contain a TNF- ⁇ convertase inhibitor.
  • samples include but are not limited to solutions of compounds obtained following organic synthesis, aliquots from purification step fractions, and extracts from cells or tissues, or from other biological or microbial materials.
  • TNF- ⁇ convertase substrates that can be used in the basic assays of the invention include polypeptides comprising the complete proTNF- ⁇ sequence, and truncated variants (polypeptides) thereof, the preferred requirement being that all substrates contain the specific Ala-Val bond, the cleavage of which characterizes the TNF- ⁇ convertases of the invention.
  • substrates including a protein (SEQ ID NO: 3) and a polypeptide (SEQ ID NO: 4) substrate.
  • Others are known in the art, such as those disclosed by Mohler et al. [Nature 570:218 ( 1995)] .
  • the term "substrate” is defined herein to mean all such materials.
  • Substrates suitable for use in the assays are preferably based on human proTNF- ⁇ , although it may be possible to use substrates from other species.
  • the substrates can be engineered so that the activity of a TNF- ⁇ convertase causes a positive or negative measurable change in the substrate. This may result in a loss or gain of a measurable signal, following cleavage of the substrate.
  • TNF- ⁇ convertase can be used in the basic screening methods of this invention, although use of a primate or human enzyme is preferred for the identification of compounds suitable for use as human therapeutics.
  • the term "TNF- ⁇ convertase” encompasses both the wild-type variants and analogs, such as truncated or substituted variants, as long as they possess substantial proteolytic activity as defined herein. Use of a wild-type, full-length human enzyme is however preferred. Whether a given analog would be suitable for use in an assay of the invention can readily be determined through routine experimentation, using the disclosed methods .
  • a mammalian TNF- ⁇ convertase could be brought together with a substrate and a test sample to identify an inhibitor, and all such methods are within the scope of this invention.
  • a mammalian cell system is employed in which one or more nucleic acids encoding a mammalian TNF- ⁇ convertase and a substrate are transfected into a host cell. These nucleic acids can be contained in a single recombinant vector or in two, as is the case in an Example below.
  • Particularly preferred mammalian host cells for use in the foregoing system inherently lack or have minimal ability to cleave proTNF- ⁇ to the mature, secreted form.
  • Examples of such a cell are the 293 human embryonic kidney cell line and clones derived therefrom.
  • the 293 line is available from the American Type Culture Collection, Rockville, MD, under Accession No. ATCC CRL 1573.
  • a clone derived from the 293 line, designated 293EBNA is available from Invitrogen.
  • TNF- ⁇ convertase inhibitors identified in the basic screens of this invention may be suitable for therapeutic administration, although they may also inhibit other metalloproteases. If it is desired to identify a specific inhibitor of a TNF- ⁇ convertase, i.e., one that will not inhibit the activity of other, more general metalloproteases, that can be done using another embodiment of the present invention.
  • the term "specific inhibitor of a TNF- ⁇ convertase” is defined to mean an inhibitor which blocks the proteolytic activity of a TNF- ⁇ convertase but does not inhibit the activity of collagenase or other matrix-degrading metalloproteases.
  • MMP- 1 Interstitial Collagenase
  • MMP-2 72-kDa Gelatinase (MMP-2) Collagens IV, V, VII and X
  • MMP-3 Collagens III, IV, V and IX
  • MMP-8 Neutrophil Collagenase
  • the screening methods of the invention further comprise:
  • a specific inhibitor of a TNF- ⁇ convertase is identified by measuring substantially undiminished cleavage of the substrate, compared to what would be measured in the absence of such inhibitor.
  • a mammalian cell system is employed in which one or more nucleic acids encoding a matrix- degrading metalloprotease and a substrate are transfected into a host cell. These nucleic acids can be contained in a single recombinant vector or in two.
  • Substantially undiminished cleavage of a substrate by a specific inhibitor of a TNF- ⁇ convertase will be observed by measuring at least about 75%, preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 99% of the cleavage measured in the absence of such an inhibitor.
  • the present invention provides methods for cloning bovine TNF- ⁇ convertase and corresponding enzymes from other mammalian species. Briefly, Southern and Northern blot analysis can be carried out to identify cells from other species expressing genes encoding the TNF- ⁇ convertases.
  • Complementary DNA (cDNA) libraries can be prepared by standard methods from mRNA isolated from such cells, and degenerate probes or PCR primers based on the amino acid sequence information provided herein can be used to identify clones encoding a TNF- ⁇ convertase. Alternatively, expression cloning methodology can be used to identify particular clones encoding a TNF- ⁇ convertase.
  • An antibody preparation which exhibits cross-reactivity with TNF- ⁇ convertases from a number of mammalian species may be useful in monitoring expression cloning.
  • a co-transfection system described more fully below is used to identify clones capable of cleaving proTNF- ⁇ to the mature, secreted form. Selected clones can then be amplified, and cDNA isolated from them can be inserted into vectors suitable for expression in prokaryotic or eukaryotic expression systems.
  • this method for identifying a nucleic acid encoding a mammalian TNF- ⁇ convertase comprises:
  • the TNF- ⁇ convertase substrate used is proTNF- ⁇ , although any of the other substrates mentioned herein could be used instead.
  • substantially increased cleavage of the substrate will be observed by measuring at least about 5 times more, preferably at least about 10 times more, more preferably at least about 25 times more, and most preferably at least about 50 times more cleavage of the substrate than would occur in the absence of a nucleic acid encoding a mammalian TNF- ⁇ convertase.
  • clones encoding TNF- ⁇ convertases from various mammalian species can be isolated and sequenced, and the coding regions can be excised and inserted into an appropriate vector.
  • Recombinant expression vectors in this invention are typically self-replicating DNA or RNA constructs comprising nucleic acids encoding one of the TNF- ⁇ convertases, usually operably linked to suitable genetic control elements that are capable of regulating expression of the nucleic acids in compatible host cells.
  • Genetic control elements may include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation.
  • Expression vectors also may contain an origin of replication that allows the vector to replicate independently of the host cell.
  • Vectors that could be used in this invention include microbial plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which may facilitate integration of the nucleic acids into the genome of the host. Plasmids are the most commonly used form of vector but all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al, Cloning Vectors: A Laboratory
  • Suitable host cells for expressing nucleic acids encoding the TNF- ⁇ convertases include prokaryotes and higher eukaryotes.
  • Prokaryotes include both gram negative and positive organisms, e.g., E. coli and B . subtilis.
  • Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and of mammalian origin, e.g., human, primates, and rodents.
  • Prokaryotic host-vector systems include a wide variety of vectors for many different species. As used herein, E. coli and its vectors will be used generically to include equivalent vectors used in other prokaryotes.
  • a representative vector for amplifying DNA is pBR322 or many of its derivatives.
  • Vectors that can be used to express the TNF- ⁇ convertases include but are not limited to those containing the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540).
  • Higher eukaryotic tissue culture cells are preferred hosts for the recombinant production of enzymatically active TNF- ⁇ convertases.
  • any higher eukaryotic tissue culture cell line might be used, including insect baculovirus expression systems, mammalian cells are preferred. Transformation or transfection and propagation of such cells has become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS) cell lines.
  • Expression vectors for such cell lines usually include an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, e.g., from such sources as adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalovirus. Representative examples of suitable expression vectors include pCDNAl , pCD [Okayama et al, Mol. Cell Biol.
  • the present invention can be illustrated by the following examples .
  • a protein-based assay was carried out essentially as described by Mohler et al, Nature 370:218 ( 1995). Briefly, a peptide-tagged recombinant human TNF- ⁇ protein substrate (Flag-TNF- ⁇ ) was cloned and expressed in E. coli. The protein was purified by affinity chromatography using an M2 (anti-Flag)- Sepharose column (Kodak), and by ion exchange chromatography using a BIOCAD equipped with an HQ10 column. The amino acid sequence of the protein substrate is defined in the Sequence Listing by SEQ ID NO: 3, concerning which the following should be noted.
  • Amino acid residues 2-9 comprise the "Flag" sequence.
  • Residues 10 and 1 1 are a Gly-Ser connector following the Flag which were added to accommodate a restriction site used in construction.
  • the histidine at position 12 corresponds to the histidine at position -25 of SEQ ID NO: 1 , after which the two sequences are identical to the carboxyl termini.
  • fifty residues of the normal leader sequence of human proTNF- ⁇ have been deleted from this substrate. Since the deleted region is not essential for use as a substrate in an assay of this invention, other truncations containing deletion of more or fewer residues could be used as well.
  • a membrane protein sample 12 ⁇ g was mixed (in 12 ⁇ l) with 2 ng of 1 25 ⁇ _p 0 ]yp C ptid e substrate (approximately 50,000 cpm) in the presence of inhibitors of other proteolytic enzymes [e.g., 200 ⁇ M 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), 2 ⁇ M pepstatin, 200 ⁇ M leupeptin, 1 mM methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (AAPV)], and with or without 2.5 mM EDTA. Following incubation at 37 C overnight, samples were fractionated in a 16% sodium dodecylsulfate (SDS) gel and subjected to SDS polyacrylamide gel electrophoresis [SDS-
  • a polypeptide-based assay for TNF- ⁇ convertase activity was also carried out essentially using the method of Mohler et al, supra . Briefly, an amino-terminal dinitrophenylated peptide substrate, amidated at the carboxyl terminus (SEQ ID NO: 4) was synthesized and purified using standard procedures. Dinitrophenylated polypeptides corresponding to cleavage products expected for TNF- ⁇ convertase (a dinitrophenylated peptide having the sequence of residues 1 -6 of SEQ ID NO: 4) and for other membrane proteases were also synthesized.
  • a membrane protein sample was mixed in 50 ⁇ l with 1 ⁇ g peptide substrate in the presence of inhibitors (0.2 mM AEBSF, 2 ⁇ M pepstatin, 200 ⁇ M leupeptin, 1 mM AAPV). Following incubation at 37 C for from 60 minutes to overnight, the protein was precipitated by cold 5% trichloroacetic acid (TCA), 20% acetonitrile, and the soluble peptide fraction was applied to a YMC 120 angstrom C-18 ODS-AQ column (4.6 x 100 mm; YMC, Inc.).
  • TCA cold 5% trichloroacetic acid
  • the column was eluted isocratically at a flow rate of 1 ml/min using 40% acetonitrile plus 0.06 % trifloroacetic acid.
  • the elution of DNP-peptide was monitored at 360 nm using a Waters 625 LC system.
  • Fig. 1 The results of a typical assay are shown in Fig. 1 , wherein the positions of the uncut peptide and an expected convertase cleavage product (DNP-APLAQA) are shown.
  • DNP-APLAQA expected convertase cleavage product
  • Bovine spleen purchased from Pel-Freeze was cut into small pieces, washed in cold PBS, and then shredded using a Black & Decker POWER PRO food processor.
  • the tissue was resuspended in lysis buffer [20 mM Tricine (N-[2-Hydroxy- l , l -bis(hydroxymethyl)-ethyl] glycine), pH 7.8, 8% sucrose] containing 0.1 % PMSF, then homogenized using a Brinkmann POLYTRON tissue homogenizer. Cell debris was removed by two centrifugations at 8,000 x g, and the membranes were isolated by ultracentrifugation at 60,000 x g.
  • the isolated membranes were washed in 10 mM Hepes, pH 7.5, then resuspended and frozen at -20°C until used.
  • the membrane fraction was thawed and resuspended to a concentration of 8 mg/ml protein in Buffer A (20 mM Tris, pH 7.5, 1 mM MgS04, 10 mM NaCl, 10 ⁇ M ZnS ⁇ 4) plus 2% Brij 35 (23 lauryl ether), and incubated at 4° C for 30 minutes.
  • the membranes were collected by ultracentrifugation at 60,000 x g, then resuspended to 4 mg/ml in buffer A plus 2% Lubrol (polyethylene glycol monododecyl ether). The insoluble protein was removed by ultracentrifugation at 60,000 x g.
  • the Brij-insoluble, lubrol-solubilized membrane protein fraction was adjusted to 0.3 M NaCl and applied to a chelating Sepharose column charged with nickel sulfate.
  • the column was washed and eluted with wash buffer (Buffer A with 0.3 M NaCl and 0.1 % octyl glucoside) plus 50 mM imidazole.
  • the eluate was concentrated by ultrafiltration, then applied to a S300 sieving column equilibrated in buffer A plus 0.1 % octylglucoside.
  • a retained fraction (corresponding to a molecular weight of approximately 60,000) was pooled, adjusted to 0.3 M NaCl and applied to a wheat germ agglutinin column.
  • the retained fraction was eluted with 0.5 M N-acetyl glucosamine in Buffer A with 0.5 M NaCl, dialyzed against 1 mM sodium phosphate buffer, pH 7, and applied to a hydroxyapatite column.
  • the unbound fraction was passed over an HQ- 10 ion exchange column (Perseptive Biosystems), and eluted with a linear gradient of from 0 to 500 mM NaCl. Fractions containing TNF- ⁇ convertase activity were pooled for further characterization.
  • the final protein fraction (approximately 10 ⁇ g) of the first bovine TNF- ⁇ convertase was applied to an 8% polyacrylamide gel in SDS-glycine buffer (Novex). Following electrophoresis, the gel was stained for 8 minutes in 10% acetic acid/50% methanol containing 0.1 % Coomassie blue, then destained for 3.5 hours in three changes of 10% acetic acid/50% methanol.
  • the polypeptide was excised and subjected to in situ tryptic cleavage, peptide isolation and microsequencing using standard methods.
  • This enzyme was a bovine TNF- ⁇ convertase, amino acid sequences of which are disclosed above.
  • bovine ADAM 10 The purification of bovine ADAM 10 was carried out by subjecting bovine spleen to the procedures described above to the point of application to the wheat germ agglutinin column. Thereafter, the retained fraction was eluted with 0.5 M N-acetyl glucosamine, dialyzed against 1 mM sodium phosphate buffer, pH 7, and applied to an hydroxyapatite column. A bound fraction from that column was passed over an HQ-10 ion exchange column. The unbound fraction, containing the TNF- ⁇ convertase activity (approximately 10 ⁇ g) was subjected to electrophoresis an 8% polyacrylamide gel u sing SDS -glycine buffer (Novex) with dithiothreitol.
  • fractionated proteins were electophoretically transferred to an IMMOBILON filter membrane in 10 mM CAPS (3-[Cyclohexylamino]- l -propanesulfonic acid) buffer, pH 10, with 10% methanol.
  • the protein band was visualized by staining the membrane 0.1 % Ponceau S in 40% methanol, 10% acetic acid, excised, and subjected to in situ tryptic cleavage, peptide isolation and microsequencing, including N-terminal analysis, using standard methods .
  • Human MT-MMP1 cDNA was cloned from THP- 1 cell (ATCC TIB 202) total RNA, which was converted to single-stranded DNA using a
  • Cloning of human MT-MMP2 was initiated by isolating total RNA from THP- 1 cells and preparing single-stranded DNA as described above. The DNA was then subjected to a two-step PCR protocol to obtain the full-length MT-MMP2 cDNA as follows.
  • PCR reactions were run that encompassed overlapping front and back halves of MT-MMP2.
  • One reaction was set up using primers designated #B5295GD (SEQ ID NO: 7; 5' forward) and "reverse internal” (SEQ ID NO: 8; internal 3').
  • a second PCR reaction was set up using primers #B5296GD (SEQ ID NO: 9; 3' reverse) and "forward internal” (SEQ ID NO: 10; internal 5').
  • PCR conditions were as described above.
  • the product of each of these reactions was isolated by agarose gel electrophoresis.
  • the two products from the PCR reactions were mixed with PCR primers B5295GD and B5296GD, and PCR was performed under the same conditions as described above.
  • the product from this reaction was cut with EcoRl/Xba l, isolated by agarose gel electrophoresis, and cloned into vector pSR ⁇ SPORT that had been cut with the same restriction enzymes.
  • Human MT-MMP3 was cloned as described for MT-MMP 1 but from aorta polyA + RNA (Clontech) using PCR primers designated #5322 (SEQ ID NO: 1 1 ; 5' forward primer) and #5323 (SEQ ID NO: 12; 3' reverse primer).
  • the PCR product was cut with Kpnl/Hindlll, isolated, and cloned into vector pSR ⁇ SPORT that had been cut with the same restriction enzymes.
  • human MMP7 (matrilysin) was cloned from human testis poly A+ RNA (Clontech) and using PCR primers #5367 (SEQ ID NO: 13; 5' forward primer) and #5369 (SEQ ID NO: 14; 3' reverse primer).
  • the PCR product was cut with Kpn l/Hindlll, isolated, and cloned into vector pSR ⁇ SPORT that had been cut with the same restriction enzymes .
  • Human MMP 12 (macrophage metalloelastase) was similarly cloned from human aorta polyA + RNA using PCR primers #A0698H03 (SEQ ID NO: 15; 5' forward primer) and #A0698H08 (SEQ ID NO: 16; 3' reverse primer).
  • the PCR product was cut with Kp n l/Xb a l and, following isolation, cloned into similarly-cut vector pSR ⁇ S PORT.
  • Bovine ADAM 10 was cloned as described above from 5 ⁇ g of total RNA isolated from bovine spleen poly A + RNA (Clontech). The resulting single-stranded DNA was then used for PCR, using primers having sequences defined in the Sequence listing by SEQ ID NO: 17 (5' forward primer) and SEQ ID NO: 18 (3' reverse primer). PCR conditions were as described above, and the PCR product was digested with Kpn l and H i n dlll and ligated into similarly-digested vector pCEP4 (Invitrogen) .
  • Human proTNF- ⁇ cDNA can be cloned from total RNA isolated from LPS (lipopolysaccharide)-stimulated THP- 1 cells and converted to single-stranded DNA as described above. This DNA can then be used for PCR, e.g., using primers designed to introduce _5 mHI cleavage sites into the PCR product.
  • the sequences of suitable 5' and 3' primers are defined in the Sequence Listing by SEQ ID NO: 19 and SEQ ID NO: 20, respectively.
  • the PCR product is then cut with BamU to produce an insert that can be cloned into a _9 ⁇ mHI-cleaved expression vector.
  • pUCTNF Vector pUCTNF was then cut with Sa ll/Hin dlll, and a fragment retaining the coding region for proTNF- ⁇ was ligated into a vector designated pSR ⁇ SPORT that had cut with the same restriction enzymes.
  • Vector pSR ⁇ SPORT had previously been constructed as follows. pSVSPORT 1 (GibcoBRL) was cut with Clal/Pst to remove the SV promoter and treated with Klenow polymerase to fill in the Cla l overhang and produce a blunt end.
  • a fragment containing the SR ⁇ promoter and SV40 t antigen was obtained following cleavage of plasmid pDSRG (ATCC 68233 ; International Patent Application Publication No. WO 91/01078) with Hindlll/Pst and filling of the H dIII overhang with Klenow polymerase. This fragment was then ligated into the cut pSVSPORT 1 to produce pSR ⁇ SPORT.
  • Vector pSR ⁇ SPORT was cleaved using NotllSall, and a 1.5 kb stuffer cDNA fragment was ligated into the cut vector.
  • the stuffer cDNA fragment which was a neomycin resistance gene, was prepared by digesting plasmid PMC lneo Poly A (Stratagene, Catalog No. 213201 ) with Xhol/Sall, and the small fragment was isolated and ligated into S ⁇ /I-digested pSL1 190 (Pharmacia, catalog No. 27-4386). The stuffer fragment was released by digesting the resulting vector pSC1 190-Neo with Sail, and the 1.5 kb fragment was isolated.
  • the construct incorporating the stuffer fragment was cleaved using No tllSa ll, and the linearized vector was separated from the insert cDNA by agarose gel electrophoresis. The cleaved vector was then repurified in a second agarose electrophoresis gel. The pure cleaved vector DNA was isolated from the agarose gel using GELZYME (Invitrogen), following the recommended conditions. This vector was used to clone library cDNA.
  • RNA was prepared by treating Mono Mac-6 cells [Zieg ler- Hei tbrock et al, Int. J. Cancer 41 :456 ( 1988)] with lipopolysaccharide at 1 ⁇ g/ l for 18 hours, and with PMA at 10 ng/ml for one hour prior to RNA isolation. Total RNA was prepared from these cells using the guanidine thiocyanate method (Sambrook et al, supra, pp. 7.19-7.22).
  • the cells were collected by centrifugation, resuspended in guanidine thiocyanate (Gibco BRL) with 2.5 grams of N- laurylsarcosine sodium salt (Sigma), 5 drops of anti-foam A (Sigma) and 0.75 ml of 2-mercaptoethanol (Biorad).
  • cDNA Five micrograms of mRNA were used to synthesize cDNA following the protocols in the SUPERSCRIPT Plasmid System for cDNA synthesis and plasmid cloning (Gibco BRL), with the following modifications. Following second strand synthesis, the cDNA was phenol/chloroform extracted, ethanol precipitated, and then treated with T4 DNA Polymerase (Pharmacia LKB ) , following the manufacturer's instructions. Following Notl digestion, the resuspended cD ⁇ A was subjected to electrophoresis in 1 % SEAPLAQUE GTG agarose (FMC) and visualized by ethidium bromide staining. The portion of the gel from 2 kb to 13 kb was excised and digested with GELZYME (Invitrogen).
  • the resulting size-enriched cD ⁇ A was ligated with the NotllSall- cleaved pS R ⁇ Sport vector overnight using a 2: 1 vector/insert concentration ratio.
  • the ligation mixture was then extracted with phenol/chloroform, precipitated with ethanol, and electroporated into ELECTROMAX DH10B cells (Gibco BRL) under the prescribed conditions.
  • the cells were plated out at a density of about 1000 colonies per plate, with a total of around 7 x 10 5 colonies for the entire library.
  • Plasmid DNA was sequenced by the Taq DyeDeoxyTM Terminator Cycle Sequencing kit and method (Perkin-Elmer/Applied Biosystems) in two directions using an Applied Biosystems Model 373A DNA Sequencer. Sequence alignment was performed using ABI SEQED Analysis and Sequence Navigator software. The results are shown in SEQ ID NO: 21 , which provides the complete open reading frame nucleotide sequence together with the predicted amino acid sequence.
  • SEQ ID NO: 21 provides the complete open reading frame nucleotide sequence together with the predicted amino acid sequence.
  • 293EBNA human embryonic kidney cell line 293 and a clone derived therefrom, designated 293EBNA, lacked the ability to cleave transfected human proTNF- ⁇ . That was true even though all of the cells when transfected with a vector expressing a human growth hormone as a control secreted that product. For that reason, 293/293EBNA cells are preferred transfection hosts for the assay described below.
  • This assay system was established using nucleic acids encoding known membrane-type matrix metalloproteases, some of which possess TNF- ⁇ convertase activity as defined herein. Using this system in conjunction with a vector(s) encoding one of the specific human membrane-type matrix metalloproteases or bovine ADAM 10, and human proTNF- ⁇ , specific TNF- ⁇ convertase inhibitors can be identified.
  • the same system can also be used to identify other nucleic acids encoding other mammalian TNF- ⁇ convertases as well. That can be accomplished by substituting nucleic acids from cDNA or other libraries for the nucleic acids encoding the exemplary matrix metalloproteases, and observing TNF- ⁇ convertase activity expressed thereby .
  • host cells were transfected in one of two ways. In one method, human 293EBNA cells (Invitrogen) at 5 x 10 6 /0.25 ml of RPMI with 10% fetal bovine serum (FBS) were placed in a 0.4 cm electroporation-cuvette with 5 ⁇ g total DNA.
  • the cells were electroporated using a GENE PULSER (BioRad) at 200V, 960 ⁇ Fd, and 100 ohms. After recovering for 5 minutes, the cells were diluted into 15 ml of medium and placed in a tissue culture flask at 37°C, 5% C0 2 .
  • transfections were carried out using Lipofectin (GibcoBRL).
  • DNA (2 ⁇ g total) was mixed with 10 ⁇ l of Lipofectin in 200 ⁇ l of serum free medium (Opti-MEM, GibcoBRL) at room temperature for 15 minutes. Then 600 ⁇ l of Opti-MEM was added and the mixture added to 10 6 293EBNA cells. After 4 hours at 37°C, 200 ⁇ l of 50% FBS were added, and the supernatant was collected 24-48 hours later for analysis. For smaller numbers of cells the conditions were scaled down proportionally.
  • the Lipofectin method was also used to assay for bovine ADAM 10 activity, whereby 1 ⁇ l of Lipofectin was diluted into 10 ⁇ l of OPTI- MEM medium and allowed to stand at room temperature for 30 minutes. The solution was mixed with 10 ⁇ l of OPTI-MEM containing 50 ng of proTNF ⁇ -S R ⁇ SPORT with or without 100 ng of ADAM 10- pCEP4, and allowed to stand at room temperature for 15 minutes. Sixty ⁇ l of OPTI-MEM were added, and the entire 80 ⁇ l were used to replace the spent growth medium in the seeded wells.
  • enzyme-linked immunosorbant assay was carried out by diluting a human TNF- ⁇ capture antibody (Pharmingen #1863 I D) to 1 ⁇ g/ml in 0.1 M NaHC0 3 , pH 8.2, and coating the solution onto a 96 well Nunc MAXISORP microtiter plate at 100 ⁇ l/well overnight at 4°C. The wells were then blocked with 200 ⁇ l/well of PBS containing 10% FBS and 0.1 % azide and stored at 4°C until used for assay.
  • ELISA enzyme-linked immunosorbant assay
  • microtiter wells were washed with PB S containing 0.05 % Tween 20 (polyoxyethylenesorbitan monolaurate). Samples and standards were diluted in PBS containing 10%- FBS, added at 100 ⁇ l/well, and incubated at 37°C for 2 hours. After washing as described above, 100 ⁇ l of biotinylated anti-TNF- ⁇ (Pharmingen #18642D) were added at 1 ⁇ g/ml in PBS with 10% FBS and incubated for 45 minutes at 22°C.
  • Tween 20 polyoxyethylenesorbitan monolaurate
  • streptavidin-HRP BioSource
  • ABTS substrate KP Labs
  • KP Labs ABTS substrate
  • the plates were read at 405 nm using a Molecular Devices microtiter plate reader.
  • DNA encoding MT-MMP l or MT-MMP3 described above was cloned into expression vector pREP8 (Invitrogen) and co-transfected with the vector encoding human proTNF- ⁇ into 293EBNA cells. Following an assay carried out as described above, it was observed that both of the matrix metalloproteases caused cleavage of the proTNF- ⁇ . This is evident in Fig. 2, where the results produced using proTNF- ⁇ alone (Bar 1 ), proTNF- ⁇ plus MT-MMPl (Bar 2), and proTNF- ⁇ plus MT-MMP3 (Bar 3) are shown. Similar activity was not shown by MMP7 or by MMP12.
  • MMP l produced a clear secreted TNF- ⁇ signal by ELISA. This was determined by varying the amount of MT-MMP l vector in the presence of a constant amount of proTNF- ⁇ (50 ng/transfection) and empty pREP8 vector (100 ng/transfection). Thus, this system is useful for identifying other TNF- ⁇ convertases by expression cloning. Although the data are not shown, similar results were obtained for MT-MMP2
  • 293EBNA cells were co- transfected with expression vectors encoding human proTNF- ⁇ and MT-MMPl as described above, and assayed in the presence or absence of varying amounts of an MMP inhibitor, designated SCH 43534, which had previously been shown to block the release of TNF- ⁇ from activated human THP- 1 cells.
  • an MMP inhibitor designated SCH 43534
  • proTNF- ⁇ alone Bar 1
  • proTNF- ⁇ plus MT-MMPl Bar 2
  • proTNF- ⁇ plus MT-MMPl plus 1 ⁇ M SCH 43534
  • proTNF- ⁇ plus MT-MMPl plus 10 ⁇ M SCH 43534
  • AGA GTT AAA AGA ATC ACA GTA GAA AAC AGT AAA GTT TTT CTG GTT CCT 1056 Arg Val Lys Arg He Thr Val Glu Asn Ser Lys Val Phe Leu Val Pro 340 345 350
  • TGT TTA CTT TCA CAA CAT TTA AAA TCA TTA GAA TAC TTG GAT CTC AGT 1104 Cys Leu Leu Ser Gin His Leu Lys Ser Leu Glu Tyr Leu Asp Leu Ser 355 360 365 370 GAA AAT TTG ATG GTT GAA GAA TAC TTG AAA AAT TCA GCC TGT GAG GAT 1152 Glu Asn Leu Met Val Glu Glu Tyr Leu Lys Asn Ser Ala Cys Glu Asp 375 380 385

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Abstract

L'invention concerne des FNT-α convertases humaines et bovines isolées, des acides nucléiques et des vecteurs recombinés les codant, des cellules hôtes comprenant ces acides nucléiques et ces vecteurs, ainsi que des procédés pour fabriquer ces convertases à l'aide des cellules hôtes. L'invention concerne d'autre part des anticorps et des fragments de fixation d'antigènes de ceux-ci, qui se lient spécifiquement aux convertases et sont utiles pour traiter des affections médicales provoquées ou induites par le FNT-α. L'invention concerne également des méthodes de criblage pour identifier des inhibiteurs spécifiques des FNT-α convertases mammaliennes, et pour identifier des acides nucléiques codant pour ces convertases.
PCT/US1997/011637 1996-07-12 1997-07-10 Fnt-alpha convertase mammalienne WO1998002557A2 (fr)

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JP10506072A JP2000500660A (ja) 1996-07-12 1997-07-10 哺乳動物ＴＮＦ―αコンベルターゼ
EP97931547A EP0912746A2 (fr) 1996-07-12 1997-07-10 Fnt-alpha convertase mammalienne
AU35149/97A AU3514997A (en) 1996-07-12 1997-07-10 Mammalian tnf-alpha convertases

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998050547A2 (fr) * 1997-05-07 1998-11-12 Schering Corporation Recepteur proteique humain du type toll, reactifs connexes et techniques afferentes
WO2000060064A1 (fr) * 1999-04-01 2000-10-12 The Procter & Gamble Company Processus de purification d'une enzyme du type fixant le calcium, par exemple une métalloprotéase, à l'aide d'un tampon exempt de calcium
US7271248B2 (en) 1997-05-07 2007-09-18 Schering Corporation Human receptor proteins; related reagents and methods
WO2019121846A1 (fr) * 2017-12-19 2019-06-27 CSL Behring Lengnau AG Protéine de purification et inactivation de virus à l'aide de glycosides d'alkyle

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024501A1 (fr) * 1994-03-07 1995-09-14 Cetus Oncology Corporation Compositions pour l'inhibition de la formation des facteurs de necrose tumorale (tnf) et leur utilisation
WO1996041624A1 (fr) * 1995-06-08 1996-12-27 Immunex Corporation Enzyme de conversion du facteur de necrose tumorale alpha

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024501A1 (fr) * 1994-03-07 1995-09-14 Cetus Oncology Corporation Compositions pour l'inhibition de la formation des facteurs de necrose tumorale (tnf) et leur utilisation
WO1996041624A1 (fr) * 1995-06-08 1996-12-27 Immunex Corporation Enzyme de conversion du facteur de necrose tumorale alpha

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
BLACK H ET AL: "Identification of a TNF converting enzyme and its validation as a target for drug development" THE FASEB JOURNAL, vol. 9, no. 4, 10 March 1995, page A1254 XP002061364 *
BLACK R A ET AL: "A METALLOPROTEINASE DISINTEGRIN THAT RELEASES TUMOUR-NECROSIS FACTER-ALPHA FROM CELLS" NATURE, vol. 385, no. 6618, 20 February 1997, pages 729-733, XP002034337 *
BLACK R ET AL: "Cloning of a membrane-bound TNF -alpha converting enzyme (TACE)." 6TH INTERNATIONAL TUMOR NECROSIS FACTOR CONGRESS, RHODES, GREECE, MAY 8-12, 1996. EUROPEAN CYTOKINE NETWORK 7 (2). 1996. 180. ISSN: 1148-5493, XP002046452 *
BLACK R ET AL: "Relaxed specificity of matrix metalloproteinases (MMPs) and TIMP insensitivity of tumor necrosis factor alpha (TNF-alpha) production suggest the major TNF-alpha converting enzyme is not an MMP" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 225, 14 August 1996, ORLANDO, FL US, pages 400-405, XP002061488 *
EMBL/GENBANK/DDBJ DATABASES Accession no T93485; Identification no HS48563 HILLIER L ET AL: "The WashU-Merck EST Project" XP002061366 *
L.HOWARD ET AL: "Molecular cloning of MADM: a catalytically active mammalian disintegrin-metalloprotease expressed in various cell types" BIOCHEMICAL JOURNAL, vol. 317, 1 July 1996, page 45-50 XP002046451 *
LUNN C A ET AL: "Purification of ADAM 10 from bovine spleen as a TNFalpha convertase." FEBS LETTERS, vol. 400, 6 January 1997, page 333-335 XP002046454 *
MCGEEHAN G M ET AL: "REGULATION OF TUMOUR NECROSIS FACTOR-ALPHA PROCESSING BY A METALLOPROTEINASE INHIBITOR" NATURE, vol. 370, no. 6490, 18 August 1994, pages 558-561, XP000574095 *
MOHLER K M ET AL: "PROTECTION AGAINST A LETHAL DOSE OF ENDOTOXIN BY AN INHIBITOR OF TUMOUR NECROSIS FACTOR PROCESSING" NATURE, vol. 370, no. 6486, 21 July 1994, pages 218-220, XP000574714 cited in the application *
ROBACHE-GALLEA S ET AL: "Partial purification of a tumor necrosis factor-alpha converting activity" EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 27, May 1997, pages 1275-1282, XP002061365 *
SATO H ET AL: "A MATRIX METALLOPROTEINASE EXPRESSED ON THE SURFACE OF INVASIVE TUMOUR CELLS" NATURE, vol. 370, no. 6484, 7 July 1994, pages 61-65, XP000578393 cited in the application *
T. WOLFSBERG ET AL: "ADAM, a novel family of membrane proteins containing a disintegrin and metalloprotease domain" JOURNAL OF CELL BIOLOGY, vol. 131, no. 2, October 1995, pages 275-278, XP002046453 cited in the application *
TAKINO T ET AL: "IDENTIFICATION OF THE SECOND MEMBRANE-TYPE MATRIX METALLOPROTEINASE (MT-MMP-2) GENE FROM A HUMAN PLACENTA CDNA LIBRARY MT-MMPS FORM A UNIQUE MEMBRANE-TYPE SUBCLASS IN THE MMP FAMILY" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 39, 29 September 1995, pages 23013-23020, XP000644492 cited in the application *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998050547A2 (fr) * 1997-05-07 1998-11-12 Schering Corporation Recepteur proteique humain du type toll, reactifs connexes et techniques afferentes
WO1998050547A3 (fr) * 1997-05-07 1999-03-11 Schering Corp Recepteur proteique humain du type toll, reactifs connexes et techniques afferentes
US7271248B2 (en) 1997-05-07 2007-09-18 Schering Corporation Human receptor proteins; related reagents and methods
US7670603B2 (en) 1997-05-07 2010-03-02 Schering Corporation Human DNAX toll-like receptor 4 proteins, related reagents and methods
WO2000060064A1 (fr) * 1999-04-01 2000-10-12 The Procter & Gamble Company Processus de purification d'une enzyme du type fixant le calcium, par exemple une métalloprotéase, à l'aide d'un tampon exempt de calcium
WO2019121846A1 (fr) * 2017-12-19 2019-06-27 CSL Behring Lengnau AG Protéine de purification et inactivation de virus à l'aide de glycosides d'alkyle
US11479578B2 (en) 2017-12-19 2022-10-25 CSL Behring Lengnau AG Protein purification and virus inactivation with alkyl glycosides

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