EP0770141A1 - PROTEASE-1 ET 2 D'APOPTOSE DE TYPE ENZYME DE CONVERSION D'INTERLEUKINE-1 $g(b) - Google Patents

PROTEASE-1 ET 2 D'APOPTOSE DE TYPE ENZYME DE CONVERSION D'INTERLEUKINE-1 $g(b)

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Publication number
EP0770141A1
EP0770141A1 EP94922009A EP94922009A EP0770141A1 EP 0770141 A1 EP0770141 A1 EP 0770141A1 EP 94922009 A EP94922009 A EP 94922009A EP 94922009 A EP94922009 A EP 94922009A EP 0770141 A1 EP0770141 A1 EP 0770141A1
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EP
European Patent Office
Prior art keywords
polypeptide
ice
lap
polynucleotide
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP94922009A
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German (de)
English (en)
Other versions
EP0770141A4 (fr
Inventor
Wei-Wu He
Craig A. Rosen
Gregg A. Apartment H HASTINGS
Peter L. Hudson
Ewen F. Apartment 406 KIRKNESS
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Human Genome Sciences Inc
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Human Genome Sciences Inc
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Application filed by Human Genome Sciences Inc filed Critical Human Genome Sciences Inc
Publication of EP0770141A1 publication Critical patent/EP0770141A1/fr
Publication of EP0770141A4 publication Critical patent/EP0770141A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C12N9/6475—Interleukin 1-beta convertase-like enzymes (3.4.22.10; 3.4.22.36; 3.4.22.63)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are interleukin-1 ⁇ converting enzyme like apoptosis protease-1 and interleukin-1 jS converting enzyme like apoptosis protease-2, sometimes hereinafter referred to collectively as "ICE-LAP-1 and 2" . The invention also relates to inhibiting the action of such polypeptides.
  • an interleukin-1/3 converting enzyme ICE
  • ICE interleukin-1/3 converting enzyme
  • the overall amino acid identity between ced-3 and ICE is 28%, with a region of 115 amino acid ⁇ (residues 246-360 of ced-3 and 164-278 of ICE) that shows the highest identity (43%).
  • This region contains a conserved pentapeptide, QACRG (residues 356-360 of ced-3 ) , which contains a cysteine known to be essential for ICE function.
  • QACRG conserved pentapeptides 356-360 of ced-3
  • the ICE-LAP-1 and 2 polypeptides of the present invention also have the same conserved pentapeptide and the cysteine residue which i ⁇ es ⁇ ential for ICE function.
  • ced-3 ⁇ uggests not only that ced-3 might function a ⁇ a cy ⁇ teine protea ⁇ e but also that ICE might act as a vertebrate programmed cell death gene, ced-3 and the vertebrate counterpart, ICE, control programmed cell death during embryonic development, (Gagliarnini, V. et al., Science, 263:826:828 (1994).
  • ICE mRNA has been detected in a variety of tissue ⁇ , including peripheral blood monocyte ⁇ , peripheral blood lymphocytes, peripheral blood neutrophils, resting and activated peripheral blood T lymphocytes, placenta, the B lymphoblastoid line CB23, and monocytic leukemia cell line THP-1 cells (Cerretti, D.P., et al., Science, 256:97-100 (1992)), suggesting that ICE may have an additional substrate in addition to pro-IL-13.
  • the sub ⁇ trate that ICE acts upon to cause cell death is presently unknown.
  • One possibility is that it may be a vertebrate homolog of the C. elegan ⁇ cell death gene ced-4 .
  • ICE might directly cause cell death by proteolytically cleaving proteins that are essential for cell viability.
  • the mammalian gene bcl-2 has been found to protect immune cells called lymphocytes from cell suicide. Also, crmA, a cow pox virus gene protein product inhibits ICE' ⁇ protein splitting activity.
  • novel mature polypeptides which are ICE-LAP-1 and 2, as well as fragments, analogs and derivatives thereof.
  • the polypeptides of the present invention are of human origin.
  • polynucleotides (DNA or RNA) which encode such polypeptides.
  • proce ⁇ for producing ⁇ uch polypeptide ⁇ by recombinant technigue ⁇ .
  • a process for utilizing such polypeptides, or polynucleotide ⁇ encoding ⁇ uch polypeptide ⁇ for therapeutic purpo ⁇ e ⁇ for example, a ⁇ an antiviral agent, an anti-tumor agent and to control embryonic development and ti ⁇ sue ho eosta ⁇ is.
  • antagoni ⁇ t/inhibitor ⁇ to such polypeptide ⁇ , which may be u ⁇ ed to inhibit the action of ⁇ uch polypeptide ⁇ , for example, in the treatment of Alzheimer's disea ⁇ e, Parkin ⁇ on' ⁇ disease, rheumatoid arthritis, septic shock and head injury.
  • Figure 1 shows ⁇ the cDNA and corre ⁇ ponding deduced amino acid sequence of ICE-LAP-1.
  • the polypeptide encoded by the amino acid sequence shown is the putative mature form of the polypeptide (minus the initial methionine residue), and the standard one-letter abbreviation for amino acids is u ⁇ ed.
  • Figure 2 ⁇ how ⁇ the cDNA and corresponding deduced amino acid sequence of ICE-LAP-2.
  • the polypeptide encoded by the amino acid sequence ⁇ hown i ⁇ the putative mature form of the polypeptide (minus the initial methionine residue).
  • Figure 3 shows an amino acid sequence compari ⁇ on of ICE-LAP-1 to ICE.
  • Figure 4 illu ⁇ trate ⁇ a gel after performing a Northern Blot analysis of ICE-LAP-1 showing the concentration in various human ti ⁇ sues.
  • isolated nucleic acids which encode the mature polypeptides having the deduced amino acid sequence of Figures 1 and 2 or for the mature polypeptide encoded by the cDNA of the clones depo ⁇ ited as ATCC Deposit No. 75772 and .
  • ATCC Deposit No. 75772 contains the cDNA encoding for ICE-
  • LAP-1 contains the cDNA encoding for
  • the polynucleotide encoding ICE-LAP-1 was discovered in a cDNA library derived from human fetal liver. It is structurally related to the Interleukin-13 converting enzyme family. It contains an open reading frame encoding a protein of approximately 377 amino acid residues. The protein exhibits the highest degree of homology to human interleukin-1/3 converting enzy e with 68 % similarity and 53% identity over the entire amino acid ⁇ equence. It ⁇ hould be pointed out that the pentapeptide QACRG i ⁇ con ⁇ erved and i ⁇ located at amino acid po ⁇ ition 256-260.
  • the polynucleotide encoding ICE-LAP-2 wa ⁇ discovered in a cDNA library derived from human Jurkat Cells. It is structurally related to the ICE family. It contains an open reading frame encoding a protein of about 435 amino acid residues. The protein exhibits the highest degree of homology to the mouse Nedd-2 protein with 91 % identity and 94 % similarity over a 128 amino acid stretch. The overall protein exhibits the highest degree of homology to C. elegan ⁇ cell death protein ced-3 with approximately 40% identity and 60% similarity over 400 amino acid residue ⁇ . It i ⁇ al ⁇ o important that the pentapeptide QACRG i ⁇ conserved and i ⁇ located at amino po ⁇ ition 301-305.
  • the polynucleotide ⁇ of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encode the mature polypeptides may be identical to the coding ⁇ equence ⁇ hown in Figure ⁇ 1 and 2 or that of the depo ⁇ ited clones or may be a different coding sequence which coding sequence, a ⁇ a re ⁇ ult of the redundancy or degeneracy of the genetic code, encode the same mature polypeptide ⁇ , and derivative ⁇ thereof, a ⁇ the DNA of Figure ⁇ 1 and 2 or the depo ⁇ ited cDNA.
  • the polynucleotide ⁇ which encode for the mature polypeptide ⁇ of Figure ⁇ 1 and 2 or for the mature polypeptides encoded by the deposited cDNAs may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non- coding ⁇ equence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompas ⁇ es a polynucleotide which includes only coding sequence for the polypeptide a ⁇ well a ⁇ a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotide ⁇ which encode for fragment ⁇ , analog ⁇ and derivative ⁇ of the polypeptide ⁇ having the deduced amino acid sequence of Figures 1 and 2 or the polypeptide ⁇ encoded by the cDNA of the depo ⁇ ited clones.
  • the variants of the polynucleotide ⁇ may be naturally occurring allelic variant ⁇ of the polynucleotide ⁇ or non-naturally occurring variants of the polynucleotides.
  • the present invention includes polynucleotides encoding the same mature polypeptides as shown in Figures 1 and 2 or the same mature polypeptides encoded by the cDNA of the deposited clones a ⁇ well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides of Figures 1 and 2 or the polypeptides encoded by the cDNA of the deposited clones.
  • Such nucleotide variants include deletion variant ⁇ , substitution variants and addition or insertion variant ⁇ .
  • the polynucleotide ⁇ may have a coding ⁇ equence which i ⁇ a naturally occurring allelic variant of the coding ⁇ equence ⁇ hown in Figure ⁇ 1 and 2 or of the coding sequence of the depo ⁇ ited clone ⁇ .
  • an allelic variant i ⁇ an alternate form of a polynucleotide sequence which may have a sub ⁇ titution, deletion or addition of nucleotide ⁇ , which doe ⁇ not ⁇ ub ⁇ tantially alter the function of the encoded polypeptide ⁇ .
  • the polynucleotides may also encode for a proprotein which i ⁇ the mature protein plu ⁇ additional 5' amino acid re ⁇ idues.
  • the polynucleotide of the pre ⁇ ent invention may encode for a mature protein, or for a protein having a pro ⁇ equence or for a protein having both a pro ⁇ equence and a pre ⁇ equence (leader sequence).
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a sequence which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa-histidine tag ⁇ upplied by a pQE-9 vector to provide for purification of the mature polypeptide ⁇ fu ⁇ ed to the marker in the ca ⁇ e of a bacterial ho ⁇ t, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian ho ⁇ t, e.g. COS-7 cell ⁇ , i ⁇ u ⁇ ed.
  • the HA tag correspond ⁇ to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)) .
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described ⁇ equence ⁇ if there i ⁇ at least 50% and preferably 70% identity between the sequence ⁇ .
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides .
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polynucleotides which hybridize to the hereinabove de ⁇ cribed polynucleotides in a preferred embodiment encode polypeptides which retain sub ⁇ tantially the same biological function or activity as the mature polypeptide ⁇ encoded by the cDNA of Figure ⁇ 1 and 2 or the depo ⁇ ited cDNA ⁇ .
  • the deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to tho ⁇ e of ⁇ kill in the art and are not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • the sequence of the polynucleotide ⁇ contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequence ⁇ herein.
  • a licen ⁇ e may be required to make, u ⁇ e or ⁇ ell the depo ⁇ ited material ⁇ , and no ⁇ uch licen ⁇ e is hereby granted.
  • the present invention further relates to ICE-LAP-1 and 2 polypeptides which have the deduced amino acid sequence of Figures 1 and 2 or which ha ⁇ the amino acid ⁇ equence encoded by the depo ⁇ ited cDNA ⁇ , a ⁇ well as fragment ⁇ , analogs and derivative ⁇ of ⁇ uch polypeptide ⁇ .
  • fragment when referring to the polypeptides of Figure ⁇ 1 and 2 or that encoded by the depo ⁇ ited cDNA, ean ⁇ polypeptide ⁇ which retain e ⁇ entially the same biological function or activity as ⁇ uch polypeptides, and wherein derivative ⁇ include polypeptides with enhanced or reduced biological function.
  • An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce active mature polypeptide ⁇ .
  • polypeptides of the present invention may be recombinant polypeptides, natural polypeptides- or synthetic polypeptide ⁇ , preferably recombinant polypeptide ⁇ .
  • the fragment, derivative or analog of the polypeptides of Figure ⁇ 1 and 2 or that encoded by the deposited cDNA ⁇ may be (i) one in which one or more of the amino acid residues are sub ⁇ tituted with a con ⁇ erved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such sub ⁇ tituted amino acid re ⁇ idue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid re ⁇ idues includes a substituent group, or (iii) one in which the mature polypeptide i ⁇ fu ⁇ ed with another compound, ⁇ uch a ⁇ a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • the polypeptide ⁇ and polynucleotide ⁇ of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material i ⁇ removed from it ⁇ original environment (e.g., the natural environment if it i ⁇ naturally occurring).
  • Such polynucleotide ⁇ could be part of a vector and/or ⁇ uch polynucleotide ⁇ or polypeptide ⁇ could be part of a compo ⁇ ition, and ⁇ till be i ⁇ olated in that ⁇ uch vector or compo ⁇ ition is not part of its natural environment.
  • the present invention also relates to vectors which include polynucleotide ⁇ of the pre ⁇ ent invention, ho ⁇ t cell ⁇ which are genetically engineered with vector ⁇ of the invention and the production of polypeptide ⁇ of the invention by recombinant technique ⁇ .
  • Ho ⁇ t cell ⁇ are genetically engineered (tran ⁇ duced or tran ⁇ formed or tran ⁇ fected) with the vector ⁇ of thi ⁇ invention which may be, for example, a cloning vector or an expre ⁇ ion vector.
  • the vector may be, for example, in the form of a pla ⁇ mid, a viral particle, a phage, etc.
  • the engineered ho ⁇ t cell ⁇ can be cultured in conventional nutrient media modified as appropriate for activating promoter ⁇ , ⁇ electing tran ⁇ formant ⁇ or amplifying the ICE-LAP-1 gene ⁇ .
  • the culture condition ⁇ , ⁇ uch a ⁇ temperature, pH and the like, are tho ⁇ e previou ⁇ ly u ⁇ ed with the ho ⁇ t cell ⁇ elected for expre ⁇ ion, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thu ⁇ , for example, the polynucleotide may be included in any one of a variety of expres ⁇ ion vector ⁇ for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and ⁇ ynthetic DNA sequences, e.g., derivative ⁇ of SV40; bacterial pla ⁇ mid ⁇ ; phage DNA; baculoviru ⁇ ; yea ⁇ t pla ⁇ mid ⁇ ; vector ⁇ derived from combination ⁇ of pla ⁇ mid ⁇ and phage DNA, viral DNA ⁇ uch a ⁇ vaccinia, adenoviru ⁇ , fowl pox viru ⁇ , and p ⁇ eudorabie ⁇ .
  • any other vector may be used a ⁇ long a ⁇ it i ⁇ replicable and viable in the ho ⁇ t.
  • the appropriate DNA ⁇ equence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease ⁇ ite( ⁇ ) by procedure ⁇ known in the art.
  • procedure ⁇ and other ⁇ are deemed to be within the ⁇ cope of tho ⁇ e ⁇ killed in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence( ⁇ ) (promoter) to direct mRNA ⁇ ynthe ⁇ i ⁇ .
  • ⁇ promoter
  • a ⁇ repre ⁇ entative example ⁇ of ⁇ uch promoter ⁇ there may be mentioned: LTR ⁇ from retroviru ⁇ e ⁇ , e.g. RSV, HIV, HTLVI, CMV or SV40 promoter, the E. coli. lac or trp. the phage lambda P L promoter and other promoter ⁇ known to control expre ⁇ ion of gene ⁇ in prokaryotic or eukaryotic cell ⁇ or their viru ⁇ e ⁇ .
  • al ⁇ o cellular ⁇ ignal ⁇ can be u ⁇ ed, for example, human- ⁇ -actin- pro oter) .
  • the expre ⁇ ion vector can contain a ribosome binding site for tran ⁇ lation initiation and a transcription terminator.
  • the vector may also include appropriate ⁇ equence ⁇ for amplifying the copy number of the gene.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells ⁇ uch a ⁇ dihydrofolate reducta ⁇ e or neomycin re ⁇ i ⁇ tance for eukaryotic cell culture, or ⁇ uch a ⁇ tetracycline or ampicillin re ⁇ i ⁇ tance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, a ⁇ well a ⁇ an appropriate promoter or control ⁇ equence, may be employed to tran ⁇ form an appropriate ho ⁇ t to permit the ho ⁇ t to expre ⁇ the protein.
  • a ⁇ repre ⁇ entative example ⁇ of appropriate ho ⁇ t ⁇ there may be mentioned: bacterial cell ⁇ , such a ⁇ E. coli- Bacillu ⁇ ⁇ ubtilis, Streptomyce ⁇ , Salmonella typhimurium: fungal cell ⁇ , such as yeast; insect cell ⁇ ⁇ uch a ⁇ Dro ⁇ ophila and Sf9; animal cells ⁇ uch as CHO, COS, HEK 293 or Bowes melanoma; plant cells, etc.
  • bacterial cell ⁇ such as a ⁇ E. coli- Bacillu ⁇ ⁇ ubtilis, Streptomyce ⁇ , Salmonella typhimurium
  • fungal cell ⁇ such as yeast
  • insect cell ⁇ ⁇ uch a ⁇ Dro ⁇ ophila and Sf9 insect cell ⁇ ⁇ uch a ⁇ Dro ⁇ ophila and Sf9
  • animal cells ⁇ uch as CHO, COS, HEK 293 or Bowes melanoma
  • plant cells etc.
  • the pre ⁇ ent invention al ⁇ o include ⁇ recombinant constructs comprising one or more of the sequence ⁇ a ⁇ broadly de ⁇ cribed above.
  • the con ⁇ truct ⁇ comprise a vector, such as a plasmid or viral vector, into which a ⁇ equence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further compri ⁇ e ⁇ regulatory ⁇ equence ⁇ , including, for example, a promoter, operably linked to the ⁇ equence.
  • Large number ⁇ of ⁇ uitable vector ⁇ and promoter ⁇ are known to tho ⁇ e of ⁇ kill in the art, and are commercially available.
  • the following vector ⁇ are provided by way of example.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pb ⁇ , pDIO, phagescript, psiX174, pblue ⁇ cript SK, pb ⁇ k ⁇ , pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
  • any other pla ⁇ mid or vector may be u ⁇ ed as long a ⁇ they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transfera ⁇ e) vector ⁇ or other vector ⁇ with selectable markers.
  • Two appropriate vector ⁇ are pKK232-8 and pCM7.
  • Particular named bacterial promoter ⁇ include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoter ⁇ include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviru ⁇ , and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described construct ⁇ .
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the ho ⁇ t cell can be effected by calcium pho ⁇ phate tran ⁇ fection, DEAE-Dextran mediated tran ⁇ fection, lipofection or electroporation. (Davi ⁇ , L., Dibner, M. , Battey, I., Ba ⁇ ic Method ⁇ in Molecular Biology, (1986)).
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant ⁇ equence.
  • the polypeptide ⁇ of the invention can be ⁇ ynthetically produced by conventional peptide ⁇ ynthe ⁇ izer ⁇ .
  • Mature proteins can be expressed in mammalian cells, yea ⁇ t, bacteria, or other cell ⁇ under the control of appropriate promoter ⁇ .
  • Cell-free tran ⁇ lation sy ⁇ tem ⁇ can al ⁇ o be employed to produce ⁇ uch protein ⁇ u ⁇ ing RNA ⁇ derived from the DNA con ⁇ tructs of the present invention.
  • Enhancers are cis-acting element ⁇ of DNA, usually about from 10 to 300 bp that act on a promoter to increase it ⁇ tran ⁇ cription. Exa ple ⁇ including the SV40 enhancer on the late ⁇ ide of the replication origin bp 100 to 270, a cytomegaloviru ⁇ early promoter enhancer, the polyo a enhancer on the late ⁇ ide of the replication origin, and adenovirus enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the ho ⁇ t cell, e.g., the ampicillin re ⁇ i ⁇ tance gene of E. coli and S. cerevi ⁇ iae TRP1 gene, and a promoter derived from a highly-expre ⁇ ed gene to direct tran ⁇ cription of a downstream structural sequence.
  • promoters can be derived from operon ⁇ encoding glycolytic enzyme ⁇ ⁇ uch a ⁇ 3- pho ⁇ phoglycerate kina ⁇ e (PGK), ⁇ -factor, acid phosphatase, or heat ⁇ hock protein ⁇ , among other ⁇ .
  • the heterologous structural sequence is as ⁇ embled in appropriate pha ⁇ e with tran ⁇ lation initiation and termination ⁇ equence ⁇ , and preferably, a leader sequence capable of directing secretion of translated protein into the peripla ⁇ mic ⁇ pace or extracellular medium.
  • the heterologou ⁇ ⁇ equence can encode a fu ⁇ ion protein including an N-terminal identification peptide imparting de ⁇ ired character- i ⁇ tic ⁇ , e.g., ⁇ tabilization or ⁇ implified purification of expre ⁇ sed recombinant product.
  • Useful expre ⁇ ion vector ⁇ for bacterial u ⁇ e are con ⁇ tructed by in ⁇ erting a ⁇ tructural DNA sequence encoding a desired protein together with suitable tran ⁇ lation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will compri ⁇ e one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the ho ⁇ t.
  • Suitable prokaryotic ho ⁇ t ⁇ for tran ⁇ formation include E. coli- Bacillu ⁇ subtilis. Salmonella typhimuriu and variou ⁇ species within the genera Pseudomona ⁇ , Streptomyces, and Staphylococcus, although other ⁇ may al ⁇ o be employed a ⁇ a matter of choice.
  • useful expression vectors for bacterial use can compri ⁇ e a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • cohausal vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Upp ⁇ ala, Sweden) and GEM1 (Promega Biotec, Madi ⁇ on, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expres ⁇ ed.
  • the ⁇ elected promoter i ⁇ induced by appropriate mean ⁇ e.g., temperature shift or chemical induction
  • cell ⁇ are cultured for an additional period.
  • Cell ⁇ are typically harve ⁇ ted by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expres ⁇ ion of proteins can be disrupted by any convenient method, including freeze-thaw cycling, ⁇ onication, mechanical di ⁇ ruption, or u ⁇ e of cell ly ⁇ ing agent ⁇ , such methods are well know to those skilled in the art.
  • Variou ⁇ mammalian cell culture ⁇ y ⁇ tem ⁇ can al ⁇ o be employed to expre ⁇ recombinant protein.
  • mammalian expres ⁇ ion sy ⁇ terns include the COS-7 lines of monkey kidney fibroblast ⁇ , de ⁇ cribed by Gluzman, Cell, 23:175 (1981), and other cell line ⁇ capable of expre ⁇ ing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell line ⁇ .
  • Mammalian expre ⁇ ion vector ⁇ may compri ⁇ e an origin of replication, a ⁇ uitable promoter and enhancer, polyadenylation site, splice donor and acceptor sites, tran ⁇ criptional termination ⁇ equence ⁇ , and 5' flanking nontran ⁇ cribed ⁇ equence ⁇ .
  • DNA ⁇ equence ⁇ derived fro the SV40 ⁇ plice and polyadenylation sites may be used to provide the required nontranscribed genetic element ⁇ .
  • the ICE-LAP-1 and 2 polypeptide ⁇ can be recovered and purified from recombinant cell culture ⁇ by method ⁇ including ammonium ⁇ ulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, pho ⁇ phocellulo ⁇ e chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, a ⁇ necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the pre ⁇ ent invention may be glyco ⁇ ylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
  • ICE-LAP-1 and 2 polypeptides may be employed to treat abnormally controlled programmed cell death.
  • Abnormally controlled programmed cell death may be an underlying cause of cancers due to an abnormal amount of cell growth. Accordingly, since ICE-LAP genes are implicated in programmed cell death, they may be used to target unwanted cells, for example, cancerous cell ⁇ .
  • ICE-LAP-1 and 2 may also be used to control vertebrate development and tis ⁇ ue homeo ⁇ ta ⁇ i ⁇ , due to it ⁇ apopto ⁇ i ⁇ ability.
  • Al ⁇ o, ICE-LAP-1 and 2 polypeptides may be used to overcome many viral infections by overcoming the ⁇ uppre ⁇ ed programmed cell death, ⁇ ince programmed cell death may be one of the primary antiviral defen ⁇ e mechani ⁇ m ⁇ of cell ⁇ .
  • ICE-LAP-1 and 2 may al ⁇ o be employed to treat immuno- suppre ⁇ ion related di ⁇ order ⁇ , ⁇ uch a ⁇ AIDS, by targeting virus infected cells for cell death.
  • the polypeptide ⁇ of the pre ⁇ ent invention may al ⁇ o be u ⁇ ed for identifying other molecule ⁇ which have ⁇ imilar biological activity.
  • Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • polypeptides may al ⁇ o be employed in accordance with the pre ⁇ ent invention by expre ⁇ ion of such polypeptides in vivo , which i ⁇ often referred to a ⁇ "gene therapy.”
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo , with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • Such method ⁇ are well-known in the art.
  • cell ⁇ may be engineered by procedure ⁇ known in the art by u ⁇ e of a retroviral particle containing RNA encoding a polypeptide of the pre ⁇ ent invention.
  • cell ⁇ may be engineered in vivo for expre ⁇ ion of a polypeptide in vivo by, for example, procedure ⁇ known in the art.
  • procedure ⁇ known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the pre ⁇ ent invention may be admini ⁇ tered to a patient for engineering cell ⁇ in vivo and expression of the polypeptide in vivo .
  • the expre ⁇ ion vehicle for engineering cell ⁇ may be other than a retroviru ⁇ , for example, an adenoviru ⁇ which may be u ⁇ ed to engineer cell ⁇ in vivo after combination with a ⁇ uitable delivery vehicle.
  • the polypeptide ⁇ of the pre ⁇ ent invention may be employed in combination with a ⁇ uitable pharmaceutical carrier.
  • a ⁇ uitable pharmaceutical carrier Such compo ⁇ ition ⁇ comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation ⁇ hould ⁇ uit the mode of admini ⁇ tration.
  • the invention al ⁇ o provide ⁇ a pharmaceutical pack or kit compri ⁇ ing one or more container ⁇ filled with one or more of the ingredient ⁇ of the pharmaceutical compo ⁇ ition ⁇ of the invention.
  • Associated with such container( ⁇ ) can be a notice in the form pre ⁇ cribed by a governmental agency regulating the manufacture, u ⁇ e or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.
  • the pharmaceutical composition ⁇ may be administered in a convenient manner such a ⁇ by the intravenou ⁇ , intraperitoneal, intramuscular, subcutaneou ⁇ , intrana ⁇ al or intradermal route ⁇ .
  • ICE-LAP-1 and 2 are admini ⁇ tered in an amount which i ⁇ effective for treating and/or prophylaxi ⁇ of the ⁇ pecific indication.
  • ICE-LAP-1 and 2 will be admini ⁇ tered in an amount of at lea ⁇ t 10 g/kg body weight, and in mo ⁇ t ca ⁇ e ⁇ they will be administered in an amount not in excess of 8 mg/Kg body weight per day.
  • the dosage is from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptom ⁇ , etc.
  • the ⁇ equence ⁇ of the present invention are also valuable for chromosome identification.
  • the sequence i ⁇ specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagent ⁇ based on actual ⁇ equence data (repeat polymorphi ⁇ m ⁇ ) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention i ⁇ an important fir ⁇ t ⁇ tep in correlating tho ⁇ e ⁇ equence ⁇ with genes associated with disease.
  • sequence ⁇ can be mapped to chromo ⁇ omes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
  • Computer analysi ⁇ of the cDNA i ⁇ u ⁇ ed to rapidly ⁇ elect primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process.
  • These primer ⁇ are then u ⁇ ed for PCR ⁇ creening of ⁇ omatic cell hybrid ⁇ containing individual human chromo ⁇ ome ⁇ . Only tho ⁇ e hybrid ⁇ containing the human gene corre ⁇ ponding to the primer will yield an amplified fragment.
  • mapping of ⁇ omatic cell hybrid ⁇ i ⁇ a rapid procedure for a ⁇ igning a particular DNA to a particular chromo ⁇ ome.
  • ⁇ ublocalization can be achieved with panel ⁇ of fragment ⁇ from ⁇ pecific chromo ⁇ ome ⁇ or pool ⁇ of large genomic clone ⁇ in an analogou ⁇ manner.
  • Other mapping strategies that can similarly be u ⁇ ed to map to its chromosome include in ⁇ itu hybridization, prescreening with labeled flow-sorted chromosome ⁇ and pre ⁇ election by hybridization to con ⁇ truct chromo ⁇ ome ⁇ pecific-cDNA librarie ⁇ .
  • Fluore ⁇ cence in situ hybridization (FISH) of a cDNA clone ⁇ to a metaphase chromosomal spread can be used to provide a preci ⁇ e chromosomal location in one step.
  • This technique can be used with cDNA a ⁇ short a ⁇ 500 or 600 base ⁇ ; however, clone ⁇ larger than 2,000 bp have a higher likelihood of binding to a unique chromo ⁇ omal location with ⁇ ufficient ⁇ ignal intensity for simple detection.
  • FISH require ⁇ use of the clones from which the EST was derived, and the longer the better.
  • the phy ⁇ ical po ⁇ ition of the sequence on the chromosome can be correlated with genetic map data.
  • genetic map data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through John ⁇ Hopkin ⁇ Univer ⁇ ity Welch Medical Library) .
  • the relation ⁇ hip between gene ⁇ and di ⁇ ea ⁇ e ⁇ that have been mapped to the ⁇ ame chromo ⁇ omal region are then identified through linkage analy ⁇ i ⁇ (coinheritance of physically adjacent genes).
  • a cDNA precisely localized to a chromosomal region as ⁇ ociated with the di ⁇ ea ⁇ e could be one of between 50 and 500 potential cau ⁇ ative gene ⁇ . (Thi ⁇ a ⁇ ume ⁇ 1 megaba ⁇ e mapping resolution and one gene per 20 kb) .
  • the polypeptides, their fragment ⁇ or other derivative ⁇ , or analog ⁇ thereof, or cell ⁇ expre ⁇ ing them can be u ⁇ ed a ⁇ an immunogen to produce antibodie ⁇ thereto.
  • the ⁇ e antibodie ⁇ can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodie ⁇ , a ⁇ well a ⁇ Fab fragment ⁇ , or the product of an Fab expre ⁇ ion library. Variou ⁇ procedure ⁇ known in the art may be used for the production of ⁇ uch antibodie ⁇ and fragment ⁇ .
  • Antibodie ⁇ generated again ⁇ t the polypeptide ⁇ corre ⁇ ponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodie ⁇ binding the whole native polypeptide ⁇ . Such antibodie ⁇ can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodie ⁇ produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trio a technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodie ⁇ and Cancer Therapy, Alan R. Li ⁇ , Inc., pp. 77-96) .
  • the present invention also relates to a diagnostic assay for detecting levels of ICE-LAP-1 and 2 in a sample taken from a ho ⁇ t, eg. blood, urine or serum sample.
  • the level of ICE- LAP-1 and 2 may be detected, for example, by an immunoa ⁇ ay technique by procedure ⁇ known in the art.
  • An example of ⁇ uch an a ⁇ ay i ⁇ a ⁇ andwich a ⁇ ay which utilize ⁇ two antibodie ⁇ ⁇ pecific to an ICE-LAP-1 or 2 antigen, preferably monoclonal antibodie ⁇ with one of the antibodies being labeled, eg. by coupling a ⁇ uitable label such a ⁇ an indicator enzyme, eg.
  • hor ⁇ eradi ⁇ h peroxida ⁇ e The unlabeled antibody i ⁇ preferably on a ⁇ olid ⁇ upport. If antigen i ⁇ present, the antigen will bind to both antibodie ⁇ . After binding of the peroxidase-coupled antibody to the antigen, the peroxida ⁇ e can be u ⁇ ed to generate a colored product that i ⁇ mea ⁇ urable and whose concentration i ⁇ related to the amount of antigen in a ⁇ ample. Becau ⁇ e of the catalytic nature of the enzyme the ⁇ y ⁇ tem greatly amplifie ⁇ the ⁇ ignal. Altered levels of ICE-LAP-1 and 2 are indicative of the particular disease ⁇ mentioned above.
  • the pre ⁇ ent invention i ⁇ also directed to antagoni ⁇ t/inhibitor ⁇ of the polypeptide ⁇ of the pre ⁇ ent invention which may be u ⁇ ed to reduce or eliminate the function of the polypeptides.
  • an antagonist is an antibody, or in some ca ⁇ e ⁇ , an oligonucleotide which bind ⁇ to the ICE-LAP-1 and 2 polypeptides.
  • An example of an inhibitor is a small molecule which bind ⁇ to and occupie ⁇ the catalytic ⁇ ite of the polypeptide ⁇ thereby making the catalytic ⁇ ite inaccessible to sub ⁇ trate ⁇ uch that normal biological activity i ⁇ prevented.
  • Example ⁇ of ⁇ mall molecule ⁇ include but are not limited to ⁇ mall peptide ⁇ or peptide-like molecule ⁇ .
  • the level ⁇ of ICE-LAP-1 and 2 in vivo may be reduced by admmi ⁇ tration of antisense constructs which inhibits production of ICE-LAP-1 and 2 in vivo by the use of antisense technology.
  • Antisen ⁇ e technology can be u ⁇ ed to control gene expre ⁇ ion through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence which encodes for the mature polypeptides of the pre ⁇ ent invention, i ⁇ u ⁇ ed to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acid ⁇ Res., 3:173 (1979); Cooney et al. Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing tran ⁇ cription and the production of ICE-LAP-l and 2 polypeptide ⁇ .
  • the anti ⁇ ense RNA oligonucleotide hybridize ⁇ to the mRNA in vivo and block ⁇ tran ⁇ lation of the mRNA molecule into the ICE-LAP-l and 2 polypeptide ⁇ (anti ⁇ ense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides a ⁇ Antisense Inhibitors of Gene Expre ⁇ ion, CRC Pre ⁇ , Boca Raton, FL (1988)).
  • the antagoni ⁇ t/inhibitor ⁇ may be u ⁇ ed to treat non- programmed necrotic cell death related to cardiova ⁇ cular di ⁇ ea ⁇ es, stroke ⁇ , trauma, and other degenerative di ⁇ ea ⁇ e ⁇ where abnormal regulation of ICE-LAP-l and 2 may lead to pathological cell death, for example, immuno ⁇ uppres ⁇ ion-related di ⁇ order ⁇ , Alzheimer' ⁇ di ⁇ ea ⁇ e, Parkin ⁇ on' ⁇ di ⁇ ea ⁇ e, rheumatoid arthriti ⁇ .
  • the antagonist/inhibitors may al ⁇ o be u ⁇ ed to treat im une-ba ⁇ ed disease ⁇ of the lung and airway ⁇ , central nervou ⁇ ⁇ yste , eyes and ear ⁇ , joints, bones, cardiovascular sy ⁇ tem and ga ⁇ trointe ⁇ tinal and urogenital system ⁇ .
  • the antagonist/inhibitors may be employed in a compo ⁇ ition with a pharmaceutically acceptable carrier, e.g., as hereinabove de ⁇ cribed.
  • the present invention is further related to a process of screening molecule ⁇ to identify antagoni ⁇ t/inhibitor ⁇ or agoni ⁇ t ⁇ of the ICE-LAP-l and 2 polypeptide ⁇ of the pre ⁇ ent invention.
  • Agoni ⁇ t ⁇ increa ⁇ e the natural biological function of ICE-LAP-l and 2, while antagonists reduce or eliminate such function.
  • “Pla ⁇ mid ⁇ ” are de ⁇ ignated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting pla ⁇ mid ⁇ herein are either commercially available, publicly available on an unre ⁇ tricted basis, or can be constructed from available pla ⁇ mid ⁇ in accord with published procedure ⁇ .
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors . and other requirements were used as would be known to the ordinarily skilled artisan.
  • For analytical purposes typically 1 ⁇ g of pla ⁇ mid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • For the purpose of isolating DNA fragment ⁇ for pla ⁇ mid con ⁇ truction typically 5 to 50 ⁇ g of DNA are dige ⁇ ted with 20 to 250 unit ⁇ of enzyme in a larger volume. Appropriate buffer ⁇ and ⁇ ub ⁇ trate amounts for particular re ⁇ triction enzyme ⁇ are specified by the manufacturer.
  • Oligonucleotide ⁇ refer ⁇ to either a ⁇ ingle stranded polydeoxynucleotide or two complementary polydeoxynucleotide strand ⁇ which may be chemically ⁇ ynthe ⁇ ized. Such ⁇ ynthetic oligonucleotide ⁇ have no 5' pho ⁇ phate and thu ⁇ will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kina ⁇ e. A ⁇ ynthetic oligonucleotide will ligate to a fragment that ha ⁇ not been depho ⁇ phorylated.
  • Ligaation refer ⁇ to the process of forming pho ⁇ phodie ⁇ ter bond ⁇ between two double ⁇ tranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("liga ⁇ e") per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • liga ⁇ e T4 DNA ligase
  • the expres ⁇ ion of a plasmid, ICE-LAP-l HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin re ⁇ i ⁇ tance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation ⁇ ite.
  • a DNA fragment encoding the entire ICE-LAP-l precursor and a HA tag fused in rame to its 3' end was cloned into the polylinker region of the vector, therefore, the recombinant protein expres ⁇ ion is directed under the CMV promoter.
  • the HA tag correspond to an epitope derived from the influenza hemagglutinin protein a ⁇ previou ⁇ ly de ⁇ cribed (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767).
  • the infusion of HA tag to our target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • AATCAAGCGTAGTCTGGGACGTCGTATGGGTAATTGCCAGGAAAGAGGTAAA 3' contain ⁇ tran ⁇ lation ⁇ top codon, HA tag and the la ⁇ t 28 nucleotide ⁇ of the ICE-LAP-l coding sequence (not including the stop codon). Therefore, the PCR product contains the ICE-LAP-l coding sequence followed by HA tag fused in frame, and a translation termination stop codon next to the HA tag.
  • the PCR amplified DNA fragment was ligated with pcDNAI/Amp by blunt end ligation. The ligation mixture was transformed into E.
  • the HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767).
  • the infusion of HA tag to our target protein allows ea ⁇ y detection of the recombinant protein with an antibody that recognize ⁇ the HA epitope.
  • the DNA ⁇ equence encoding for ICE-LAP-2, ATCC # was constructed by PCR on the full-length ICE-LAP-2 using two primer ⁇ : the 5' primer 5' AGCTGATGGCCGCTGACAGGGG 3' contains the ICE-LAP-2 translational initiation ⁇ ite, ATG, followed by 14 nucleotides of ICE-LAP-2 coding sequence starting from the initiation codon; the 3' sequence 5'
  • AATCAAGCGTAGTCTGGGACGTCGTATGGGTATGTGGGAGGGTGTCCTGGGA 3' contains translation ⁇ top codon, HA tag and the la ⁇ t 20 nucleotide ⁇ of the ICE-LAP-2 coding ⁇ equence (not including the ⁇ top codon). Therefore, the PCR product contain ⁇ the ICE-LAP-2 coding ⁇ equence followed by HA tag fu ⁇ ed in frame, and a tran ⁇ lation termination ⁇ top codon next to the HA tag.
  • the PCR amplified DNA fragment wa ⁇ ligated with pcDNAI/Amp by blunt end ligation.
  • the ligation mixture wa ⁇ tran ⁇ formed into E.
  • RNAzolTM B sy ⁇ tem Biotecx Laboratorie ⁇ , Inc. 6023 South Loop East, Houston, TX 77033. About lO ⁇ g of total RNA isolated from each human tissue specified was separated on 1% agarose gel and blotted onto a nylon filter. (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press, (1989)). The labeling reaction was done according to the Stratagene Prime-It kit with 50ng DNA fragment. The labeled DNA was purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc.
  • the filter was then hybridized with radioactive labeled full length ICE-LAP- 1 gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65'C. After wa ⁇ h twice at room temperature and twice at 60"C with 0.5 x SSC, 0.1% SDS, the filter wa ⁇ then expo ⁇ ed at -70°C overnight with an intensifying screen.
  • the me ⁇ age RNA for ICE-LAP-l i ⁇ abundant in liver. ( Figure 5).
  • Northern blot analy ⁇ i ⁇ i ⁇ carried out to examine the level ⁇ of expre ⁇ ion of ICE-LAP-2 in human tissue ⁇ .
  • Total cellular RNA ⁇ ample ⁇ were isolated with RNAzolTM B sy ⁇ tem (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033).
  • the labeling reaction wa ⁇ done according to the Stratagene Prime-It kit with 50ng DNA fragment.
  • the labeled DNA wa ⁇ purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303).
  • the filter wa ⁇ then hybridized with radioactive labeled full length ICE-LAP- 2 gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65°C. After wa ⁇ h twice at room temperature and twice at 60"C with 0.5 x SSC, 0.1% SDS, the filter wa ⁇ then expo ⁇ ed at -70°C overnight with an intensifying ⁇ creen.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • CAAATATCCC CCAATAAAAA AGCTCATCCG AATATGGAGG CTGGACCACC TGAGTCAGGA 360
  • CAGTCATCTG AGAACCTGGA GGAAGATGCT GTTTACAAGA CCCACGTGGA GAAGGACTTC 960 ATTGCTTTCT GCTCTTCAAC GCCACACAAC GTGTCCTGGA GAGACAGCAC AATGGGCTCT 1020
  • Phe Phe A ⁇ n lie Asp Gin lie Ser Phe Asn Ly ⁇ Ly ⁇ Ala Hi ⁇ Pro
  • Gly lie Leu Glu Gly lie Cys Gly Thr Val His Asp Glu Lys Lys
  • a ⁇ p Met lie Cy ⁇ Gly Tyr Ala Cy ⁇ Leu Ly ⁇ Gly Thr Ala Ala Met

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Abstract

L'invention concerne des protéases-1 et 2 d'apoptose de type enzyme de conversion d'interleukine-1 β et l'ADN (ARN) codant ces polypeptides. L'invention concerne également une procédure de production de ces polypeptides par génie génétique ainsi que des anticorps et des antagonistes/inhibiteurs contre lesdits polypeptides. De plus, l'invention concerne des procédés d'utilisation des polypeptides, par exemple, en tant qu'agent antitumeur, et agent antiviral, ainsi que des anticorps et des antagonistes/inhibiteurs contre lesdits polypeptides, par exemple, dans le traitement de la maladie d'Alzheimer, de la maladie de Parkinson, de la polyarthrite rhumatoïde et de lésions de la tête.
EP94922009A 1994-06-23 1994-06-23 PROTEASE-1 ET 2 D'APOPTOSE DE TYPE ENZYME DE CONVERSION D'INTERLEUKINE-1 -g(b) Withdrawn EP0770141A4 (fr)

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JPH08511690A (ja) * 1993-06-24 1996-12-10 ザ・ジェネラル・ホスピタル・コーポレーション プログラムされた細胞死遺伝子及びタンパク質
US6087160A (en) * 1993-06-24 2000-07-11 The General Hospital Corporation Programmed cell death genes and proteins
FR2723378B1 (fr) * 1994-08-02 1996-10-31 Roussel Uclaf Sequence d'adn codant pour une proteine humaine tx apparentee a l'enzyme de conversion de l'interleukine-1beta, proteine tx, procede de production, compositions pharmaceutiques et leurs applications
US6288037B1 (en) 1996-01-29 2001-09-11 Basf Aktiengesellschaft Substrates and inhibitors for cysteine protease ICH-1
WO1997046663A1 (fr) * 1996-05-20 1997-12-11 Smithkline Beecham Corporation Protease-6 de l'apoptose semblable a une enzyme de conversion d'interleukine-1 beta
AU3514197A (en) 1996-07-03 1998-01-21 Genetics Institute Inc. Protease fmh-1, an ice/ced-like protease
US6512104B1 (en) 1996-10-01 2003-01-28 Amgen Inc. Interleukin-1β converting enzyme like cysteine protease
EP0842665A3 (fr) * 1996-11-14 2002-12-18 Smithkline Beecham Corporation Protéases d'apoptose de type enzyme de conversion d'interleukine-1-bèta et leurs agonistes
US5929042A (en) * 1997-03-03 1999-07-27 The Trustees Of Columbia University In The City Of New York Antisense compounds which prevent cell death and uses thereof
US7223856B2 (en) 1997-03-03 2007-05-29 The Trustees Of Columbia University In The City Of New York Antisense compounds which prevent cell death and uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015577A1 (fr) * 1990-04-04 1991-10-17 Black, Roy, A. INTERLEUKIN 1'beta' PROTEASE
WO1995000160A1 (fr) * 1993-06-24 1995-01-05 The General Hospital Corporation Genes regulateurs de la mort cellulaire programmee et proteines
WO1995027793A1 (fr) * 1994-04-08 1995-10-19 Merck Frosst Canada Inc. ADN CODANT UN PRECURSEUR DE LA CYSTEINE PROTEINASE II (ICErel-II) APPARENTEE A L'ENZYME DE CONVERSION DE L'INTERLEUKINE-1$g(b)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015577A1 (fr) * 1990-04-04 1991-10-17 Black, Roy, A. INTERLEUKIN 1'beta' PROTEASE
WO1995000160A1 (fr) * 1993-06-24 1995-01-05 The General Hospital Corporation Genes regulateurs de la mort cellulaire programmee et proteines
WO1995027793A1 (fr) * 1994-04-08 1995-10-19 Merck Frosst Canada Inc. ADN CODANT UN PRECURSEUR DE LA CYSTEINE PROTEINASE II (ICErel-II) APPARENTEE A L'ENZYME DE CONVERSION DE L'INTERLEUKINE-1$g(b)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9600297A1 *

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