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Definitions

• the present invention relates to tumor necrosis factor alpha (TNF ⁇ ), and more specifically to the enzyme TNF ⁇ convertase (TNF ⁇ -con) that can proteolytically convert TNF ⁇ precursor to mature TNF ⁇ .
• TNF ⁇ tumor necrosis factor alpha
• TNF ⁇ -con enzyme TNF ⁇ convertase
• the present invention provides DNA sequences encoding mammalian TNF ⁇ -con and functional
• TNF ⁇ -con purification of TNF ⁇ -con, and methods for treating diseases or conditions resulting from abnormal levels of TNF ⁇ in a mammalian subject.
• TNF ⁇ also known as cachectin
• cachectin is a mammalian protein that is produced primarily by activated monocytes and macrophages.
• TNF ⁇ is a potent cytokine that plays a pivotal role in host defense against invasion by microorganisms by mediating cellular responses to infection.
• TNF ⁇ is generally not present in measurable amounts in normal mammalian sera, but appears rapidly in response to several types of stimuli, including infection by viruses or bacteria, trypanosoma and plasmodia, and the cytokine IL-1 (Beutler and Cerami, 1989, Ann. Rev. Immunol. 7:625-655).
• the most potent known stimulus of TNF ⁇ production is bacterial lipopolysaccharide (LPS).
• TNF ⁇ is an important endogenous factor in the pathogenesis of septic shock (Williams and Summers, 1994, Exp. Opin. Invest. Drugs 3:1051-1056), and in chronic wasting (cachexia) associated with acute inflammatory or malignant diseases (Vassali, 1992, Ann. Rev. Immunol. 10:411-452; Beutler and Cerami, 1989, above).
• TNF ⁇ has been recognized as manifesting a dose dependent toxicity. For example, if TNF ⁇ is present at high levels even for a short period of time, it may trigger septic shock. If TNF ⁇ is present at low levels for too long a period of time, it may result in cachexia.
• TNF ⁇ vascular endothelial fibroblasts
• systemic inflammatory response syndrome reperfusion injury
• cardiovascular disease infectious disease
• obstetrical or gynecological disorders inflammatory disease or
• Human TNF ⁇ is initially synthesized as a membrane- bound precursor of approximately 26 kDa. A soluble mature 17 kDa peptide is released from the precursor after enzymatic cleavage by TNF ⁇ -con of the bond between TNF ⁇ precursor residues Ala 76 and Val 77 .
• the TNF ⁇ precursor lacks a standard signal sequence. However, secretory vesicle transport events may be coupled to processing since mutation of the Ala 76 -Val 77 cleavage site prevents secretion (Perez et al., 1990, Cell 63:251-258).
• TNF ⁇ -con partially purified TNF ⁇ -con from a human monocytic cell line, THP-1, and showed it to be a Zn 2+ -dependent metalloproteinase.
• U.S. Pat. No. 5,344,915 discloses the isolation of soluble TNF ⁇ binding proteins from human urine.
• TNF ⁇ anti- sense mRNA in cells that otherwise produce TNF ⁇ to bind to and prevent the translation of TNF ⁇ precursor mRNA. See No. EP 0 414 607 A2.
• U.S. Patent No. 5,385,901 discloses the use of thalidomide and related compounds to specifically inhibit the production of TNF ⁇ .
• TNF ⁇ -con Screening for compounds that inhibit TNF ⁇ -con would be facilitated by a readily available and abundant source of purified TNF ⁇ -con. Such inhibitors would be useful to reduce or otherwise modulate TNF ⁇ levels in a mammalian subject, thereby treating diseases or conditions resulting from abnormal levels of TNF ⁇ .
• the availability of large quantities of TNF ⁇ -con would facilitate the development and identification of derivatives, analogs and peptides of TNF ⁇ -con that could serve to modulate TNF ⁇ levels in a mammalian subject in need of such treatment. Accordingly, it would be useful to provide compositions and methods with which to produce large quantities of isolated TNF ⁇ -con.
• the present invention is directed to TNF ⁇ , and more specifically to TNF ⁇ -con having biological activity to convert TNF ⁇ precursor to mature TNF ⁇ .
• the present invention provides cDNA sequences encoding enzymatically active
• mammalian TNF ⁇ -con and more specifically a cDNA sequence encoding human TNF ⁇ -con.
• the present invention also provides DNA sequences encoding derivatives, analogs or peptides of mammalian TNF ⁇ -con polypeptides that are substantially similar to mammalian TNF ⁇ -con and that exhibit biological activity.
• the present invention further provides recombinant expression vectors comprising said DNA sequences, host cell lines comprising said DNA sequences or expression vectors, and recombinantly expressed, enzymatically active TNF ⁇ -con, or functional equivalents thereof.
• the present invention further provides compounds that inhibit the biological activity of TNF ⁇ -con, which may be useful for treating diseases or conditions related to abnormal levels of TNF ⁇ .
• the present invention further provides novel modified inhibitors for use as ligands in the affinity purification of TNF ⁇ -con.
• FIG. 1 cDNA sequence (SEQ ID NO 1) encoding human
• TNF ⁇ -con and corresponding deduced amino acid sequence (SEQ ID NO 2). Asterisks show the termination codon.
• Amino acid residues 405-409 represent the conserved sequence of the zinc-binding motif of metalloproteinases.
• FIG. 2A Enzyme activity, as determined by
• FIG. 2B SDS-PAGE analysis of fractions collected from the glycerol gradient corresponding to those tested in FIG. 2A.
• FIG. 3 Reducing SDS-PAGE analysis of porcine TNF ⁇ -con before and after deglycosylation.
• FIG. 4A Enzyme activity, as determined by
• FIG. 4B SDS-PAGE analysis of fractions collected from the glycerol gradient corresponding to those tested in FIG. 4A.
• FIG. 5 A contiguous mapping of clones psC-1, psC- 2, psC-3 and psC-5, obtained by screening a porcine spleen cDNA library with probes specific to the porcine TNF ⁇ -con coding sequence. Sequence comparison showed that the four clones overlap. The map represents the entire length of 2,414 bases from the four clones.
• FIG. 6 cDNA sequence (SEQ ID NO 9) encoding a portion of the major open reading frame of porcine TNF ⁇ -con and corresponding deduced partial amino acid sequence (SEQ ID NO 10). Asterisks show the termination codon. Amino acids 8-12 represent the conserved sequence of the zinc-binding motif of metalloproteinases.
• FIG. 7 Contiguous map of positive clones isolated from cDNA libraries from human leukocytes (hc7, hc9, hell), and from human monocytes (3'#1, 3'#4, 3'#5, 5'#4, 5'#7). hc7 contains two internal deletions at base pairs 525-613 and 2156-2294.
• FIG. 8 Structure of a biotinylated hydroxamic acid-related compound useful in the affinity purification of TNF ⁇ -con.
• FIG. 9 Stepwise (A-I) synthesis of a biotinylated hydroxamic acid-related compound (I) .
• FIG. 10 Stepwise synthesis of reagent C used in the synthesis procedure shown in FIG. 9.
• FIG. 11 Sequence of RACE14 clone (SEQ ID NO 39).
• nucleic acid sequences which can be used in accordance with the invention include but are not limited to any nucleic acid sequence that encodes a TNF ⁇ -con or a functional equivalent thereof, including: (a) any nucleic acid sequence that encodes a TNF ⁇ -con or a functional equivalent thereof, including: (a) any nucleic acid sequence that encodes a TNF ⁇ -con or a functional equivalent thereof, including: (a) any nucleic acid sequence that encodes a TNF ⁇ -con or a functional equivalent thereof, including: (a) any nucleic acid sequence that encodes a TNF ⁇ -con or a functional equivalent thereof, including: (a) any nucleic acid sequence that encodes a TNF ⁇ -con or a functional equivalent thereof, including: (a) any nucleic acid sequence that encodes a TNF ⁇ -con or a functional equivalent thereof, including: (a) any nucleic acid sequence that encodes a TNF ⁇ -con or a functional equivalent thereof, including: (a) any nucleic acid sequence that encode
• nucleotide sequence that is complementary to a nucleotide sequence that hybridizes to a mammalian TNF ⁇ -con coding sequence under highly stringent conditions, e.g., washing in 0.1 x SSC/0.1 % SDS at 68°C (Ausubel et al., eds.,
• nucleotide sequence that hybridizes to a nucleotide
• TNF ⁇ -con refers to a naturally occurring mammalian TNF ⁇ -con polypeptide and all functional equivalents thereof, unless otherwise noted.
• TNF ⁇ -con encompass all derivatives, analogs and peptides, as those terms are used in the art, of a mammalian TNF ⁇ -con that are
• polypeptide and exhibit a biological activity of a mammalian TNF ⁇ -con.
• a peptide or polypeptide is considered to be substantially similar to a mammalian TNF ⁇ -con if its amino acid
• sequence is at least about 50% homologous to the
• a polypeptide that is substantially similar to a mammalian TNF ⁇ -con will also preferably exhibit at least one type of biological activity characteristic of a mammalian TNF ⁇ -con.
• biological activity as applied to TNF ⁇ -con encompasses: (1) the ability to proteolytically cleave a mammalian TNF ⁇ precursor at a cleavage site corresponding to the peptide bond between residues Ala 76 and Val 77 of human TNF ⁇
• TNF ⁇ precursor or (2) the ability to cleave an equivalent peptide bond in a synthetic substrate; or (3) the ability to detectably bind to TNF ⁇ precursor polypeptide, to TNF ⁇ , or to a synthetic substrate comprising an
• Any mammalian tissue or cell can serve as a source of a nucleotide sequence encoding TNF ⁇ -con for use in molecular cloning. Since TNF ⁇ -con activity is
• TNF ⁇ precursor to mature TNF ⁇ any tissues or cells that produce mature TNF ⁇ will serve as a source for TNF ⁇ -con mRNA and TNF ⁇ -con polypeptide.
• TNF ⁇ is produced in a wide range of tissues or cells that produce mature TNF ⁇ .
• mammalian tissues and cells in response to various types of stimulation, including exposure to LPS.
• various types of stimulation including exposure to LPS.
• mammalian tissues include but are not limited to spleen and thymus.
• Specific mammalian cell types include but are not limited to macrophages, monocytes, T-lymphocytes, ⁇ -lymphocytes, mast cells, polymorphonuclear leukocytes, keratinocytes, astrocytes, microglial cells, smooth muscle cells, intestinal paneth cells, and tumor cells including fibrosarcomas, epithelial tumor lines,
• TNF ⁇ -con mRNA and polypeptides can be purified from LPS-stimulated cells of murine macrophage cell line RAW 264.7, which can be obtained from the
• any human cell can serve as a source of a nucleotide sequence encoding TNF ⁇ -con for molecular cloning.
• TNF ⁇ -con mRNA and polypeptide can be obtained from human monocytes which can be purified from human blood, as for example by density centrifugation (Kriegler et al., 1988, Cell 53:45-53).
• An established human monocytic cell line that can be used as a source of TNF ⁇ -con mRNA and polypeptide is THP-1, which can be obtained from the ATCC (Accession No. TIB 202).
• TNF ⁇ in any of the above cell lines or tissues can be determined either
• anti-TNF ⁇ antibodies which may be produced by standard methods, or by utilizing any of several bioassays known in the art including, for
• a nucleotide sequence encoding TNF ⁇ -con may be isolated from any of the above described tissues or cells by known methods, including but not limited to reverse transcriptase-polymerase chain reaction (RT-PCR) from total mRNA to produce cDNA, or by screening cloned genomic DNA (e.g., a DNA library) with probes unique to the TNF ⁇ -con gene sequence.
• RT-PCR reverse transcriptase-polymerase chain reaction
• genomic DNA e.g., a DNA library
• a labelled probe derived from a TNF ⁇ -con cDNA may be used to isolate a TNF ⁇ -con related gene by screening a genomic library.
• total mRNA can be isolated from any of the aforementioned tissues or cell types and reverse transcribed to produce cDNA, which is then screened, for example, with a
• Such a probe can be designed based, for example, on a partial amino acid sequence of a TNF ⁇ -con polypeptide from the same or a different mammalian species, taking into account the genetic code and its known degeneracy.
• TNF ⁇ -con can be isolated from any of the above- listed tissues or cells such as, for example, from mammalian spleen tissue according to procedures described in Section 6.1 below. Briefly, mammalian spleen tissue is ground up in. an appropriate buffer containing protease inhibitors at 4°C to obtain, through a series of steps, a membrane preparation which is then passed through a concanavalin A (conA) column, which binds TNF ⁇ -con.
• conA concanavalin A
• Further purification of the eluted enzyme typically requires one or more affinity chromatography steps such as, for example, by contacting a partially purified TNF ⁇ - con preparation with a modified inhibitor of TNF ⁇ -con under conditions that allow the binding of TNF ⁇ -con to the inhibitor, and isolating the TNF ⁇ -con-inhibitor conjugate.
• a novel modified inhibitor is an hydroxamic acid-related inhibitor having the formula shown in FIG. 8, where R comprises a moiety that can be used for binding to a further compound so as to isolate any TNF ⁇ - con that binds to the inhibitor.
• the moiety may be biotin.
• R may further comprise a spacer arm between the inhibitor and the moiety.
• the spacer arm can be any chain length and may incorporate a disulfide bond between the hydroxamic acid inhibitor and the biotin moiety.
• Biotinylation of the inhibitor can be carried out by known methods. Preparation of the specific biotinylated inhibitor shown in FIG. 8 is described below (Section 7). Additionally, the present invention encompasses the use of derivatives of biotin that may improve solubility or increase affinity for streptavidin.
• TNF ⁇ -con can be isolated by photoaffinity chromatography using known methods and utilizing, for example, a photoreactive crosslinking derivative of an inhibitor of TNF ⁇ -con, such as Gl
• TNF ⁇ -con isolated from porcine spleen has an apparent mass of about 85 kDa, which drops to about 62 kDa after deglycosylation.
• polypeptide Once the polypeptide is isolated, a complete or partial amino acid sequence of the polypeptide may be obtained, e.g., by the Edman degradation procedure (see Creighton, 1983, Proteins, Structures And Molecular
• Degenerate oligonucleotide primers may then be designed based on the complete or partial amino acid sequence, and used in a PCR to amplify a portion of the TNF ⁇ -con coding sequence from either a genomic or cDNA library. The amplified portion may then be used as a probe to obtain the full nucleotide coding sequence for TNF ⁇ -con.
• oligonucleotide PCR primers designed as based on a 41 amino acid fragment of porcine TNF ⁇ -con, are useful to amplify a portion of a mammalian TNF ⁇ -con coding
• primer conv-1 having sequence 5'-GTI CA(A/G) GA(T/C) GT(A/G) AT(T/C/A) GA-3'(SEQ ID NO 3); primer conv-2, having sequence 5'-GTI CA(A/G) GA(T/C) GT(T/C) AT(T/C/A) GA-3'(SEQ ID NO 4); and primer conv-3, having sequence 5'-CC IAC (A/G/T)AT (A/G)TT (A/G)TC (T/C)GC-3' (SEQ ID NO 5).
• primer conv-1 SEQ ID NO 3
• primer conv-2 SEQ ID NO 4
• primer conv-3 SEQ ID NO 5
• 89 bp fragment SEQ ID NO 8
• PCR amplification may be carried out by known methods. See, for example, the techniques described in Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc., San Diego; and Erlich (ed) 1992, PCR Technology, Oxford University Press, New York, which are incorporated herein by reference.
• a PCR mixture comprising any suitable primers, the nucleotide sequence to be
• amplified, and appropriate enzymes and buffers are processed according to standard PCR protocols to amplify the DNA sequence.
• Amplification may be carried out, for example, on a cDNA library that has been prepared by reverse transcription of porcine spleen poly(A+) mRNA using commercially available reagents such as, for example, a cDNA cycle kit from Invitrogen.
• the sequence of any amplified product may be determined to confirm that it corresponds to the complete or partial amino acid sequence of a TNF ⁇ -con.
• Such amplified sequences may then be used to screen for TNF ⁇ -con coding sequences in any mammalian cDNA or genomic library, including human. Once obtained, the full TNF ⁇ -con sequence can be
• DNA fragments are typically generated, some of which will encode the desired TNF ⁇ -con sequence.
• the DNA may be cleaved at specific sites using various restriction enzymes.
• the linear DNA fragments can then be
• identification of one or more specific DNA fragments containing the TNF ⁇ -con DNA sequence may be accomplished in a number of ways. For example, if an amount of a TNF ⁇ -con gene or its specific RNA, or a portion thereof, is detectably labeled, the generated DNA fragments may be screened by hybridization to the labeled probe.
• DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of a TNF ⁇ -con protein.
• DNA sequences include those capable of hybridizing to a nucleotide sequence that is complementary to the TNF ⁇ -con coding sequence under highly or moderately stringent conditions, as defined above.
• Stringency conditions may be adjusted in a number of ways. For example, when performing PCR, the temperature at which annealing of primers to template takes place and/or the concentration of MgCl 2 in the reaction buffer may be adjusted.
• stringency may be adjusted by changes in the ionic strength of the wash solutions and/or by careful control of the temperature at which the washes are carried out.
• Altered nucleotide sequences which may be used in accordance with the invention include those comprising deletions, additions or substitutions of different nucleotides resulting in a sequence that encodes the same or a functionally equivalent gene product. Alterations in the nucleotide sequence may result in changes, i.e., deletions, additions, substitutions or truncations, in the amino acid sequence that may or may not be silent, but which produce a product that exhibits a biological activity characteristic of TNF ⁇ -con. Such nucleotide changes may be made taking into account similarities in the polarity, charge, solubility, hydrophobicity,
• negatively charged amino acids include aspartic acid and glutamic acid;
• positively charged amino acids include lysine and
• Amino acids with uncharged polar head groups or nonpolar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
• a DNA sequence encoding TNF ⁇ -con is isolated, it can be amplified by any methods known in the art, including by chemical synthesis, PCR amplification, or cloning in a host cell. See, for example, the techniques described in Maniatis et al . , 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
• a cDNA encoding human TNF ⁇ -con has now been cloned and sequenced (SEQ ID NO 1) (FIG. 1) that encodes the amino acid sequence (SEQ ID NO 2) also shown therein.
• SEQ ID NO 1 FIG. 1
• the cDNA sequence for human TNF ⁇ -con was obtained by first purifying porcine TNF ⁇ -con, determining a partial amino acid sequence thereof, synthesizing
• PCR primers based on the partial porcine amino acid sequence, amplifying a fragment of the porcine TNF ⁇ - con DNA coding region, and using the fragment as a probe to screen human leukocyte and monocyte cDNA libraries so as to isolate a full length cDNA sequence encoding human TNF ⁇ -con.
• TNF ⁇ -con DNA sequence of the invention can be analyzed by known methods, including but not limited to Southern hybridization, Northern
• Southern hybridization with a TNF ⁇ - con specific probe can allow the detection of the TNF ⁇ - con gene, either natural or introduced, in various cell types.
• Northern hybridization analysis can be used to determine the expression of the TNF ⁇ -con gene in
• hybridization conditions for both Southern and Northern hybridization can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific TNF ⁇ -con probe used.
• Restriction endonuclease mapping can be used to roughly determine the genetic structure of the TNF ⁇ -con gene, and the extent of homology between the TNF ⁇ -con gene and other genes. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis.
• DNA sequence analysis can be performed by any techniques known in the art, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol.
• a DNA sequence encoding TNF ⁇ -con may be transferred directly into a host cell, or first inserted into an appropriate expression vector which is then transferred to a host cell, for propagation and expression.
• a vector is preferably constructed so that the TNF ⁇ -con coding sequence is in operative association with one or more regulatory elements.
• regulatory element includes but is not limited to inducible and non- inducible promoters, enhancers, operators and other elements known in the art that serve to drive and/or regulate expression. Also, as used herein, a DNA coding sequence is in "operative association" with one or more regulatory elements where the regulatory elements
• TNF ⁇ -con DNA sequence constructs containing the TNF ⁇ -con DNA sequence in operative association with appropriate regulatory elements.
• methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Maniatis, et al., 1989, above; Ausubel et al., 1989, above; and Sambrook et al., 1989, above.
• host expression vector systems preferably those which contain the necessary regulatory elements for directing the replication, transcription, and translation of a TNF ⁇ -con coding sequence, may be utilized equally well by those skilled in the art to express the TNF ⁇ -con coding sequence.
• expression vector systems include but are not limited to microorganisms such as bacteria transformed with
• insect cell systems infected with recombinant virus expression vectors e.g., baculovirus, containing the TNF ⁇ -con coding sequence
• animal cell systems infected with recombinant virus expression vectors e.g., adenovirus or vaccinia virus containing the TNF ⁇ -con sequence
• the regulatory elements of these vectors may vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements may be used. For instance, when cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells, e.g., mouse metallothionein promoter, or from viruses that grow in these cells, e.g., vaccinia virus 7.5K promoter or Moloney murine sarcoma virus long terminal repeat, may be used. Promoters obtained by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted sequences.
• Illustrative transcriptional regulatory regions or promoters include for bacteria, the 0-gal promoter, the T7 promoter, the TAC promoter, ⁇ left and right promoters, trp and lac promoters, trp-lac fusion
• glycolytic enzyme promoters such as ADH-I and -II promoters, GPK promoter, PGI promoter, TRP promoter, etc, for mammalian cells, SV40 early and late promoters, adenovirus major late
• Specific initiation signals are also required for sufficient translation of inserted protein coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire TNF ⁇ -con gene, including its own initiation codon and adjacent sequences, are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including the ATG
• initiation codon must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the TNF ⁇ -con coding sequences to ensure in-frame translation of the entire insert.
• exogenous translational control signals and initiation codons can be obtained from a variety of sources, both natural and synthetic.
• the efficiency of expression may be enhanced by the inclusion of transcription attenuation sequences, enhancer elements, etc .
• the TNF ⁇ -con coding sequence may be ligated to an adenovirus transcription/ translation control complex, e.g., the late promoter and tripartite ladder sequence.
• This chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome, e.g., region E3 or E4, will result in a recombinant virus that is viable and capable of expressing TNF ⁇ -con in infected hosts. Similarly, the vaccinia 7.5K promoter may be used.
• TNF ⁇ -con is an insect system such as, for example, where Autographa californica nuclear
• polyhidrosis virus is used as a vector to express the foreign sequence.
• the virus grows in Spodoptera frugiperda cells.
• the TNF ⁇ -con coding sequence may be cloned into a non-essential region, e .g. , the polyhedrin gene of the virus, and placed under the control of an AcNPV promoter such as, for example, the polyhedrin promoter.
• Successful insertion of the TNF ⁇ -con coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus, i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene.
• These recombinant viruses may then be used to infect Spodoptera frugiperda cells in which the inserted gene is to be expressed.
• retroviral vectors prepared in amphotropic packaging cell lines permit high efficiency expression in numerous cells types. This method allows the assessment of cell-type specific processing,
• a host cell strain may be chosen which modulates the expression of the inserted sequence, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers, e.g., zinc and cadmium ions for metallothionein promoters.
• inducers e.g., zinc and cadmium ions for metallothionein promoters.
• TNF ⁇ -con may thus be controlled. This is important if the protein product of the cloned foreign gene is lethal to host cells. Furthermore, modifications such as, for example, phosphorylation or glycosylation, and processing, such as cleavage of protein products may be important for the biological activity of the protein. Different host cells have characteristic and specific mechanisms for the post- translational modification and processing of an expressed polypeptide. For example, modifications in the
• glycosylation pattern may be important for different functions of the protein.
• TNF ⁇ -con polypeptide expressed in a particular cell type will retain the ability to bind to, but lack the ability to cleave, TNF ⁇ precursor.
• TNF ⁇ -con polypeptide expressed in a particular cell type will retain the ability to bind to, but lack the ability to cleave, TNF ⁇ precursor.
• polypeptides fall within the scope of the present invention.
• the cell line or host system may be chosen to ensure the desired modification and processing of the expressed protein.
• Fusion protein expression vectors may be used to express a TNF ⁇ -con fusion protein.
• the purified TNF ⁇ - con fusion protein may be used to raise antisera against the TNF ⁇ -con protein, to study the biochemical properties of the TNF ⁇ -con protein, to engineer TNF ⁇ -con fusion proteins with different enzymatic activities, or to aid in the identification or purification of the expressed protein.
• Possible fusion protein expression vectors include but are not limited to vectors that express ⁇ - galactosidase and trpE fusions, maltose-binding protein fusions, glutathione-S-transferase fusions and
• polyhistidine fusions carrier regions.
• Methods known in the art can be used to construct expression vectors coding for such TNF ⁇ -con fusion proteins. See, for example, the techniques described in Maniatis, et al ., 1989, above; Ausubel et al., 1989, above; and Sambrook et al ., 1989, above.
• the TNF ⁇ -con fusion protein may comprise a region that may be used for purification.
• amylose resin may be used for purification of maltose binding protein fusions
• glutathione-agarose beads may be used for purification of glutathione-S-transferase fusion proteins
• divalent nickel resin can be used for the purification of polyhistidine fusions.
• antibodies against a carrier protein or peptide may be used for affinity chromatography
• a nucleotide sequence coding for the target epitope of a monoclonal antibody may be engineered into the expression vector in operative association with the regulatory elements and situated so that the expressed epitope is fused to the TNF ⁇ -con polypeptide.
• a nucleotide sequence coding for the FLAGTM epitope tag International Biotechnologies
• TNF ⁇ -con-FLAGTM epitope fusion product may then be detected and affinity-purified using commercially available anti-FLAGTM antibodies (IBI).
• the expression vector may also be engineered to contain polylinker sequences that encode specific
• protease cleavage sites so that any cloned protein may be released from the carrier region by treatment with a specific protease.
• DNA sequences encoding the thrombin or factor Xa cleavage sites may be included in the fusion protein vectors.
• a signal sequence upstream from and in reading frame with the polypeptide coding sequence may be
• Non-limiting examples of signal sequences include those from ⁇ -factor, immunoglobulins, outer membrane proteins, penicillinase and T-cell receptors, among others.
• the expression vector may be engineered to further comprise a coding sequence for a reporter gene product or other selectable marker.
• a coding sequence should preferably be in operative
• Reporter genes which may be useful in the invention are well-known in the art and include those encoding chloramphenicol acetyltransferase (CAT), firefly luciferase, and human growth hormone, among others. Coding sequences that encode selectable markers useful in the invention are also well-known in the art and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those that encode thymidine kinase activity or resistance to methotrexate, among others.
• CAT chloramphenicol acetyltransferase
• Coding sequences that encode selectable markers useful in the invention are also well-known in the art and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those that encode thymidine kinase activity or resistance to methotrexate, among others.
• the expression vector can be additionally engineered according to known methods to enhance or optimize polypeptide expression, such as by mutating DNA regulatory elements to increase promoter strength or to alter the polypeptide coding sequence itself. Other modifications may include deleting intron sequences or excess non-coding sequences from the 5' and/or 3' ends ;J . the polypeptide coding sequence in order to minimize sequence- or distance-associated negative effects on expression of the polypeptide, e.g., by minimizing or eliminating message destabilizing sequences.
• vectors can be engineered to contain a unique protease cleavage sequence downstream of the 5' end.
• a protease sequence such as the thrombin cleavage sequence could be placed such that cleavage will produce an active, truncated TNF ⁇ -con.
• the recombinant expression vector comprising a DNA sequence encoding TNF ⁇ -con is preferably transformed or transfected into one or more cells of a substantially homogeneous culture of a suitable host microorganism or insect or mammalian cell line.
• the expression vector may be introduced into the host cell in accordance with known techniques, including but not limited to transformation using calcium phosphate-precipitated DNA, microinjection of DNA, electroporation, transfection by contacting the cells with a virus, liposome-mediated transfection, DEAE- dextran transfection, transduction, conjugation,
• the integration and maintenance of the polypeptide coding sequence into the host cell genome, or episomally, can be confirmed by standard techniques, e.g., by Southern hybridization analysis, PCR analysis, including reverse transcriptase-PCR (RT-PCR), or by immunological assays for the expected protein products.
• standard techniques e.g., by Southern hybridization analysis, PCR analysis, including reverse transcriptase-PCR (RT-PCR), or by immunological assays for the expected protein products.
• Host cells containing the recombinant TNF ⁇ -con coding sequence and that express biologically active product may be identified by at least four general approaches: (i) DNA-DNA, DNA-RNA or RNA-antisense RNA hybridization; (ii) detecting the presence or absence of "marker" gene functions; (iii) assessing the level of transcription as measured by the expression of TNF ⁇ -con mRNA transcripts in the host cell; and (iv) detecting the presence of mature gene product as measured, for example, by immunoassay or by the presence of biological activity.
• the presence of the TNF ⁇ -con DNA sequence can be detected by nucleotide hybridization using labeled probes that are homologous to the TNF ⁇ -con coding sequence.
• expression vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions, e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.
• marker gene functions e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.
• a marker gene can be placed in tandem with the TNF ⁇ -con sequence under the control of the same or different promoter used to control the expression of the TNF ⁇ -con coding sequence.
• Expression of the marker in response to induction or selection indicates expression of the TNF ⁇ -con coding sequence.
• expression of the FLAGTM epitope is detectable in cell extracts using anti-FLAG M2 monoclonal antibodies (IBI) in conjunction, for example, with the Western ExposureTM chemi-luminescent detection system (Clontech).
• transcriptional activity of the TNF ⁇ -con coding region can be assessed by
• total cellular mRNA can be isolated and analyzed by Northern blot using a probe that is homologous to the TNF ⁇ -con coding sequence or to particular portions thereof.
• the expression of the mature protein product can be assessed immunologically, as for example by Western blots, radio- immunoprecipitation., enzyme-linked immunoassays and the like.
• protein expression can be confirmed and further characterized by histochemical localization using known methods. See, for example. Bullock and
• cells or tissues transformed with an expression vector of the invention can be sectioned, and the sections probed with either polyclonal or
• Bound primary antibodies may then be detected by standard techniques, e . g. , using the
• biotinylated protein A-alkaline phosphatase-conjugated streptavidin technique or a secondary antibody bearing a detectable label that binds to the primary antibody.
• TNF ⁇ -con biological activity involves the detection of TNF ⁇ -con exhibiting a biological activity.
• One of the biological activities associ .ated with yTNF ⁇ -con protei.n i.s i.ts ability to enzymatically convert TNF ⁇ precursor to mature TNF ⁇ .
• Another is the ability of TNF ⁇ -con to cleave a synthetic substrate.
• non-limiting methods for detecting the presence of biologically active TNF ⁇ - con include detecting the conversion of TNF ⁇ precursor to mature TNF ⁇ or the cleavage of a synthetic substrate.
• TNF ⁇ -con biological activity can be followed by
• This synthetic substrate spans the cleavage site of human TNF ⁇ precursor. Enzyme activity may be assayed by incubating an enzyme
• the buffer may also contain the following protease
• leupeptin (10 ⁇ M), pepstatin (1 ⁇ M), phosphoramidon (10 ⁇ M), AEBSF (1 mM) and E-64 (10 ⁇ M) (Sigma or Calbiochem).
• the assay is preferably carried out at 37°C for 15 min to 3 hr. The reaction may then be quenched by addition of an equal volume of 1% hepta- fluorobutyric acid (HFBA).
• HFBA hepta- fluorobutyric acid
• Analysis of proteolytic activity may be carried out by separating and detecting substrate and products, for example, by HPLC on a C-18 Vyadac column using a water/acetonitrile gradient from 22 to 35% acetonitrile, with both the water and acetonitrile containing 0.1% HFBA.
• TNF ⁇ -con activity may be
• a fluorescent tag detected by tracking a fluorescent tag after cleavage of a synthetic substrate, such as Z-Ser-Pro-Leu-Ala-Gln-Ala- Val-Arg-Ser-Lys(X)-Ser-Arg (SEQ ID NO 17), where Z is a fluorophore such as NBD (6-(N-(7-nitrobenz-2-oxa-1,3- diazol-4-yl) amino) or rhodamine, and X, which is linked as a side group to Lys, is, for example, dimethyl
• TNF ⁇ -con activity may be
• TNF ⁇ precursor polypeptide may be radiolabelled by incorporation of 3S S-cysteine using an in vitro
• radiolabelled substrate is preferably incubated for 1-3 hr at 37°C with the TNF ⁇ -con preparation in a buffer containing 0.1-1.0% NP-40, 0.25M sucrose, 10 mM HEPES, pH 7.5, and protease inhibitors (10 ⁇ M leupeptin, 10 ⁇ M phosphoramidon, 1 mM AEBSF, 10 ⁇ M E-64 , 1 ⁇ M pepstatin, 10 ⁇ M diprotinin A, 10 ⁇ M amstatin, 10 ⁇ M bestatin and 10 ⁇ M diprotinin B).
• protease inhibitors 10 ⁇ M leupeptin, 10 ⁇ M phosphoramidon, 1 mM AEBSF, 10 ⁇ M E-64 , 1 ⁇ M pepstatin, 10 ⁇ M diprotinin A, 10 ⁇ M amstatin, 10 ⁇ M bestatin and 10 ⁇ M diprotinin B).
• the reaction may be quenched by adding loading buffer containing 4% SDS, 200 mM dithiothreitol, 20% glycerol, and 0.2% bromophenol blue. Samples are boiled, loaded onto polyacrylamide gels to separate substrate from product, and visualized using a
• TNF ⁇ -con activity may be
• bioassay that detects the presence of TNF ⁇ . Any one of several bioassays known in the art can be used, such as a
• the host cells may be grown under conditions conducive to maximum production of a biologically active TNF ⁇ -con. Such conditions will typically include growing cells to high density.
• induction conditions may be employed such as, for example, temperature change, exhaustion of nutrients, accumulation of excess metabolic by-products, or the like, as appropriate to induce expression.
• the expressed protein is retained in the host cells, the cells are harvested, lysed and the product isolated and purified from the lysate under extraction conditions known in the art to minimize protein degradation, such as, for example, at 4°C, or with protease inhibitors, or both.
• the expressed protein is secreted, the exhausted nutrient medium may simply be collected and the product isolated therefrom.
• the expressed protein may be purified using standard methods, including but not limited to any combination of the following methods: ammonium sulfate precipitation, size fractionation, ion exchange
• TNF ⁇ -con may be affinity-purified by binding the
• TNF ⁇ -con to: (a) a monoclonal antibody raised against the polypeptide; (b) a lectin, such as conA; (c) TNF ⁇ or its precursor; or (d) a TNF ⁇ -con inhibitor, for example, a hydroxamic acid-related inhibitor such as Gl 129471 (see McGeehan et al., 1994, above), among others.
• TNF ⁇ -con can be affinity-purified using a biotinylated hydroxamic acid- related inhibitor prepared as described below (see
• Section 7 Briefly, a cell lysate, exhausted culture medium or partially purified enzyme preparation
• TNF ⁇ -con comprising TNF ⁇ -con may be contacted with a biotinylated TNF ⁇ -con inhibitor under conditions conducive to binding of TNF ⁇ -con to the biotinylated inhibitor to form a TNF ⁇ - con-inhibitor-biotin conjugate.
• the TNF ⁇ -con-inhibitor- biotin conjugate may then be isolated by contacting it with streptavidin bound to a solid phase matrix, such as ULTRALINKTM Immobilized Neutravidin Plus on 3M EMPHAZETM Biosupport Medium ABI (Pierce), under conditions
• the enzyme may then be eluted, for example, by incubation of the support medium overnight in a low salt buffer such as 10 mM NaCl, with protease inhibitors, such as leupeptin (10 ⁇ M), pepstatin (1 ⁇ M), phosphoramidon (10 ⁇ M), AEBSF (1 mM) and E-64 (10 ⁇ M).
• a low salt buffer such as 10 mM NaCl
• protease inhibitors such as leupeptin (10 ⁇ M), pepstatin (1 ⁇ M), phosphoramidon (10 ⁇ M), AEBSF (1 mM) and E-64 (10 ⁇ M).
• Increasing purity of the enzyme preparation can be monitored at each step of the purification procedure by known methods, such as by determining protein yield versus enzymatic activity after each successive purification step. Purity can be assessed by electrophoretic or .chromatographic techniques.
• TNF ⁇ -con polypeptide of sufficient purity may be characterized by standard methods, such as by determining its enzyme kinetics and substrate specificity. For example, the ability of a purified TNF ⁇ -con to cleave short peptides may be
• These peptides will preferably span the cleavage sequence of TNF ⁇ precursor, but may be modified, for example, by the presence of amino acid substitutions, deletions or derivatizations, or by differences in substrate length (see Section 8, below).
• the amino acid sequence of the TNF ⁇ -con protein can be deduced from the cDNA sequence or, alternatively, by direct sequencing of the protein, e.g., with an automated amino acid sequencer.
• the deduced amino acid sequence of human TNF ⁇ -con (SEQ ID NO 2) is depicted in FIG 1.
• the TNF ⁇ -con protein sequence can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824), which can be used to identify hydrophobic and hydrophilic regions of the TNF ⁇ -con protein and, accordingly, the corresponding regions of the gene sequence which encode such regions.
• a hydrophilicity analysis Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824
• the present invention includes other methods for identifying the specific site(s) on the TNF ⁇ -con polypeptide that interact with TNF ⁇ precursor. For example, site-directed mutagenesis of DNA encoding the TNF ⁇ -con protein may be used to destroy, inhibit or otherwise alter the interaction between TNF ⁇ -con and TNF ⁇ precursor, thus producing variants such as TNF ⁇ -con antagonists.
• a series of deletion mutants in the TNF ⁇ -con coding region may be constructed and analyzed to determine the minimum amino acid sequence requirements for binding to and
• Deletion mutants of the TNF ⁇ -con coding sequence may be constructed using methods known in the art which include but are not limited to use of nucleases and/or restriction enzymes, site-directed mutagenesis techniques, etc.
• the mutated polypeptides may be assayed for their ability to bind to the TNF ⁇ precursor or to a synthetic substrate, for example, by gel filtration assays.
• derivatives, analogs and peptides related to TNF ⁇ -con can be chemically synthesized
• a peptide corresponding to a portion of TNF ⁇ -con that exhibits a desired biological activity can be made using a peptide synthesizer.
• a peptide synthesizer for example, a peptide synthesizer.
• ANALOGS AND PEPTIDES OF TNF ⁇ -CON The production and use of derivatives, analogs and peptides related to TNF ⁇ -con are also within the scope of the invention and can be used, for example, in immunoassays, for immunizations, therapeutically, etc .
• Such molecules which retain, inhibit, or otherwise modulate a desired TNF ⁇ -con biological activity property can be used as agonists, antagonists, inhibitors or, more generally, as modulators of such an activity.
• agonist e.g., acetylcholine
• antagonist e.g., acetylcholine
• inhibitor e.g., acetylcholine
• modulator e.g., acetylcholine
• the derivatives, analogs and peptides of the invention can be produced by various methods known in the art.
• the manipulations which result in their production can occur either at the gene or protein level, or both.
• the cloned TNF ⁇ -con DNA sequence can be modified in vitro by any of numerous strategies known in the art. See Maniatis, et al . , 1989, above; Ausubel et al . , 1989, above; and Sambrook et al . , 1989, above. Such modifications include but are not limited to endonuclease digestion, mutations to create or destroy translation, initiation, and/or termination sequences, or that create variations in the coding region, or any combination thereof. Any technique for mutagenesis known in the art can be used, including but not limited to in vitro site-directed mutagenesis (see, for example, Hutchinson et al . , 1978, J. Biol. Chem.
• the expressed polypeptide may contain deletions, additions or substitutions of amino acids which may or may not result in a silent change within the sequence to produce a biologically active variant.
• TNF ⁇ -con DNA sequence may also be made at the protein level. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to: substitution of one or more L-amino acids of the TNF ⁇ -con polypeptide with corresponding D-amino acids, amino acid analogs or amino acid mimics, e.g., so as to produce carbazates or tertiary centers; or specific chemical modification, as for example with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.
• TNF ⁇ -con An example of a peptide of TNF ⁇ -con would be a truncated version of TNF ⁇ -con which, for example, may be produced by removal of a transmembrane domain normally present in the native protein, so as to produce a
• the TNF ⁇ -con polypeptide, or a peptide or analog thereof may be derivatized by conjugation to the protein of other chemical groups, including but not limited to acetyl groups, glycosyl groups, lipids, and phosphates, among others. Such conjugation is preferably by covalent linkage at TNF ⁇ -con amino acid side chains and/or at the N-terminus or C-terminus of the
• Water soluble polymers especially polyethylene glycol, may be conjugated to TNF ⁇ -con to provide
• additional desirable properties while retaining, at least in part, a desired biological activity, such as TNF ⁇ antagonism.
• additional desirable properties include, for example, increased solubility in aqueous solutions, increased stability in storage, reduced immunogenicity, increased resistance to proteolytic degradation, and increased in vivo half-life.
• Water soluble polymers suitable for use with the peptides of the invention include polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ethers, and ⁇ , ⁇ -poly[ (2-hydroxyethyl)-DL-aspartamide]. Polyethylene glycol is particularly preferred. Methods of making water-soluble polymer conjugates of proteins are described in, among other places, U.S. Pat. No.
• Such derivatives, analogs and peptides may be used to compete with full length wild-type TNF ⁇ -con protein for binding to TNF ⁇ precursor, and in so doing serve to inhibit or antagonize TNF ⁇ -con activity.
• TNF ⁇ -con derivatives, analogs and peptides that are capable of binding to mature TNF ⁇ may be used to neutralize excess levels of TNF ⁇ in the serum or tissues of a mammalian subject in need of such
• Recombinantly expressed TNF ⁇ -con may be used to screen for molecules that reduce or otherwise modulate TNF ⁇ levels by inhibiting or otherwise modulating one or more biological activities of TNF ⁇ -con.
• Such molecules may include small organic or inorganic compounds, antibodies, peptides, or other molecules that inhibit or otherwise modulate the ability of TNF ⁇ -con to: (1) bind to TNF ⁇ precursor or to a synthetic substrate; (2) convert TNF ⁇ precursor to mature TNF ⁇ ; or (3) cleave a synthetic substrate.
• hydroxamic acid-related compounds several of which have been shown to inhibit TNF ⁇ -con activity. See Mohler et al., 1994, above; Gearing et al., 1994, above; McGeehan et al., 1994, above; and WO 95/06031.
• Synthetic compounds, natural products, and other potential sources of modulatory compounds can be screened in a number of ways.
• the ability of a test molecule to inhibit the activity of TNF ⁇ -con may be measured using standard biochemical methods such as gel filtration assays to detect an effect on binding, or assays that detect an effect on the cleavage of precursor peptides, or using in vitro bioassays that detect the presence of TNF ⁇ .
• One non-limiting method by which a compound capable of binding to TNF ⁇ -con may be isolated and identified comprises: (a) conjugating TNF ⁇ -con, or a portion thereof, to a solid phase matrix; (b) contacting the TNF ⁇ -con-solid phase matrix conjugate with a material comprising a test compound for an interval and under conditions sufficient to allow the compound to bind to the conjugated enzyme; (c) washing away unbound material from the solid phase matrix; (d) detecting the presence of compound bound to the conjugated TNF ⁇ -con; (e) eluting the bound compound from the immobilized enzyme, and (f) collecting and thereby isolating the compound.
• the compound may first be eluted from the immobilized enzyme and then detected and characterized. Once isolated, the compound can be tested for its ability to inhibit or otherwise modulate one or more biological activities of TNF ⁇ -con.
• Random peptide libraries consisting of all possible combinations of amino acids may be used to identify peptides that are able to bind to TNF ⁇ -con.
• Identification of peptides that are able to bind to TNF ⁇ - con may be accomplished by screening such a peptide library with recombinant TNF ⁇ -con proteins.
• any binding domains of TNF ⁇ -con may be separately expressed and used to screen peptide
• One non-limiting way to identify and isolate a peptide that interacts and forms a complex with TNF ⁇ -con involves attaching a detectable label to TNF ⁇ -con protein to facilitate the identification of such a complex.
• TNF ⁇ -con may be conjugated to an enzyme such as alkaline phosphatase or horseradish peroxidase, or to a fluorescent tag, such as fluorescein isothylocynate
• TNF ⁇ -con expression vectors may be
• an epitope for which a commercially available antibody exists such as the FLAGTM epitope as described above.
• the epitope-specific antibody may be tagged using methods well known in the art including, for example, by labeling with enzymes, fluorescent dyes or colored or magnetic beads, or the epitope-specific antibody may be detected using a labelled secondary antibody.
• a DNA sequence encoding a peptide that interacts with TNF ⁇ -con to form a complex may be cloned into an appropriate expression vector for overexpression in either bacteria or eukaryotic cells.
• the peptide may be purified from cell extracts by known methods. Alternatively, the peptide may be synthesized by solid phase techniques followed by cleavage from resin and purification by HPLC. Once isolated, the peptide can be tested for its ability to inhibit or otherwise modulate the biological activity of TNF ⁇ -con.
• Antibodies to TNF ⁇ -con may be useful, for example, as affinity reagents to purify native or recombinant TNF ⁇ -con, or to detect the presence of TNF ⁇ - con, for example, in histological sections, in cell or tissue extracts, in culture medium, or in enzyme
• TNF ⁇ -con polypeptide Either the entire TNF ⁇ -con polypeptide or a sub-sequence thereof may be used as immunogen against which antibodies can be raised.
• the entire TNF ⁇ -con polypeptide or a sub-sequence thereof may be used as immunogen against which antibodies can be raised.
• catalytic domain of the enzyme may be isolated and used as an immunogen against which antibodies can be raised.
• various host animals including but not limited to rabbits, mice, rats, etc . , may be immunized by injection with TNF ⁇ -con or a portion thereof. Immunizations are carried out according to known methods.
• Various adjuvants may be used to increase the immunological response, depending on the host species, including Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum, among others.
• BCG Bacille Calmette-Guerin
• corynebacterium parvum among others.
• Monoclonal antibodies to TNF ⁇ -con may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al ., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. USA, 80:2026-2030) and the EBV-hybrid ⁇ ma technique (Cole et al ., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
• Antibody fragments which contain specific binding sites for the TNF ⁇ -con protein may be generated by known techniques.
• fragments include but are not limited to: F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule, and Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
• oligonucleotide sequences that include anti-sense
• oligonucleotides phosphorothioates, and ribozymes that function to bind to, degrade and/or inhibit the
• Anti-sense oligonucleotides act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
• antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the cDNA sequence encoding TNF ⁇ -con can be synthesized, for example, by conventional phosphodiester techniques.
• Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
• ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
• Engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of TNF ⁇ -con RNA sequences are also within the scope of the invention.
• ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays, for example. Both the anti-sense oligonucleotides and ribozymes of the invention may be prepared by known methods. These include techniques for chemical
• anti- sense RNA molecules may be generated by in-vitro or in- vivo transcription of DNA sequences encoding the RNA molecule.
• DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
• Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxyribo- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
• Compounds that inhibit the conversion of TNF ⁇ precursor to mature TNF ⁇ or otherwise modulate the effective levels of TNF ⁇ in the sera or tissues of a mammalian subject can be used to treat diseases or conditions related to elevated or otherwise abnormal levels of TNF ⁇ in the subject.
• treatment as used herein with reference to a disease or condition is used broadly and is not limited to a method of curing the disease or condition.
• treatment includes any method that serves to reduce one or more of the pathological effects or symptoms of a disease or condition, or to reduce the rate of progression of one or more of such pathological effects or symptoms.
• Diseases or conditions that may be treated by the methods of the invention are diseases characterized by one or more of the following criteria: elevated or otherwise abnormal levels of TNF ⁇ in serum or tissues of a mammalian subject; the development of septic shock; or the development of cachexia.
• elevated and abnormal as used herein are relative terms and are used to describe the levels of TNF ⁇ in a subject in need of treatment as compared to a normal subject of similar age, gender, weight, etc.
• the present invention provides methods for treating such diseases or conditions in a mammalian subject in need of such treatment, comprising
• TNF ⁇ TNF ⁇
• TNF ⁇ -con by inhibiting the ability of TNF ⁇ -con to convert TNF ⁇ precursor to TNF ⁇ , or by inhibiting the cellular secretion of TNF ⁇ , or by binding to and thereby reducing the effective concentration of soluble TNF ⁇ in the serum or tissues of the subject.
• Diseases or conditions that can be treated according to the method of the present invention include systemic inflammatory response syndrome, reperfusion injury, cardiovascular disease, infectious disease, obstetrical or gynecological disorders, inflammatory disease or autoimmunity, allergic or atopic diseases, malignancies, transplants, among others. More
• diseases or conditions which may be treated by the method of the present invention include but are not limited to septic shock, cachexia, AIDS, graft- versus-host disease, cerebral malaria, Crohn's disease, diabetes, osteoporosis, restenosis, psoriasis and
• the method of the present invention may also be used to prevent or reduce the extent of infarction due, for example, to an ischemic event.
• the present invention further contemplates the use of combination therapy, wherein one or more compounds that inhibit or otherwise modulate a biological activity of TNF ⁇ -con, as disclosed above, can be used in
• Such other reagents may include, for example, small organic or inorganic molecules or antibodies directed to TNF ⁇ -con or to another component or factor underlying a disease or condition in a mammalian subject, in which combination therapy will serve to increase or otherwise improve the efficacy of treatment of the disease or condition.
• TNF ⁇ -con inhibitors may be used in conjunction with an antibody directed against a component of the inflammatory response such as that involved in an autoimmune disease, such as, for example, rheumatoid arthritis, to increase the efficacy of treatment.
• one or more TNF ⁇ -con inhibitors may be used in conjunction with an anti-CD4 antibody or with an anti-CD23 antibody to treat an autoimmune disease.
• a humanized anti-CD4 antibody is disclosed in PCT GB 91/01578.
• Anti-CD23 antibodies are described in Dougall et al., 1994, Tibtech 12:372-379.
• one or more TNF ⁇ -con inhibitors may be used in conjunction with more conventional treatments, such as with methotrexate or cyclosporin A, among others.
• the present invention further contemplates a complex comprising a TNF ⁇ -con and a therapeutic agent capable of modulating the activity of the enzyme for use in treating a disease or condition associated with TNF ⁇ .
• a complex may further comprise the enzyme's
• the present invention further provides
• compositions or formulations for use in a method of treatment comprising one or more compounds that reduce or otherwise modulate the effective level of TNF ⁇ in sera or tissues of a mammalian subject and a pharmaceutically acceptable carrier.
• the invention further encompasses formulations for a combination therapeutic comprising one or more compounds that inhibit the biological activity of TNF ⁇ -con, one or more
• aqueous carriers may be used in the pharmaceutical formulation of the invention, such as water, buffered water, 0.4% saline, 0.3% glycine, and the like.
• the pharmaceutical formulations may also comprise additional components that serve to extend the shelf-life of pharmaceutical formulations, including preservatives, protein stabilizers, and the like.
• the formulations are preferably sterile and free of particulate matter (for injectable forms). These compositions may be sterilized by conventional, well-known sterilization techniques.
• compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate
• physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium
• formulations of the invention may be adapted for various forms of administration, including orally,
• the present invention further provides formulations for the sustained release of one or more compounds that reduce or otherwise modulate the total or effective TNF ⁇ levels in a subject by inhibiting or otherwise modulating the biological activity of TNF ⁇ -con.
• sustained release formulations include composites of biocompatible polymers, such as poly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.
• biocompatible polymers such as poly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.
• degradable polymers in drug delivery vehicles have been reviewed in several publications, including A. Domb et al ., 1992, Polymers for Advanced Technologies 3:279-292. Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in the text by M. Chasin and R. Langer (eds.), 1990, "Biodegradable Polymers as Drug Delivery Systems", in: Drugs and the Pharmaceutical Sciences, Vol 45, M. De
• Liposomes may also be used to provide for the sustained release of TNF ⁇ -con antagonists or other modulating compounds. Details concerning how to use and make liposomal formulations of drugs of interest can be found in, among other places, U.S. Pat. No 4,944,948;
• TNF ⁇ -con antagonist for example, at the site of an infection, etc.
• a purified TNF ⁇ -con inhibitor or other modulating compound may be combined with compatible, nontoxic pharmaceutical excipients and administered to a mammalian subject, e.g., to treat a disease or condition characterized by an elevated or otherwise abnormal level of TNF ⁇ in the serum or tissues of the subject.
• mammalian subject is intended to include humans and animals. In the case of administration to animals, it may be preferable to incorporate the drug into the animal's feed, possibly in a prepared combination of drug and nutritional material ready for use by the farmer.
• the compound may be administered orally, rectally, transdermally, by pulmonary infiltration, insufflation or parenterally (including intravenously, subcutaneously and intramuscularly) to humans, in any suitable
• An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Primary among these is the level of TNF ⁇ in serum or tissue of the subject. Additional factors include the size of the subject, the age of the subject, the general condition of the subject, the particular disease or condition being treated, the severity of the disease, the presence of other drugs in the subject, the in vivo activity of the antagonist or modulating compound and the like. The trial dosages would preferably be chosen after
• a typical daily human dose of a TNF ⁇ -con antagonist or other, modulating compound may be in an amount, for example, of from about 0.1 mg to about 200 mg per kilogram of body weight, more preferably from about 1 mg to about 100 mg per kilogram body weight, and most preferably about 5 mg to about 50 mg per kilogram body weight.
• gene therapy to replace mutated TNF ⁇ -con with a wild type complement of the gene, or to transfer nucleotide sequences that are anti-sense to a portion of TNF ⁇ -con into a subject.
• gene therapy are useful to treat any disease or condition resulting from an elevated or otherwise abnormal level of TNF ⁇ or TNF ⁇ -con in the subject.
• Methods for transferring the wild type TNF ⁇ -con gene into the targeted tissue may include reconstitution of recombinant TNF ⁇ -con molecules into liposomes for delivery into target cells.
• recombinant viral vectors may be engineered to express wild type TNF ⁇ -con.
• Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes virus or bovine papilloma virus, may be used to deliver wild type TNF ⁇ -con into the targeted cell
• Porcine TNF ⁇ -con was isolated from pig spleen. All steps were conducted at 4°C. Approximately 10 to 20 fresh pig spleens, total 3-6 kilograms (kg), were cut into small pieces and placed in a beaker containing cold grinding buffer (10 mM HEPES, pH 7.5, 0.25 M sucrose, 2 mM MgCl 2 ) with protease inhibitors (1 mM AEBSF, 1 ⁇ M pepstatin, 10 ⁇ M E-64, 10 ⁇ M leupeptin, 10 ⁇ M
• the pellet was resuspended in 1/6 the volume of the supernatant in buffer (10 mM HEPES, pH 7.5, 0.25 M sucrose) with protease inhibitors (1 mM AEBSF, 1 ⁇ M pepstatin, 10 ⁇ M E-64, 10 ⁇ M leupeptin, 10 ⁇ M
• Buffer B (0.05% NP-40, 10 mM HEPES, pH 7.5, 200 mM NaCl) with protease inhibitors (1 mM AEBSF, 1 ⁇ M pepstatin, 10 ⁇ M E-64, 10 ⁇ M leupeptin, 10 ⁇ M
• the membranes were pelleted by centrifugation in a TFA 20.250 rotor for 1 hr at 20,000 rpm.
• the pellet was combined with 6 liters of Buffer C (Buffer B with 1% NP-40) with protease inhibitors (1 mM AEBSF, 1 ⁇ M pepstatin, 10 ⁇ M E-64, 10 ⁇ M leupeptin, 10 ⁇ M phosphoramidon) for every liter of initial membrane suspension.
• the solution was stirred for 30 min at 4°C followed by centrifugation in the TFA 20.250 rotor for 1 hr at 20,000 rpm.
• the supernatant was loaded onto a 1 liter conA column (Pharmacia) equilibrated in the same buffer as the supernatant at a flow rate of 20 ml per min.
• the column was washed with 5 volumes of Buffer C without protease inhibitors, followed by 5 volumes of Buffer D (same as Buffer C (without protease inhibitors) except that the NaCl concentration was .10 mM.
• Convertase activity as determined by HPLC monitoring of the cleavage of a synthetic DNP substrate, was eluted off with 10 column volumes of Buffer E (Buffer D containing 250 mM methyl mannopyranoside) with protease inhibitors (1 mM AEBSF, 1 ⁇ M pepstatin, 10 ⁇ M E-64, 10 ⁇ M leupeptin).
• Buffer E Buffer D containing 250 mM methyl mannopyranoside
• protease inhibitors (1 mM AEBSF, 1 ⁇ M pepstatin, 10 ⁇ M E-64, 10 ⁇ M leupeptin).
• the eluant was loaded directly onto a 500 ml Q fast flow column (Pharmacia) .
• the column was washed with Buffer F (0.5% NP-40, and 10 mM HEPES, pH 7.5).
• protease inhibitors 1 mM AEBSF, 1 ⁇ M pepstatin, 10 ⁇ M E-64, 10 ⁇ M leupeptin, 10 ⁇ M phosphoramidon.
• the eluted protein was dialyzed against Buffer F without NaCl, but with protease
• the material from the Q-fast flow column was additionally purified with Cibacron Blue 3000 (Sigma) .
• the eluant was loaded onto a 300 ml column at a flow rate of 10 ml per min.
• the column was washed with 3 column volumes of Buffer F containing 200 mM NaCl without protease inhibitors.
• the activity was then eluted from the column with Buffer F containing 1.5 M NaCl and protease inhibitors (1 mM AEBSF, 1 ⁇ M pepstatin, 10 ⁇ M E- 64, 10 ⁇ M leupeptin).
• the eluted protein was then dialyzed as above.
• a biotinylated hydroxamic acid inhibitor prepared as described below (Section 7) was added to the dialyzed protein to a final concentration of 1 ⁇ M. After 10-30 min incubation at 4°C, affinity beads (ULTRALINKTM Immobilized Neutravidin Plus on 3M EMPHAZETM Biosupport Medium ABI (Pierce)) were added (0.4 ml of slurry per 1 ml of enzyme solution). After incubating for 10 min with gentle rocking, the slurry was pelleted by centrifugation in a microfuge.
• the beads were washed three times with 1.5 ml of Buffer F containing 0.5 M NaCl.
• the enzyme was eluted off the beads by gentle rocking overnight in Buffer F containing protease inhibitors (1 mM AEBSF, 1 ⁇ M pepstatin, 10 ⁇ M E- 64, 10 ⁇ M leupeptin) without NaCl at 4°C.
• the TNF ⁇ -con was subjected to a final separation step consisting of sedimentation through a glycerol gradient under centrifugal force as follows.
• the material eluted from the affinity resin was layered on top of an 8 to 27.8% glycerol gradient made as follows. The following
• Reagents for deglycosylation were obtained as a kit from New England Biolabs (Beverly, Mass.). An aliquot (25 ⁇ l) of TNF ⁇ -con preparation obtained from pooled fractions 16-20 from the glycerol gradient step was incubated for 10 min at 100°C in the presence of 2.5 ⁇ l 10x denaturing buffer. The following were then added: NP-40 (3.5 ⁇ l); 10x G7 buffer (3.5 ⁇ l); and pure PNGase F (0.5 ⁇ l; 10 units).
• porcine TNF ⁇ -con prepared as above was assessed by SDS-PAGE under reducing conditions (FIG. 2B).
• Isolated porcine TNF ⁇ -con, prepared as above, has an apparent molecular weight of about 85 kDa, as shown by the correlation of enzyme activity with the 85 kDa band throughout the glycerol gradient fractions (FIG. 2A). After deglycosylation, the molecular weight drops to about 62 kDa (FIG. 3).
• Isolated human TNF ⁇ -con has a very similar apparent molecular weight of about 86.5 kDa (Fig. 4B), as shown by the correlation of enzyme activity with the 86.5 kDa band throughout the glycerol gradient fractions (FIG. 4A).
• Affinity-purified material was either sequenced directly, or analyzed by SDS-PAGE using two 8-16% Novox (San Diego, CA) mini-gels (100 x 100 x 1mm) in Tris- glycine buffer. Electrophoretically separated proteins were detected using ISS Pro-Green (Natick, MA) according to manufacturer's directions. A prominent band at about 85 kDa was excised .and the protein was electroeluted directly onto a Hewlett-Packard C18 sequencing column, as described by Moyer et al ., 1994, in: Crabb, J. (ed), Techniques in Protein Chemistry, Vol. V. pp. 195-204, Academic Press, San Diego, CA. In situ reduction, alkylation, and digestion with Lys-con (Wako) were performed according to Burkhart et al . , 1993, in:
• oligonucleotide PCR primers were designed based on the partial amino acid sequence of porcine TNF ⁇ -con
• primer conv-1 has
• primer conv-2 has sequence 5'-GTI CA(A/G) GA(T/C) GT(A/G) AT(T/C/A) GA-3' (SEQ ID NO 3); primer conv-2 has sequence 5'-GTI CA(A/G) GA(T/C) GT(T/C) AT/T/C/A) GA-3' (SEQ ID NO 4); primer conv-3 has sequence 5'-CC IAC (A/G/T)AT (A/G)TT (A/G)TC (T/C)GC-3' (SEQ ID NO 5); and primer conv-4 has sequence 5'-CC IAC (A/G/T)AT (A/G)TT (A/G)TC (A/G)GC-3' (SEQ ID NO 6).
• Primers conv-1 and conv-2 represent the 5' nucleotide sequences and the two primers only differ at one base position as shown above.
• Conv-3 (SEQ ID NO 5) and conv-4 (SEQ ID NO 6) are the 3' primers as shown.
• Reverse transcriptase PCR was performed on porcine spleen poly (A+) RNA according to the
• Each of four PCRs used a pair of primers, one 5' primer i.e., either conv-1 (SEQ ID NO 3) or conv-2 (SEQ ID NO 4), and one 3' primer, i.e., either conv-3 (SEQ ID NO 5) or conv-4 (SEQ ID NO 6), at a final concentration of 2 mM each.
• the expected 89 bp PCR fragment (SEQ ID NO 8) was obtained when primer conv-3 (SEQ ID NO 5) was used with primer conv-1 (SEQ ID NO 3) or conv-2 (SEQ ID NO 4) in the reaction.
• primer conv-4 (SEQ ID NO 6) together with conv-1 (SEQ ID NO 3) or conv-2 (SEQ ID NO 4) gave rise to a fragment of approximately 300 bp.
• the two PCR fragments thus obtained were made blunt-ended, subcloned into Bluescript II SK at the Smal site, and subjected to DNA sequencing using the Taq dideoxy terminator method.
• DNA sequence analysis revealed that the 89 bp fragment (SEQ ID NO. 8) encoded an amino acid sequence (SEQ ID NO 15) identical to the known partial peptide sequence of porcine TNF ⁇ -con on which the primers were based, as shown below.
• the 300 bp fragment was highly homologous to a human actin-binding protein (filamin) according to a sequence comparison with the GenBank
• the 89 bp fragment (SEQ ID NO 8) was used as a probe to screen a porcine spleen cDNA library constructed in ⁇ gt10.
• the probe was labeled by random priming (BRL kit) in the presence of a 32 P-dCTP.
• a single-stranded oligonucleotide of 55 bp was also synthesized as a probe according to the sequence of the 89 bp fragment.
• the 55 bp oligomer was located
• the 55 bp oligomer was labeled by kinase-end labeling using ⁇ 32 P-ATP. Hybridization of the screening filters was performed in a 40% formamide buffer at 39°C, and the final wash was in 1 x SSC (55 bp oligomer probe) or 0.2 x ssc (89 bp probe) at 46°C. 6.2.2. RESULTS
• the initial screening resulted in the isolation of four positive clones out of 2.5 x 10 5 recombinants. These four clones (psC-1, psC-2, psC-3 and psC-5) ranged from 1.1 kb to 2.3 kb in size and were sequenced after subcloning into the EcoRI site of Bluescript II SK. The clone psC-3 was completely sequenced in both directions by subcloning the smaller restriction fragments into Bluescript II SK and using flanking T3 and T7 sequences as sequencing primers. Sequence comparison showed that the four clones are
• FIG. 5 shows a contiguous mapping of the entire length of 2,414 bases covered by the four clones. Sequence analysis demonstrated that the 2,414 bp domain contained the coding sequence of the known 41 amino acid peptide sequence obtained from purified porcine spleen TNF ⁇ - con, but did not contain the coding region for the N- terminus of the porcine TNF-con. The conserved Zn 2+ -binding motif of metalloproteinases was also lacking.
• Two ⁇ gt10 cDNA libraries constructed either from human leukocyte poly(A+) RNA (Clontech) or human monocyte poly(A+) RNA were used to screen for the full length human TNF ⁇ -con cDNA.
• the 120 bp EcoRI-Pstl fragment and its flanking 3' 690 bp Pstl-BamHI fragment from the porcine psC-2 clone were labeled by random priming (BRL kit) and used to screen both libraries.
• Replicate filters were hybridized in 50% formamide at 42°C, and the final wash was in 0.2 x SSC with 0.1% SDS at 55°C. Of approximately 2.5 x 10 5 clones from each library, six of 14 positive clones
• Synthesis of a first strand was carried out using oligonucleotide primer RACE1 (22-mer) having the following sequence: S'-CCTAGAGTCAGGCTCACCAACC-3' (SEQ ID NO 32), which is complementary to bp no. 541-520 of TNF ⁇ -con (FIG. 1) in sequence with Met start at bp no. 164-166.
• RACE1 22-mer
• SEQ ID NO 32 S'-CCTAGAGTCAGGCTCACCAACC-3'
• Met start at bp no. 164-166 The following were combined: 1 ⁇ l RACEl Oligonucleotide (2.5 pmoles/ ⁇ l); 1 ⁇ l Poly(A+) RNA from THP1-5A cells treated for 3 hr with TNF ⁇ and dibutyryl cyclic AMP (0.84 ⁇ g) ; and 9.5 ⁇ l DEPC- treated dH 2 O.
• the mixture was incubated at 70°C for 10 min and then chilled on ice
• RNase H (1 ⁇ l, 2 U) was added, and the mixture incubated at 55°C for 10 min, and then chilled on ice.
• the cDNA was purified using GLASSMAXTM DNA isolation cartridges according to manufacturers protocol [Gibco/BRL, catalog no. 18374-025]. Samples were partially dried in vacuo to bring volume to approximately 30 ⁇ l.
• reaction buffer 3.0 ⁇ l 25 mM MgCl 2 ; 1.0 ⁇ l 10 mM dNTPs; 1 ⁇ l RACE2B Oligonucleotide (10 pmoles/ ⁇ l); and 1 ⁇ l Anchor Oligo-nucleotide (10 pmoles/ ⁇ l).
• RACE2B oligonucleotide (48-mer) has the following sequence (SEQ ID NO 33):
• Anchor Oligonucleotide from Gibco/ BRL, has the following sequence (SEQ ID NO 34):
• reaction mixtures were incubated at 80°C for 5 min.
• Five ⁇ l of HOT TUBTM Polymerase (Amersham Inc.) in IX reaction buffer (1 U/5 ⁇ l) was added, and the mixtures cycled in a Perkin Elmer 9600 thermal cycler 34 times (1 min at 94°C, 30 sec at 50°C, 2 min at 72°C); and 1 time (1 min at 94°C, 30 sec at 50°C, and 10 min at 72°C). All 15 reaction mixtures were combined and the DNA was precipitated at -20°C with 2 M ammonium acetate and 2 volumes of 100% EtOH. DNA precipitate was collected by centrifugation, rinsed with cold 70% EtOH, dried in vacuo, and resuspended in 20 ⁇ l of dH 2 O.
• DNA was electrophoresed through a 1.5% agarose gel in 0.5X TBE buffer. DNA migrating as a stained smear from approximately 375-700 bp was collected by electro-elution into a well containing 100 mM ammonium acetate. The DNA was extracted once with phenol/chloroform (1:1), and
• DNA precipitate was collected by centrifugation, rinsed with cold 70% EtOH, dried in vacuo , and resuspended in 21.5 ⁇ l of Dh 2 O.
• DH5 ⁇ MAX EFFICIENCYTM Cells (Gibco/BRL) (100 ⁇ l) were transformed with 1.5 ⁇ l of the ligation mixture
• Plasmid DNA from white colonies was isolated by standard methods and analyzed for cDNA inserts by digestion with Spel and electrophoresis through a 1.0% agarose gel in 0.5X TBE buffer. Clones containing cDNA inserts were sequenced and positive clones were identified by comparison to the 5'-nucleotide sequence of TNFc clone hell.
• the DNA sequence (SEQ ID NO 39) of the RACE14 clone is shown in FIG. 11. constructing A cDNA Encoding The Entire
• RACE14 cDNA (SEQ ID NO 39) (FIG. 11) was cloned into the Spel site of Bluescript plasmid, pBS-SKII+ with the 3 '-end of the cDNA oriented closest to the T7 promoter site on the vector.
• RACE14/pBS-SKII (3,485 bp) was cut with Bglll and Hindlll and the ends were dephosphorylated.
• hc-11 cDNA was cloned into the EcoRI site of Bluescript plasmid pBS-SKII+ with the 3'-end of the cDNA oriented closest to the T7 promoter site on the vector.
• hc-11/pBS-SKII (4,323 bp) was digested with Bglll and Hindlll to excise the hc-11 cDNA.
• the Bglll/HindlII digested hc-11 cDNA was ligated into the Bglll/HindlII digested RACE14/pBS-SKII.
• the resulting plasmid (4,677 bp) was designated RACE14/hcll-pBS- SKII.
• RACE14/hc11-pBS-SKII was digested with EcoRI and
• hc-7 cDNA (FIG. 7) was cloned into the EcoRI site of Bluescript plasmid, pBS-SKII+ with the 3'-end of the cDNA oriented closest to the T3 promoter site on the vector.
• hc-7/pBS-SKII (4,813 bp) was cut with Eael and Ncol. DNA fragments were separated on a 1% agarose, 0.5X TBE gel and the 750 bp Eael/Ncol fragment of hc-7 was isolated using FMC SPINBINDTM cartridges as above, hc-9 cDNA (FIG.
• TNFC-4 oligonucleotide sequence (SEQ ID NO 35) is:
• TNFC-3 oligonucleotide sequence (SEQ ID NO 36) is: 5'-CAGGAAGTTGCGGCCGCTGACCAGCATCTGCTAAGTCACTTCCCAGTCTTCAC-3'
• TNF ⁇ -con cDNA bp no. 2754-2719 of TNF ⁇ -con cDNA (FIG. 1).
• the mixture was incubated at 80°c for 5 min, an d 5 ⁇ l (1 U) HOT TUBTM Polymerase in IX buffer (Amersham) was then added.
• the mixture was cycled in a Perkin Elmer 9600 thermal cycler 25 times (1 min at 94°C, 2 min at 55°C, 2 min at 72°C), and 1 time (l min at 94°C, 2 min at 55°C, 10 min at 72°C).
• the PCR reaction mixture was extracted once with phenol/chloroform (1:1), and precipitated at -20°C with 2 M ammonium acetate and 2 volumes of 100% EtOH.
• DH5 ⁇ MAX EFFICIENCYTM cells (Gibco/BRL) (67 ⁇ l) were transformed with 1.5 ⁇ l each of the ligation mixtures from above according to manufacturers protocol.
• Cells were plated onto LB plates containing 50 ⁇ g/ml ampicillin, overlayed with 75 ⁇ l Bluo-Gal and 10 ⁇ l 100 mM IPTG, and incubated overnight at 37°C. Plasmid DNA from "white” colonies was isolated by standard methods and analyzed for cDNA inserts by digestion with Pstl, and duplicate aliquots were digested with BamHI and Ncol and electrophoresed through a 1.0% agarose gel in 0.5X TBE buffer.
• pBS/TNFC-1 DNA from one of the two clones, was correctly assembled as determined by its DNA sequence.
• pBS/TNFC-l corresponds to bp 164-2754 of the cDNA sequence encoding TNF ⁇ -con shown in FIG. 1, but with BamHI and Notl ends added to the sequence.
• the full-length cDNA encoding the human TNF ⁇ -con was subcloned into a baculovirus expression vector,
• pFastBacl (Gibco/BRL), as follows: pBS/TNFC-1 (5 ⁇ g) was digested with 1 ⁇ l of BamHI (Promega, 10 U/ ⁇ l); 1 ⁇ l of Notl (Promega, 10 U/ ⁇ l); 1 ⁇ l of Pvul (Gibco/BRL, 10 U/ ⁇ l) (note: Pvul was added to further cut pBluescriptSK and facilitate band identification and isolation from gel); 3 ⁇ l of 10x New England Biolabs (NEB) restriction buffer no. 3; and ad iusted to a final volume of 30 ⁇ l with water.
• the mixture was incubated for 2 hr at 37°C and then run on a 1% agarose-TAE gel.
• the approx. 2.9 kb BamHI - Notl insert band on the gel was excised in a gel piece.
• the gel piece was frozen at -70°C for 15 min, incubated at 37°C for 15 min, placed inside the upper chamber of a Millipore spin filter unit (ULTRAFREE TM Probind), and then centrifuged in a
• Ligated material (10 ⁇ l) was used to transform 100 ⁇ l of DH5 ⁇ MAX EFFICIENCYTM competent cells (Gibco/BRL) by calcium chloride precipitation according to supplier's instructions. Transformation mixture (100 ⁇ l) was plated onto a 2x YT/agar plate containing 100 ⁇ g/ml ampicillin. The plate was incubated overnight at 37°C. Colonies were picked up from the plate and diluted in 2 ml of the same medium without agar. These cultures were incubated at 37°C overnight with vigorous shaking. Plasmids were isolated using the WIZARDTM miniprep DNA purification kit (Promega), according to manufacturer's instructions.
• each isolated plasmid was incubated with 1 ⁇ l of 10X NEB buffer no. 4, 0.5 ⁇ l of Notl (Promega; 10 U/ ⁇ l), and 0.5 ⁇ l of BamHI (Promega; 10 U/ ⁇ l), and adjusted to a final volume of 10 ⁇ l with water. After 1 hr of incubation at 37°C, samples were run in a 1% agarose-TAE gel, and one candidate with the correct restriction pattern was identified. A larger plasmid preparation was made out of this isolate (100 ml in the same culture medium as described above), and the plasmid was extracted and purified using the Qiagen plasmid Midi kit, according to manufacturer's directions. Sequence of the pure plasmid was confirmed by sequencing analysis at the Glaxo Wellcome Core DNA Sequencing Facility. This plasmid was designated pFBPH/TNFC.
• An expression vector containing a partial TNF ⁇ -con cDNA was also constructed in pFastBac, starting at codon 164 (Met) and ending at codon 651 (Arg) .
• This region encodes a polypeptide that spans from before the catalytic domain to before the transmembrane domain.
• This construct was made as follows. The oligonucleotides M3 and M7 were used to obtain the desired cDNA fragment from PBS/hc-7 by PCR:
• M7 5'-CGCGCGCGCGGGATCCCTATCGTTCAATTACATCCTGTAC- TCGTTTCTCAC-3' (SEQ ID NO. 38).
• M3 and M7 were diluted after purification to a final concentration of 20 ⁇ M in water.
• Each primer (1 ⁇ l) was added to a tube containing 11.5 ⁇ l of water, followed by the addition of 2 ⁇ l of Stratagene OPTIPREPTM buffer no. 3, 2 ⁇ l of 10 mg/ml bovine serum albumin, 1 ⁇ l (0.1 ⁇ g) of pBS/hc-7, 0.5 ⁇ l of a 100 mM deoxynucleotides mixture
• the reaction mixture was cycled in a Perkin-Elmer model 9600 thermal cycler using the following PCR protocol for a total of 30 cycles: 30 sec at 94°C, 30 sec at 50oC, and 2 min at 72°C. After reaction completion, 2 ⁇ l were run on a 1% agarose-TAE buffer to confirm product size and quality. The remaining material was diluted to 85 ⁇ l with water, followed by the addition of 10 ⁇ l of 10x NEB buffer no. 4 and 5 ⁇ l of BamHI (New England Biolabs, 20 U/ ⁇ l). The restriction reaction was incubated overnight at 37°C, and the sample was resolved in a 1% agarose-TAE preparative gel. The DNA band corresponding to the restricted PCR product was excised, and the gel piece was purified by spin filtration as described above.
• the eluate collected from the bottom of the centrifuge tube was used in a ligation reaction to Pfastbac1 (Gibco/BRL) containing an inverted multiple cloning site cut with Stul and BamHI. as follows: 10 ⁇ l (1 ⁇ g) of PfastBacl were mixed with 5 ⁇ l of 10x NEB buffer no. 4, 2.5 ⁇ l of Stu I (New England Biolabs, 10 U/ ⁇ l), and 30 ⁇ l of water. The Stul cleavage reaction was allowed to proceed for 3 hr at 37°C, and then 2.5 ⁇ l of BamHI (New England Biolabs, 20 U/ ⁇ l) were added with further incubation at 37°C for 3 hr. The restricted plasmid was purified as described for the PCR product. Ligations were done in a final volume of 20 ⁇ l, and transformation and colony screening were done as
• the ligation reaction consisted of 2 ⁇ l (approx. 50 ng) of restricted Pfastbacl, 10 ⁇ l (approx. 150 ng) of restricted PCR product, 2 ⁇ l of 10x T4 DNA ligase buffer (Promega), and 2 ⁇ l of T4 DNA ligase-HC (Promega). The reaction was incubated at 12°C overnight.
• Ligated material (10 ⁇ l) was used to transform 100 ⁇ l of DH5 ⁇ MAX EFFICIENCYTM competent cells (Gibco/BRL) by calcium chloride precipitation following the supplier's instructions. 100 ⁇ l of the transformation mixture were plated onto a 2x YT/agar plate containing 100 ⁇ g/ml
• This mutation was fixed by replacing the Ncol to Xhol fragment of pFBN2 (containing the mutation) with a Ncol to Xhol fragment from clone hc-11 (wild type T at position 1,512), in the multi-step procedure described below.
• an additional Xhol site in pFBN2's multiple cloning site was outcloned.
• 2 ⁇ l (4 ⁇ g) of pFBN2 were incubated at 37°C for 3 hr with 2 ⁇ l of NEB buffer no. 4, 1 ⁇ l of Notl (Promega, 10 U/ ⁇ l), 1 ⁇ l of Hindlll (Promega, 10 U/ ⁇ l), and 14 ⁇ l of water.
• the plate was incubated for 24 hr, and colonies were picked and plasmid minipreps prepared as described above. Restriction analysis was carried out using Xhol, HinDIII, Notl and Xhol- Accl combined in order to confirm the loss of the HinDIII, Notl and the extra Xhol site.
• One construct with the correct restriction patterns was identified, its plasmid DNA prepared as described before, and sequenced. This plasmid was designated pFBN2/ ⁇ XhoI, and its sequence is identical to pFBN2, except for the deletion of the HinDIII to Notl segment of the multiple cloning site.
• both pFBN2/ ⁇ XhoI and pBS/hc-11 were digested with Ncol and Xhol as follows. 10 ⁇ g of either plasmid was mixed with 6 ⁇ l of NEB buffer no. 4, 3 ⁇ l of Ncol (Promega, 10 U/ ⁇ l), 3 ⁇ l of Xhol (Gibco/BRL, 10 U/ ⁇ l), and adjusted to a final volume of 60 ⁇ l with water. These mixtures were then incubated for 2 hr at 37°C, and loaded on a 1% agarose-TAE preparative gel. The backbone fragment of pFBN2/ ⁇ XhoI and the approx.
• 380 bp fragment from PBS/hc-11 were excised and purified by spin- filtration as described above. 50 ng (4 ⁇ l) of the backbone fragment were mixed with 12 ng (12 ⁇ l) of the small hc-11 fragment, 2 ⁇ l of 10x T4 DNA ligase buffer (Promega) and 2 ⁇ l of T4 DNA ligase (Promega, HC) , and incubated at 12°C overnight.
• Stbl-2 MAX EFFICIENCYTM competent cells 200 ⁇ l; Gibco/BRL were added, and the mixture was incubated at 0°C for 30 min, followed by a 30 sec pulse at 42°C and plating onto a 2x YT-agar plate containing 100 ⁇ g/ml ampicillin. The plate was incubated at 30°C for 24 hr. Colonies were picked and plasmid minipreps were performed as described above. Isolated plasmids were analyzed by double digestion with Xhol (Gibco/BRL, 10 U/ ⁇ l) and Accl (New England
• pFBN2/hc-11 and its sequence is identical to that of pFBN2/ ⁇ XhoI, except for the presence of the wild type T residue at position 1,512.
• pFBN2/hc-11 was used to repair pFBPH/TNFC. Both pFBN2/hc-11 (10 ⁇ g) and pFBPH/TNFC (5 ⁇ g) were mixed with 5 ⁇ l of NEB buffer no. 4, 2.5 ⁇ l of Nsil (New England Biolabs, 10 U/ ⁇ l), 2.5 ⁇ l of Ncol (Promega, 10 U/ ⁇ l), and adjusted to 50 ⁇ l with water. Mixtures were incubated for 2 hr at 37°C, and the digests were resolved in a 1% agarose-TAE preparative gel. The backbone fragment of pFBPH/TNFC and the approx.
• 600 bp fragment from pFBN2/hc-11 were excised and purified as described above.
• 50 ng (2 ⁇ l) of pFBPH/TNFC backbone fragment were then mixed with 50 ng (10 ⁇ l) of pFBN2/hc-11, 2 ⁇ l of 10x T4 DNA ligase buffer (Promega), 2 ⁇ l of T4 DNA ligase (Promega, HC) and 4 ⁇ l of water.
• the reaction was incubated overnight at 12°C, and then 200 ⁇ l of Stbl-2 competent cells were transformed with this ligate as described above. Transformant colonies were isolated and the DNA purified and analyzed as described above by
• the .new full-length expression vector was designated pFBPH/TNFCA, and differs from pFBPH/TNFC by the presence of the correct T residue at position 1512.
• Human TNF ⁇ -con is characterized by the cDNA sequence (SEQ ID NO 1) and deduced amino acid sequence (SEQ ID NO 2) shown in FIG. 1.
• the cDNA contains a 163 bp untranslated region at the 5' end, followed by a protein coding region of 2,475 bp, and a 304 bp untranslated region at the 3' end.
• the first methionine residue (presented as the first amino acid residue in FIG. 1) is the initiation methionine, as indicated by the presence upstream of this residue of a termination codon (TAG) at bases 62 to 64.
• TAG termination codon
• TNF ⁇ -con comprises 824 amino acids. TNF ⁇ -con begins with amino acid Met and ends with amino acid Cys. Based on the deduced amino acid sequence, the
• predicted molecular weight of the protein is 93.02 kDa.
• Hydropathy plots reveal 2 hydrophobic segments representing a putative signal peptide at amino acid residues 1-17, and a transmembrane region at amino acid residues 672-691.
• a search of GeneBank identified the following motifs that are unique to the metalloproteinase family: cysteine switch motif (aa 181-185), zinc-binding motif (aa 405-409), and Met-turn motif (aa 435-437).
• a biotinylated inhibitor of TNF ⁇ -con useful for the affinity purification of TNF ⁇ -con as described above (Section 6.1.1), was prepared as follows.
• Reagent (C) (FIG. 9C) was prepared by the following procedure. To a solution of concentrated sulfuric acid (75 ml) in distilled water (350 ml) were added D- leucine (FIG. 10A) (50.0 g, 0.381 mol) and potassium bromide (158 g, 1.33 mol), and the solution was cooled to just below 0°C. To the solution was added sodium nitrite (34.8 g, 0.504 mol) as a solution in distilled water (100 ml), dropwise, over a period of 1 hr. After the addition was complete, the mixture was allowed to stir at 0°C for 1 hr. Dichloromethane was added to the solution and the mixture was stirred for several minutes. The layers were separated and the aqueous layer was extracted using dichloromethane. The layers were separated and the combined organic layer was dried (MgSO 4 ) and filtered, and the filtrate was
• the biotin derivative of Gl 193463A (FIG. 91), was prepared as follows. Gl 193463A (20 mg) was dissolved in 0.6 ml dimethyl formamide and treated with 9 ⁇ l
• Partially purified human TNF ⁇ -con was generated from microsomal fractions containing convertase activity as follows.
• Mono Mac 6 cells (Ziegler-Heitbrook et al . , 1988, Int. J. Cancer, 41:456-461) were grown in RPMI 1640 medium containing 10% fetal bovine serum, 0.1% pluronic,
• protease inhibitors (1 mM AEBSF, 10 ⁇ M E-64, 10 ⁇ M
• leupeptin 1 ⁇ M pepstatin, 10 ⁇ M phosphoramidon, and 50 ⁇ M DCI.
• the suspension was pressurized under 1,000 psi N 2 in a cavitator for 30 min with stirring at 4°C. Pressure was released over 1-2 min with collection of approx. 90% of the lysate. Cell breakage was greater than 80% as determined by trypan blue exclusion.
• the broken cell suspension was sedimented in a GS-3 rotor at 3,500 rpm for 10 min.
• the supernatant (400 ml) was centrifuged in a TFA 20.25 rotor at 20,000 rpm for 45 min at 4°C.
• the resulting pellet (50 ml) was resuspended to a total volume of 750 ml in 10 mM HEPES buffer, pH 7.5, containing 0.2 M NaCl, 1.2% NP-40, and protease inhibitors as above, except DCI, via dounce homogenization and stirring for 30 min at 4°C.
• the resuspended material was then centrifuged in a TFA 20.25 rotor at 20,000 rpm for 45 min at 4°C.
• the resulting pellet had a volume of less than 10 ml.
• the supernatant (approx. 750 ml) was passed at 10 ml/min over a column containing 100 ml packed conA-sepharose (Pharmacia) which had been previously equilibrated with 10 mM HEPES buffer, pH 7.5, containing 0.2 M NaCl and 1% NP-40.
• the loaded column was washed with 200 ml of equilibration buffer, followed by 800 ml of the same buffer but without NaCl. Material was retained overnight at 4°C on the conA column.
• Protein was eluted from the conA column by passing 850 ml of 10 mM HEPES buffer, pH 7.5, containing 1% NP-40, 0.25 M methyl- ⁇ -D-mannopyranoside, and protease inhibitors as above, except DCI, over the column at 10 ml/min.
• the eluate was collected and passed over a column containing 2 ml packed POROSTM HQ anion exchanger at 5 ml/min which had been previously equilibrated with 10 mM HEPES buffer, pH 7.5, containing 1% NP-40.
• the column was washed with 10 ml of equilibration buffer.
• the buffer level over the packed bed was lowered to just cover the matrix and the inlet tube was flushed with 10 mM HEPES buffer, pH 7.5, containing 0.5 M NaCl, 1 % NP-40, and protease inhibitors as above, except DCI (high salt buffer) .
• High salt buffer was passed over the column at 1 ml/min, and ten 1 ml fractions were collected. Fractions 2 and 3, which had a distinct coloration, were pooled and contained 15.4 mg/ml protein (Micro BCA Kit;
• the enzyme was desalted by dialysis in 10 mM HEPES, pH 7.5, 1% ddm and protease inhibitors. The enzyme was then loaded onto a 200 ⁇ l POROSTM HQ column, and eluted off with the above buffer containing 0.5 M NaCl.
• Synthetic peptides were prepared that had substitutions at P1' (Val) or P2' (Arg) in the following substrate (SEQ ID NO 16):
• NBD-Ser-Pro- Leu-Ala-Gln-Ala-Val-Arg-Ser-Lys(DMC)-Ser-Arg-NH 2 (SEQ ID NO 17) (10 ⁇ M) was added to follow the reaction by monitoring fluorescence at 370 Nm (excitation), 460 Nm (emission) .
• cleavage of substrates could be compared to the standard substrate, Biotin-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser- Lys-(DNP)-NH 2 (SEQ ID NO 16), which was included at 1 ⁇ M.
• the substrates were incubated with enzyme preparation at 37°C for 0.5-4 hr and the reaction quenched by adding an equal volume of 1% HFBA. Samples were passed over a POROSTM avidin column and the products subjected to LC/MS to

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