PROTEASE-1 and 2

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 β 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.

It has recently been discovered that an interleukin-1/3 converting enzyme (ICE) is responsible for cleaving pro-lL-ljS into mature and active IL-ljδ and is also responsible for programmed cell death (or apoptosis), which is a process through which organisms get rid of unwanted cells. The present invention

is directed to ICE-LAP-1 and 2 which are structurally related to ICE.

In the nematode caenorhabditis eleganε , a genetic pathway of programmed cell death has been identified (Ellis, R.E., et al. Annu. Rev. Cell Biol., 7:663-698 (1991)). Two genes, ced-3 and ced-4 , are essential for cells to undergo programmed cell death in C. eleganε (Ellis, H.M., and Horvitz, H.R., Cell, 44:817-829 (1986)). Recessive mutations that eliminate the function of these two genes prevent normal programmed cell death during the development of C. eleganε . The known vertebrate counterpart to ced-3 protein is ICE. The overall amino acid identity between ced-3 and ICE is 28%, with a region of 115 amino acidε (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. The ICE-LAP-1 and 2 polypeptides of the present invention also have the same conserved pentapeptide and the cysteine residue which iε esεential for ICE function.

The εimilarity between ced-3 and ICE εuggests not only that ced-3 might function aε a cyεteine proteaεe 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 tissueε, including peripheral blood monocyteε, 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 subεtrate that ICE acts upon to cause cell death is presently unknown. One possibility is that it may be a vertebrate homolog of the C. eleganε cell death gene ced-4 . Alternatively, 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'ε protein splitting activity.

In accordance with one aspect of the present invention, there are provided 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.

In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with yet a further aspect of the present invention, there iε provided a proceεε for producing εuch polypeptideε by recombinant technigueε.

In accordance with yet a further aεpect of the preεent invention, there iε provided a process for utilizing such polypeptides, or polynucleotideε encoding εuch polypeptideε for therapeutic purpoεeε, for example, aε an antiviral agent, an anti-tumor agent and to control embryonic development and tiεsue ho eostaεis.

In accordance with yet a further aspect of the present invention, there iε provided an antibody against such polypeptideε.

In accordance with yet another aεpect of the preεent invention, there are provided antagoniεt/inhibitorε to such polypeptideε, which may be uεed to inhibit the action of εuch polypeptideε, for example, in the treatment of Alzheimer's diseaεe, Parkinεon'ε disease, rheumatoid arthritis, septic shock and head injury.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

Figure 1 εhowε the cDNA and correεponding 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 uεed.

Figure 2 εhowε the cDNA and corresponding deduced amino acid sequence of ICE-LAP-2. The polypeptide encoded by the amino acid sequence εhown iε the putative mature form of the polypeptide (minus the initial methionine residue).

Figure 3 shows an amino acid sequence compariεon of ICE-LAP-1 to ICE.

Figure 4 illuεtrateε a gel after performing a Northern Blot analysis of ICE-LAP-1 showing the concentration in various human tiεsues.

In accordance with an aεpect of the present invention, there are provided isolated nucleic acids (polynucleotides) 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 depoεited as ATCC Deposit No. 75772 and . ATCC Deposit No. 75772 contains the cDNA encoding for ICE-

LAP-1, and ATCC Deposit No. contains the cDNA encoding for

ICE-LAP-2.

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 εequence. It εhould be pointed out that the pentapeptide QACRG iε conεerved and iε located at amino acid poεition 256-260.

The polynucleotide encoding ICE-LAP-2 waε 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. eleganε cell death protein ced-3 with approximately 40% identity and 60% similarity over 400 amino acid residueε. It iε alεo important that the pentapeptide QACRG iε conserved and iε located at amino poεition 301-305.

The polynucleotideε 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 εequence εhown in Figureε 1 and 2 or that of the depoεited clones or may be a different coding sequence which coding sequence, aε a reεult of the redundancy or degeneracy of the genetic code, encode the same mature polypeptideε, and

derivativeε thereof, aε the DNA of Figureε 1 and 2 or the depoεited cDNA.

The polynucleotideε which encode for the mature polypeptideε of Figureε 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 εequence 5' and/or 3' of the coding sequence for the mature polypeptide.

Thuε, the term "polynucleotide encoding a polypeptide" encompasεes a polynucleotide which includes only coding sequence for the polypeptide aε well aε a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotideε which encode for fragmentε, analogε and derivativeε of the polypeptideε having the deduced amino acid sequence of Figures 1 and 2 or the polypeptideε encoded by the cDNA of the depoεited clones. The variants of the polynucleotideε may be naturally occurring allelic variantε of the polynucleotideε or non-naturally occurring variants of the polynucleotides.

Thuε, 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 aε 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 variantε, substitution variants and addition or insertion variantε.

Aε hereinabove indicated, the polynucleotideε may have a coding εequence which iε a naturally occurring allelic variant of the coding εequence εhown in Figureε 1 and 2 or of the coding sequence of the depoεited cloneε. Aε known in the art, an allelic variant iε an alternate form of a polynucleotide sequence which may have a subεtitution, deletion or addition of nucleotideε, which doeε not εubεtantially alter the function of the encoded polypeptideε. The polynucleotides may also encode for a proprotein which iε the mature protein pluε additional 5' amino acid reεidues. A mature protein having a proεequence iε a proprotein and is an inactive form of the protein. Once the prosequence iε cleaved an active mature protein remainε.

Thuε, for example, the polynucleotide of the preεent invention may encode for a mature protein, or for a protein having a proεequence or for a protein having both a proεequence and a preεequence (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 εupplied by a pQE-9 vector to provide for purification of the mature polypeptideε fuεed to the marker in the caεe of a bacterial hoεt, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian hoεt, e.g. COS-7 cellε, iε uεed. The HA tag correspondε 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 εequenceε if there iε at least 50% and preferably 70% identity between the sequenceε. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove deεcribed polynucleotides in a preferred embodiment encode polypeptides which retain subεtantially the same biological function or activity as the mature polypeptideε encoded by the cDNA of Figureε 1 and 2 or the depoεited cDNAε.

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 thoεe of εkill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotideε 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 sequenceε herein. A licenεe may be required to make, uεe or εell the depoεited materialε, and no εuch licenεe 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 haε the amino acid εequence encoded by the depoεited cDNAε, aε well as fragmentε, analogs and derivativeε of εuch polypeptideε.

The terms "fragment," "derivative" and "analog" when referring to the polypeptides of Figureε 1 and 2 or that encoded by the depoεited cDNA, eanε polypeptideε which retain eεεentially the same biological function or activity as εuch polypeptides, and wherein derivativeε 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 polypeptideε.

The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides- or synthetic polypeptideε, preferably recombinant polypeptideε.

The fragment, derivative or analog of the polypeptides of Figureε 1 and 2 or that encoded by the deposited cDNAε may be

(i) one in which one or more of the amino acid residues are subεtituted with a conεerved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such subεtituted amino acid reεidue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid reεidues includes a substituent group, or (iii) one in which the mature polypeptide iε fuεed with another compound, εuch aε 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. Such fragmentε, derivativeε and analogs are deemed to be within the scope of those εkilled in the art from the teachingε herein.

The polypeptideε and polynucleotideε of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. The term "isolated" means that the material iε removed from itε original environment (e.g., the natural environment if it iε naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide preεent in a living animal iε not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural εyεtem, is isolated. Such polynucleotideε could be part of a vector and/or εuch polynucleotideε or polypeptideε could be part of a compoεition,

and εtill be iεolated in that εuch vector or compoεition is not part of its natural environment.

The present invention also relates to vectors which include polynucleotideε of the preεent invention, hoεt cellε which are genetically engineered with vectorε of the invention and the production of polypeptideε of the invention by recombinant techniqueε.

Hoεt cellε are genetically engineered (tranεduced or tranεformed or tranεfected) with the vectorε of thiε invention which may be, for example, a cloning vector or an expreεεion vector. The vector may be, for example, in the form of a plaεmid, a viral particle, a phage, etc. The engineered hoεt cellε can be cultured in conventional nutrient media modified as appropriate for activating promoterε, εelecting tranεformantε or amplifying the ICE-LAP-1 geneε. The culture conditionε, εuch aε temperature, pH and the like, are thoεe previouεly uεed with the hoεt cell εelected for expreεεion, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thuε, for example, the polynucleotide may be included in any one of a variety of expresεion vectorε for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and εynthetic DNA sequences, e.g., derivativeε of SV40; bacterial plaεmidε; phage DNA; baculoviruε; yeaεt plaεmidε; vectorε derived from combinationε of plaεmidε and phage DNA, viral DNA εuch aε

vaccinia, adenoviruε, fowl pox viruε, and pεeudorabieε. However, any other vector may be used aε long aε it iε replicable and viable in the hoεt.

The appropriate DNA εequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease εite(ε) by procedureε known in the art. Such procedureε and otherε are deemed to be within the εcope of thoεe εkilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(ε) (promoter) to direct mRNA εyntheεiε. Aε repreεentative exampleε of εuch promoterε, there may be mentioned: LTRε from retroviruεeε, e.g. RSV, HIV, HTLVI, CMV or SV40 promoter, the E. coli. lac or trp. the phage lambda P _L promoter and other promoterε known to control expreεεion of geneε in prokaryotic or eukaryotic cellε or their viruεeε. However, alεo cellular εignalε can be uεed, for example, human-β-actin- pro oter) . The expreεεion vector can contain a ribosome binding site for tranεlation initiation and a transcription terminator. The vector may also include appropriate εequenceε for amplifying the copy number of the gene.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells εuch aε dihydrofolate reductaεe or neomycin reεiεtance for eukaryotic cell culture, or εuch aε tetracycline or ampicillin reεiεtance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, aε well aε an appropriate promoter or control εequence, may be employed to tranεform an appropriate hoεt to permit the hoεt to expreεε the protein.

Aε repreεentative exampleε of appropriate hoεtε, there may be mentioned: bacterial cellε, such aε E. coli- Bacilluε εubtilis, Streptomyceε, Salmonella typhimurium: fungal cellε, such as yeast; insect cellε εuch aε Droεophila and Sf9; animal cells εuch as CHO, COS, HEK 293 or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachingε herein.

More particularly, the preεent invention alεo includeε recombinant constructs comprising one or more of the sequenceε aε broadly deεcribed above. The conεtructε comprise a vector, such as a plasmid or viral vector, into which a εequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further compriεeε regulatory εequenceε, including, for example, a promoter, operably linked to the εequence. Large numberε of εuitable vectorε and promoterε are known to thoεe of εkill in the art, and are commercially available. The following vectorε are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbε, pDIO, phagescript, psiX174, pblueεcript SK, pbεkε, 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) . However, any other plaεmid or vector may be uεed as long aε they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferaεe) vectorε or other vectorε with selectable markers. Two appropriate vectorε are pKK232-8 and pCM7. Particular named bacterial promoterε include lad, lacZ, T3, T7, gpt, lambda P _R , P _L and trp. Eukaryotic promoterε include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviruε, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructε. 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 hoεt cell can be effected by calcium phoεphate tranεfection, DEAE-Dextran mediated tranεfection, lipofection or electroporation. (Daviε, L., Dibner, M. , Battey, I., Baεic Methodε 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 εequence. Alternatively, the polypeptideε of the invention can be εynthetically produced by conventional peptide εyntheεizerε.

Mature proteins can be expressed in mammalian cells, yeaεt, bacteria, or other cellε under the control of appropriate promoterε. Cell-free tranεlation syεtemε can alεo be employed to produce εuch proteinε uεing RNAε derived from the DNA conεtructs of the present invention. Appropriate cloning and expresεion vectorε for uεe with prokaryotic and eukaryotic hoεtε are deεcribed by Sambrook, et al.. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which iε hereby incorporated by reference.

Tranεcription of the DNA encoding the polypeptideε of the preεent invention by higher eukaryoteε iε increaεed by inεerting an enhancer sequence into the vector. Enhancers are cis-acting elementε of DNA, usually about from 10 to 300 bp that act on a promoter to increase itε tranεcription. Exa pleε including the SV40 enhancer on the late εide of the replication origin bp 100 to 270, a cytomegaloviruε early promoter enhancer, the polyo a enhancer on the late εide of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the hoεt cell, e.g., the ampicillin reεiεtance gene of E. coli and S. cereviεiae TRP1 gene, and a promoter derived from a highly-expreεεed gene to direct tranεcription of a downstream structural sequence. Such promoters can be derived from operonε encoding glycolytic enzymeε εuch aε 3- phoεphoglycerate kinaεe (PGK), α-factor, acid phosphatase, or

heat εhock proteinε, among otherε. The heterologous structural sequence is asεembled in appropriate phaεe with tranεlation initiation and termination εequenceε, and preferably, a leader sequence capable of directing secretion of translated protein into the periplaεmic εpace or extracellular medium. Optionally, the heterologouε εequence can encode a fuεion protein including an N-terminal identification peptide imparting deεired character- iεticε, e.g., εtabilization or εimplified purification of expreεsed recombinant product.

Useful expreεεion vectorε for bacterial uεe are conεtructed by inεerting a εtructural DNA sequence encoding a desired protein together with suitable tranεlation initiation and termination signals in operable reading phase with a functional promoter. The vector will compriεe 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 hoεt. Suitable prokaryotic hoεtε for tranεformation include E. coli- Bacilluε subtilis. Salmonella typhimuriu and variouε species within the genera Pseudomonaε, Streptomyces, and Staphylococcus, although otherε may alεo be employed aε a matter of choice.

Aε a repreεentative but nonlimiting example, useful expression vectors for bacterial use can compriεe 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). Such

co mercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppεala, Sweden) and GEM1 (Promega Biotec, Madiεon, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expresεed.

Following tranεformation of a εuitable hoεt εtrain and growth of the hoεt εtrain to an appropriate cell denεity, the εelected promoter iε induced by appropriate meanε (e.g., temperature shift or chemical induction) and cellε are cultured for an additional period.

Cellε are typically harveεted by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expresεion of proteins can be disrupted by any convenient method, including freeze-thaw cycling, εonication, mechanical diεruption, or uεe of cell lyεing agentε, such methods are well know to those skilled in the art.

Variouε mammalian cell culture εyεtemε can alεo be employed to expreεε recombinant protein. Examples of mammalian expresεion syεterns include the COS-7 lines of monkey kidney fibroblastε, deεcribed by Gluzman, Cell, 23:175 (1981), and other cell lineε capable of expreεεing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lineε. Mammalian expreεεion vectorε may compriεe an origin of replication, a εuitable promoter and enhancer, polyadenylation site, splice donor and acceptor sites, tranεcriptional termination εequenceε, and 5' flanking nontranεcribed εequenceε. DNA εequenceε derived

fro the SV40 εplice and polyadenylation sites may be used to provide the required nontranscribed genetic elementε.

The ICE-LAP-1 and 2 polypeptideε can be recovered and purified from recombinant cell cultureε by methodε including ammonium εulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phoεphocelluloεe chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, aε necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The 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 preεent invention may be glycoεylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The 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 cellε. ICE-LAP-1 and 2 may also be used to control vertebrate development and tisεue homeoεtaεiε, due to itε apoptoεiε ability.

Alεo, ICE-LAP-1 and 2 polypeptides may be used to overcome many viral infections by overcoming the εuppreεεed programmed cell death, εince programmed cell death may be one of the primary antiviral defenεe mechaniεmε of cellε.

ICE-LAP-1 and 2 may alεo be employed to treat immuno- suppreεεion related diεorderε, εuch aε AIDS, by targeting virus infected cells for cell death.

The polypeptideε of the preεent invention may alεo be uεed for identifying other moleculeε which have εimilar biological activity. An example of a εcreen for thiε compriεeε iεolating the coding region of the ICE-LAP geneε by uεing the known DNA εequence to synthesize an oligonucleotide probe. 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.

The polypeptides may alεo be employed in accordance with the preεent invention by expreεεion of such polypeptides in vivo , which iε often referred to aε "gene therapy."

Thuε, for example, 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. Such

methodε are well-known in the art. For example, cellε may be engineered by procedureε known in the art by uεe of a retroviral particle containing RNA encoding a polypeptide of the preεent invention.

Similarly, cellε may be engineered in vivo for expreεεion of a polypeptide in vivo by, for example, procedureε known in the art. Aε known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the preεent invention may be adminiεtered to a patient for engineering cellε in vivo and expression of the polypeptide in vivo . These and other methods for administering a polypeptide of the present invention by εuch method εhould be apparent to thoεe εkilled in the art from the teachingε of the preεent invention. For example, the expreεεion vehicle for engineering cellε may be other than a retroviruε, for example, an adenoviruε which may be uεed to engineer cellε in vivo after combination with a εuitable delivery vehicle.

The polypeptideε of the preεent invention may be employed in combination with a εuitable pharmaceutical carrier. Such compoεitionε comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation εhould εuit the mode of adminiεtration.

The invention alεo provideε a pharmaceutical pack or kit compriεing one or more containerε filled with one or more of the ingredientε of the pharmaceutical compoεitionε of the invention. Associated with such container(ε) can be a notice in the form preεcribed by a governmental agency regulating the manufacture, uεe or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositionε may be administered in a convenient manner such aε by the intravenouε, intraperitoneal, intramuscular, subcutaneouε, intranaεal or intradermal routeε. ICE-LAP-1 and 2 are adminiεtered in an amount which iε effective for treating and/or prophylaxiε of the εpecific indication. In general, ICE-LAP-1 and 2 will be adminiεtered in an amount of at leaεt 10 g/kg body weight, and in moεt caεeε they will be administered in an amount not in excess of 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptomε, etc.

The εequenceε of the present invention are also valuable for chromosome identification. The sequence iε specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome.

Few chromosome marking reagentε based on actual εequence data (repeat polymorphiεmε) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention iε an important firεt εtep in correlating thoεe εequenceε with genes associated with disease.

Briefly, sequenceε can be mapped to chromoεomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysiε of the cDNA iε uεed to rapidly εelect primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primerε are then uεed for PCR εcreening of εomatic cell hybridε containing individual human chromoεomeε. Only thoεe hybridε containing the human gene correεponding to the primer will yield an amplified fragment.

PCR mapping of εomatic cell hybridε iε a rapid procedure for aεεigning a particular DNA to a particular chromoεome. Using the present invention with the same oligonucleotide primerε, εublocalization can be achieved with panelε of fragmentε from εpecific chromoεomeε or poolε of large genomic cloneε in an analogouε manner. Other mapping strategies that can similarly be uεed to map to its chromosome include in εitu hybridization, prescreening with labeled flow-sorted chromosomeε and preεelection by hybridization to conεtruct chromoεome εpecific-cDNA librarieε.

Fluoreεcence in situ hybridization (FISH) of a cDNA cloneε to a metaphase chromosomal spread can be used to provide a

preciεe chromosomal location in one step. This technique can be used with cDNA aε short aε 500 or 600 baseε; however, cloneε larger than 2,000 bp have a higher likelihood of binding to a unique chromoεomal location with εufficient εignal intensity for simple detection. FISH requireε use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 iε better, and more than 4,000 iε probably not neceεεary to get good reεultε a reasonable percentage of the time. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence haε been mapped to a preciεe chromoεomal location, the phyεical poεition of the sequence on the chromosome can be correlated with genetic map data.. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johnε Hopkinε Univerεity Welch Medical Library) . The relationεhip between geneε and diεeaεeε that have been mapped to the εame chromoεomal region are then identified through linkage analyεiε (coinheritance of physically adjacent genes).

Next, it iε neceεεary to determine the differenceε in the cDNA or genomic εequence between affected and unaffected individualε. If a mutation iε obεerved in some or all of the affected individualε but not in any normal individualε, then the mutation iε likely to be the cauεative agent of the diεeaεe.

With current reεolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region asεociated with the diεeaεe could be one of between 50 and 500 potential cauεative geneε. (Thiε aεεumeε 1 megabaεe mapping resolution and one gene per 20 kb) .

The polypeptides, their fragmentε or other derivativeε, or analogε thereof, or cellε expreεεing them can be uεed aε an immunogen to produce antibodieε thereto. Theεe antibodieε can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodieε, aε well aε Fab fragmentε, or the product of an Fab expreεεion library. Variouε procedureε known in the art may be used for the production of εuch antibodieε and fragmentε.

Antibodieε generated againεt the polypeptideε correεponding 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 antibodieε binding the whole native polypeptideε. Such antibodieε can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodieε, any technique which provides antibodieε 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 Antibodieε and Cancer Therapy, Alan R. Liεε, Inc., pp. 77-96) .

Techniqueε deεcribed for the production of εingle chain antibodies (U.S. Patent 4,946,778) can be adapted to produce εingle chain antibodieε to immunogenic polypeptide products of this invention.

The present invention also relates to a diagnostic assay for detecting levels of ICE-LAP-1 and 2 in a sample taken from a hoεt, eg. blood, urine or serum sample. The level of ICE- LAP-1 and 2 may be detected, for example, by an immunoaεεay technique by procedureε known in the art. An example of εuch an aεεay iε a εandwich aεεay which utilizeε two antibodieε εpecific to an ICE-LAP-1 or 2 antigen, preferably monoclonal antibodieε with one of the antibodies being labeled, eg. by coupling a εuitable label such aε an indicator enzyme, eg. horεeradiεh peroxidaεe. The unlabeled antibody iε preferably on a εolid εupport. If antigen iε present, the antigen will bind to both antibodieε. After binding of the peroxidase-coupled antibody to the antigen, the peroxidaεe can be uεed to generate a colored product that iε meaεurable and whose concentration iε related to the amount of antigen in a εample. Becauεe of the catalytic nature of the enzyme the εyεtem greatly amplifieε the εignal.

Altered levels of ICE-LAP-1 and 2 are indicative of the particular diseaseε mentioned above.

The preεent invention iε also directed to antagoniεt/inhibitorε of the polypeptideε of the preεent invention which may be uεed to reduce or eliminate the function of the polypeptides.

An example of an antagonist is an antibody, or in some caεeε, an oligonucleotide which bindε to the ICE-LAP-1 and 2 polypeptides. An example of an inhibitor is a small molecule which bindε to and occupieε the catalytic εite of the polypeptideε thereby making the catalytic εite inaccessible to subεtrate εuch that normal biological activity iε prevented. Exampleε of εmall moleculeε include but are not limited to εmall peptideε or peptide-like moleculeε.

The levelε of ICE-LAP-1 and 2 in vivo may be reduced by admmiεtration of antisense constructs which inhibits production of ICE-LAP-1 and 2 in vivo by the use of antisense technology. Antisenεe technology can be uεed to control gene expreεεion through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the preεent invention, iε uεed 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. Acidε Res., 6:3073 (1979); Cooney et al. Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing tranεcription and the production of ICE-LAP-l and 2 polypeptideε. The antiεense RNA oligonucleotide hybridizeε to the mRNA in vivo and blockε tranεlation of the mRNA molecule into the ICE-LAP-l and 2 polypeptideε (antiεense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides aε Antisense Inhibitors of Gene Expreεεion, CRC Preεε, Boca Raton, FL (1988)).

The antagoniεt/inhibitorε may be uεed to treat non- programmed necrotic cell death related to cardiovaεcular diεeaεes, strokeε, trauma, and other degenerative diεeaεeε where abnormal regulation of ICE-LAP-l and 2 may lead to pathological cell death, for example, immunoεuppresεion-related diεorderε, Alzheimer'ε diεeaεe, Parkinεon'ε diεeaεe, rheumatoid arthritiε.

The antagonist/inhibitors may alεo be uεed to treat im une-baεed diseaseε of the lung and airwayε, central nervouε εyste , eyes and earε, joints, bones, cardiovascular syεtem and gaεtrointeεtinal and urogenital systemε. The antagonist/inhibitors may be employed in a compoεition with a pharmaceutically acceptable carrier, e.g., as hereinabove deεcribed.

The present invention is further related to a process of screening moleculeε to identify antagoniεt/inhibitorε or agoniεtε of the ICE-LAP-l and 2 polypeptideε of the preεent invention. Agoniεtε increaεe the natural biological function of ICE-LAP-l and 2, while antagonists reduce or eliminate auch

function. An example of such an asεay compriεeε combining ICE- LAP-l and 2 and a potential antagoniεt or agoniεt compound with their natural εubεtrate under conditionε allowing for action upon the εubεtrate and determining whether the compound preventε ICE- LAP-l or 2 from cleaving the εubstrate or enhances the cleavage.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amountε, unleεε otherwiεe εpecified, are by weight.

In order to facilitate underεtanding of the following exampleε certain frequently occurring methodε and/or termε will be deεcribed.

"Plaεmidε" are deεignated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plaεmidε herein are either commercially available, publicly available on an unreεtricted basis, or can be constructed from available plaεmidε in accord with published procedureε. In addition, 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

μg of plaεmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragmentε for plaεmid conεtruction, typically 5 to 50 μg of DNA are digeεted with 20 to 250 unitε of enzyme in a larger volume. Appropriate bufferε and εubεtrate amounts for particular reεtriction enzymeε are specified by the manufacturer. Incubation times of about 1 hour at 37'C are ordinarily used, but may vary in accordance with the supplier's instructionε. After digeεtion the reaction iε electrophoreεed directly on a polyacrylamide gel to iεolate the deεired fragment.

Size separation of the cleaved fragmentε iε performed uεing 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980), or agarose gelε (0.5 - 1.5%) .

"Oligonucleotideε" referε to either a εingle stranded polydeoxynucleotide or two complementary polydeoxynucleotide strandε which may be chemically εyntheεized. Such εynthetic oligonucleotideε have no 5' phoεphate and thuε will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinaεe. A εynthetic oligonucleotide will ligate to a fragment that haε not been dephoεphorylated.

"Ligation" referε to the process of forming phoεphodieεter bondε between two double εtranded 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 ("ligaεe") per 0.5 μg

of approximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Example 1 Expression of Recombinant ICE-LAP-l in COS cells

The expresεion of a plasmid, ICE-LAP-l HA, is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin reεiεtance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation εite. 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 expresεion is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein aε previouεly deεcribed (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.

The plasmid construction strategy iε deεcribed aε followε:

The DNA εequence encoding for ICE-LAP-l, ATCC # 75772, waε constructed by PCR on the full-length ICE-LAP-l using two primerε: the 5' primer 5' GTTCACTATGGCAGAAGGCAAGGACAG 3' containε the ICE-LAP-l tranεlational initiation εite ATG followed by 17 nucleotideε of ICE-LAP-l coding εequence εtarting from the initiation codon; the 3' εequence 5'

AATCAAGCGTAGTCTGGGACGTCGTATGGGTAATTGCCAGGAAAGAGGTAGAAA 3' containε tranεlation εtop codon, HA tag and the laεt 28 nucleotideε 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. coli strain SURE (available from Stratagene Cloning Syεtemε, 11099 North Torrey Pineε Road, La Jolla, CA 92037) the tranεformed culture waε plated on ampicillin media plateε and reεiεtant colonieε were εelected. Plaεmid DNA waε iεolated from tranεformantε and examined by reεtriction analyεiε for the preεence of the correct fragment. For expreεεion of the recombinant ICE-LAP-l, COS cellε were tranεfected with the expreεεion vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritεch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε, (1989)). The expreεεion of the ICE-LAP-l HA protein waε detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodieε: A

Laboratory Manual, Cold Spring Harbor Laboratory Preεε, (1988)). Cellε were labelled for 8 hourε with ³⁵ S-cyεteine two dayε post tranεfection. Culture media were then collected and cellε were lyεed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Triε, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media were precipitated with a HA specific monoclonal antibody. Proteinε precipitated were analyzed on 15% SDS-PAGE gelε.

Example 2 Expreεεion of Recombinant ICE-LAP-2 in COS cellε

The expreεεion of a plaεmid, ICE-LAP-2 HA, iε derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin reεiεtance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation εite. A DNA fragment encoding the entire ICE-LAP-2 precurεor and a HA tag fuεed in frame to itε 3' end waε cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. 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 eaεy detection of the recombinant protein with an antibody that recognizeε the HA epitope.

The plaεmid conεtruction εtrategy iε deεcribed as followε:

The DNA εequence encoding for ICE-LAP-2, ATCC # , was constructed by PCR on the full-length ICE-LAP-2 using two primerε: the 5' primer 5' AGCTGATGGCCGCTGACAGGGG 3' contains the ICE-LAP-2 translational initiation εite, ATG, followed by 14 nucleotides of ICE-LAP-2 coding sequence starting from the initiation codon; the 3' sequence 5'

AATCAAGCGTAGTCTGGGACGTCGTATGGGTATGTGGGAGGGTGTCCTGGGA 3' contains translation εtop codon, HA tag and the laεt 20 nucleotideε of the ICE-LAP-2 coding εequence (not including the εtop codon). Therefore, the PCR product containε the ICE-LAP-2 coding εequence followed by HA tag fuεed in frame, and a tranεlation termination εtop codon next to the HA tag. The PCR amplified DNA fragment waε ligated with pcDNAI/Amp by blunt end ligation. The ligation mixture waε tranεformed into E. coli εtrain SURE (available from Stratagene Cloning Syεtemε, 11099 North Torrey Pineε Road, La Jolla, CA 92037) the tranεformed culture waε plated on ampicillin media plateε and reεiεtant colonieε were εelected. Plaεmid DNA waε iεolated from tranεformantε and examined by reεtriction analyεiε for the preεence of the correct fragment. For expreεεion of the recombinant ICE-LAP-2, COS cellε were transfected with the expresεion vector by the DEAE-DEXTRAN method. (J. Sambrook, E. Fritεch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε, (1989)). The expreεεion of the ICE-LAP-2 HA protein was detected by

radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodieε: A Laboratory Manual, Cold Spring Harbor Laboratory Preεε, (1988)). Cellε were labelled for 8 hourε with ³⁵ S-cyεteine two dayε poεt tranεfection. Culture media were then collected and cellε were lyεed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Triε, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media were precipitated with a HA εpecific monoclonal antibody. Proteinε precipitated were analyzed on 15% SDS-PAGE gelε.

Example 3 Expression pattern of ICE-LAP-l in human tissue

Northern blot analysis was carried out to examine the levels of expresεion of ICE-LAP-l in human tissues. Total cellular RNA samples were isolated with RNAzol™ B syεtem (Biotecx Laboratorieε, Inc. 6023 South Loop East, Houston, TX 77033). About lOμg 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. 5603 Arapahoe Road, Boulder, CO 80303). 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 waεh twice at room temperature and twice at 60"C with 0.5 x SSC, 0.1% SDS, the filter waε then expoεed at -70°C overnight with an intensifying screen. The meεεage RNA for ICE-LAP-l iε abundant in liver. (Figure 5).

Example 4 Expreεεion pattern of ICE-LAP-2 in human tiεεue

Northern blot analyεiε iε carried out to examine the levelε of expreεεion of ICE-LAP-2 in human tissueε. Total cellular RNA εampleε were isolated with RNAzol™ B syεtem (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033). About lOjtxg of total RNA isolated from each human tisεue εpecified waε εeparated on 1% agarose gel and blotted onto a nylon filter. (Sambrook, Fritεch, and Maniatiε, Molecular Cloning, Cold Spring Harbor Preεε, (1989)). The labeling reaction waε done according to the Stratagene Prime-It kit with 50ng DNA fragment. The labeled DNA waε purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303). The filter waε 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 waεh twice at room temperature and twice at 60"C with 0.5 x SSC, 0.1% SDS, the filter waε then expoεed at -70°C overnight with an intensifying εcreen.

Numerouε modificationε and variations of the present invention are posεible in light of the above teachingε and, therefore, within the εcope of the appended claimε, the invention may be practiced otherwiεe than aε particularly deεcribed.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: HE, ET AL.

(ii) TITLE OF INVENTION: Interleukin-1/3 Converting Enzyme

Like Apoptoεiε Proteaεe-1 and 2

(iii) NUMBER OF SEQUENCES: 4

(iV) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART & OLSTEIN

(B) STREET: 6 BECKER FARM ROAD

(C) CITY: ROSELAND

(D) STATE: NEW JERSEY

(E) COUNTRY: USA

(F) ZIP: 07068

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 INCH DISKETTE

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WORD PERFECT 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: submitted herewith

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: FERRARO, GREGORY D.

(B) REGISTRATION NUMBER: 36,134

(C) REFERENCE/DOCKET NUMBER: 325800-184

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1318 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TTTCATTTTT AACTCTGAGG CTCTTTCCAA CGCTGTAAAA AAGGACAGAG GCTGTTCCCT 60

ATGGCAGAAG GCAACCACAG AAAAAAGCCA CTTAAGGTGT TGGAATCCCT GGGCAAAGAT 120

TTCCTCACTG GTGTTTTGGA TAACTTGGTG GAACAAAATG TACTGAACTG GAAGGAAGAG 180

GAAAAAAAGA AATATTACGA TGCTAAAACT GAAGACAAAG TTCGGGTCAT GGCAGACTCT 240

ATGCAAGAGA AGCAACGTAT GGCAGGACAA ATGCTTCTTC AAACCTTTTT TAACATAGAC 300

CAAATATCCC CCAATAAAAA AGCTCATCCG AATATGGAGG CTGGACCACC TGAGTCAGGA 360

GAATCTACAG ATGCCCTCAA GCTTTGTCCT CATGAAGAAT TCCTGAGACT ATGTAAAGAA 420

AGAGCTGAAG AGATCTATCC AATAAAGGAG AGAAACAACC GCACACGCCT GGCTCTCATC 480

ATATGCAATA CAGAGTTTGA CCATCTGCCT CCGAGGAATG GAGCTGACTT TGACATCACA 540

GGGATGAAGG AGCTACTTGA GGGTCTGGAC TATAGTGTAG ATGTAGAAGA GAATCTGACA 600

GCCAGGGATA TGGAGTCAGC GCTGAGGGCA TTTGCTACCA GACCAGAGCA CAAGTCCTCT 660

GACAGCACAT TCTTGGTACT CATGTCTCAT GGCATCCTGG AGGGAATCTG CGGAACTGTG 720

CATGATGAGA AAAAACCAGA TGTGCTGCTT TATGACACCA TCTTCCAGAT ATTCAACAAC 780

CGCAACTGCC TCAGTCTGAA GGACAAACCC AAGGTCATCA TTGTCCAGGC CTGCAGAGGT 840

GCAAACCGTG GGGAACTGTG GGTCAGAGAC TCTCCAGCAT CCTTGGAAGT GGCCTCTTCA 900

CAGTCATCTG AGAACCTGGA GGAAGATGCT GTTTACAAGA CCCACGTGGA GAAGGACTTC 960

ATTGCTTTCT GCTCTTCAAC GCCACACAAC GTGTCCTGGA GAGACAGCAC AATGGGCTCT 1020

ATCTTCATCA CACAACTCAT CACATGCTTC CAGAAATATT CTTGGTGCTG CCACCTAGAG 1080

GAAGTATTTC GGAAGGTACA GCAATCATTT GAAACTCCAA GGGCCAAAGC TCAAATGCCC 1140

ACCATAGAAC GACTGTCCAT GACAAGATAT TTCTACCTCT TTCCTGGCAA TTGAAAATGG 1200

AAGCCACAAG CAGCCCAGCC CTCCTTAATC AACTTCAAGG AGCACCTTCA TTAGTACAGC 1260

TTGCATATTT AACATTTTGT ATTTCAATAA AAGTGAAGAC AAAAAAAAAA AAAAAAAA 1318

(3) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 377 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS:

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ala Glu Gly Asn His Arg Lys Lys Pro Leu Lys Val Leu Glu

5 10 15

Ser Leu Gly Lys Asp Phe Leu Thr Gly Val Leu Asp Asn Leu Val

20 25 30

Glu Gin Asn Val Leu Asn Trp Lys Glu Glu Glu Lys Lys Lyε Tyr

35 40 45 Tyr Aεp Ala Lyε Thr Glu Aεp Lyε Val Arg Val Met Ala Aεp Ser

50 55 60

Met Gin Glu Lyε Gin Arg Met Ala Gly Gin Met Leu Leu Gin Thr

65 70 75

Phe Phe Aεn lie Asp Gin lie Ser Phe Asn Lyε Lyε Ala Hiε Pro

80 85 90

Aεn Met Glu Ala Gly Pro Pro Glu Ser Gly Glu Ser Thr Aεp Ala

95 100 105

Leu Lyε Leu Cyε Pro His Glu Glu Phe Leu Arg Leu Cys Lys Glu

110 115 120

Arg Ala Glu Glu lie Tyr Pro lie Lys Glu Arg Asn Asn Arg Thr

125 130 135

Arg Leu Ala Leu lie lie Cyε Asn Thr Glu Phe Asp His Leu Pro

140 145 150

Pro Arg Asn Gly Ala Asp Phe Aεp lie Thr Gly Met Lyε Glu Leu

155 160 165

Leu Glu Gly Leu Aεp Tyr Ser Val Aεp Val Glu Glu Aεn Leu Thr

170 175 180

Ala Arg Asp Met Glu Ser Ala Leu Arg Ala Phe Ala Thr Arg Pro

185 190 195

Glu His Lyε Ser Ser Aεp Ser Thr Phe Leu Val Leu Met Ser Hiε

200 205 210

Gly lie Leu Glu Gly lie Cys Gly Thr Val His Asp Glu Lys Lys

215 220 225

Pro Asp Val Leu Leu Tyr Aεp Thr lie Phe Gin lie Phe Aεn Aεn

230 235 240

Arg Asn Cys Leu Ser Leu Lys Aεp Lyε Pro Lyε Val lie lie Val

245 250 255

Gin Ala Cyε Arg Gly Ala Aεn Arg Gly Glu Leu Trp Val Arg Aεp

260 265 270

Ser Pro Ala Ser Leu Glu Val Ala Ser Ser Gin Ser Ser Glu Aεn

275 280 285

Leu Glu Glu Asp Ala Val Tyr Lys Thr His Val Glu Lys Aεp Phe

290 295 300 lie Ala Phe Cyε Ser Ser Thr Pro Hiε Aεn Val Ser Trp Arg Aεp

305 310 315

Ser Thr Met Gly Ser lie Phe lie Thr Gin Leu lie Thr Cyε Phe

320 325 330

Gin Lyε Tyr Ser Trp Cys Cys His Leu Glu Glu Val Phe Arg Lyε

335 340 345

Val Gin Gin Ser Phe Glu Thr Pro Arg Ala Lyε Ala Gin Met Pro

350 355 360

Thr lie Glu Arg Leu Ser Met Thr Arg Tyr Phe Tyr Leu Phe Pro

365 370 375 Gly Aεn

(4) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1430 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CGAGCGGCGC CGAGCGCGGG GTCTTGGTCC ACCTTCCAGC ACAAGGAGCT GATGGCCGCT 60

GACAGGGGAC GCAGGATATT GGGAGTGTGT GGCATGCATC CTCATCATCA GGAAACTCTA 120

AAAAAGAACC GAGTGGTGCT AGCCAAACAG CTGTTGTTGA GCGAATTGTT AGAACATCTT 180

CTGGAGAAGG ACATCATCAC CTTGGAAATG AGGGAGCTCA TCCAGGCCAA AGTGGGCAGT 240

TTCAGCCAGA ATGTGGAACT CCTCAACTTG CTGCCTAAGA GGGGTCCCCA AGCTTTTGAT 300

GCCTTCTGTG AAGCACTGAG GGAGACCAAG CAAGGCCACC TGGAGGATAT GTTGCTCACC 360

ACCCTTTCTG GGCTTCAGCA TGTACTCCCA CCGTTGAGCT GTGACTACGA CTTGAGTCTC 420

CCTTTTCCGG TGTGTGAGTC CTGTCCCCTT TACAAGAAGC TCCGCCTGTC GACAGATACT 480

GTGGAACACT CCCTAGACAA TAAAGATGGT CCTGTCTGCC TTCAGGTGAA GCCTTGCACT 540

CCTGAATTTT ATCAAACACA CTTCCAGCTG GCATATAGGT TGCAGTCTCG GCCTCGTGGC 600

CTAGCACTGG TGTTGAGCAA TGTGCACTTC ACTGGAGAGA AAGAACTGGA ATTTCGCTCT 660

GGAGGGGATG TGGACCACAG TACTCTAGTC ACCCTCTTCA AGCTTTTGGG CTATGACGTC 720

CATGTTCTAT GTGACCAGAC TGCACAGGAA ATGCAAGAGA AACTGCAGAA TTTTGCACAG 780

TTACCTGCAC ACCGAGTCAC GGACTCCTGC ATCGTGGCAC TCCTCTCGCA TGGTGTGGAG 840

GGCGCCATCT ATGGTGTGGA TGGGAAACTG CTCCAGCTCC AAGAGGTTTT TCAGCTCTTT 900

GACAACGCCA ACTGCCCAAG CCTACAGAAC AAACCAAAAA TGTTCTTCAT CCAGGCCTGC 960

CGTGGAGATG AGACTGATCG TGGGGTTGAC CAACAAGATG GAAAGAACCA CGCAGGATCC 1020

CCTGGGTGCG AGGAGAGTGA TGCCGGTAAA GAAAAGTTGC CGAAGATGAG ACTGCCCACG 1080

CGCTCAGACA TGATATGCGG CTATGCCTGC CTCAAAGGGA CTGCCGCCAT GCGGAACACC 1140

AAACGAGGTT CCTGGTACAT CGAGGCTCTT GCTCAAGTGT TTTCTGAGCG GGGTTGTGAT 1200

ATGCACGTGG CCGACATGCT GGTTAAGGTG AACGCACTTA TCAAGGATCG GGAAGGTTAT 1260

GCTCCTGGCA CAGAATTCCA CCGGTGCAAG GAGATGTCTG AATACTGCAG CACTCTGTGC 1320

CGCCACCTCT ACCTGTTCCC AGGACACCCT CCCACATGAT GTCACCTCCC CATCATCCAC 1380

GCCAAGTGGA AGCCACTGGA CCACAGGAGG TGTGATAGAG CCTTTGATCT 1430

(5) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 435 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS:

(D) TOPOLOGY: LINEAR 2

(ii) MOLECULE TYPE: PROTEIN

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Ala Ala Aεp Arg Gly Arg Arg lie Leu Gly Val Cyε Gly Met

5 10 15

His Pro His His Gin Glu Thr Leu Lys Lys Aεn Arg Val Val Leu

20 25 30

Ala Lys Gin Leu Leu Leu Ser Glu Leu Leu Glu Hiε Leu Leu Glu

35 40 45

Lyε Aεp lie lie Thr Leu Glu Met Arg Glu Leu lie Gin Ala Lyε

50 55 60

Val Gly Ser Phe Ser Gin Aεn Val Glu Leu Leu Asn Leu Leu Pro

65 70 75

Lys Arg Gly Pro Gin Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg

80 85 90

Glu Thr Lyε Gin Gly His Leu Glu Asp Met Leu Leu Thr Thr Leu

95 100 105

Ser Gly Leu Gin His Val Leu Pro Pro Leu Ser Cys Asp Tyr Aεp

110 115 120

Leu Ser Leu Pro Phe Pro Val Cyε Glu Ser Cyε Pro Leu Tyr Lyε

125 130 135

Lyε Leu Arg Leu Ser Thr Asp Thr Val Glu His Ser Leu Asp Asn

140 145 150

Lys Asp Gly Pro Val Cys Leu Gin Val Lys Pro Cys Thr Pro Glu

155 160 165

Phe Tyr Gin Thr His Phe Gin Leu Ala Tyr Arg Leu Gin Ser Arg

170 175 180

Pro Arg Gly Leu Ala Leu Val Leu Ser Asn Val Hiε Phe Thr Gly

185 190 195 Glu Lyε Glu Leu Glu Phe Arg Ser Gly Gly Aεp Val Aεp Hiε Ser

200 205 210 Thr Leu Val Thr Leu Phe Lyε Leu Leu Gly Tyr Aεp Val Hiε Val

215 220 225

Leu Cyε Aεp Gin Thr Ala Gin Glu Met Gin Glu Lyε Leu Gin Asn

230 235 240

Phe Ala Gin Leu Pro Ala His Arg Val Thr Asp Ser Cys lie Val

245 250 255

Ala Leu Leu Ser His Gly Val Glu Gly Ala lie Tyr Gly Val Aεp

260 265 270

Gly Lyε Leu Leu Gin Leu Gin Glu Val Phe Gin Leu Phe Asp Asn

275 280 285

Ala Asn Cys Pro Ser Leu Gin Asn Lys Pro Lyε Met Phe Phe lie

290 295 300

Gin Ala Cyε Arg Gly Aεp Glu Thr Aεp Arg Gly Val Aεp Gin Gin

305 310 315

Aεp Gly Lyε Aεn Hiε Ala Gly Ser Pro Gly Cyε Glu Glu Ser Aεp

320 325 330

Ala Gly Lyε Glu Lyε Leu Pro Lyε Met Arg Leu Pro Thr Arg Ser

335 340 345

Aεp Met lie Cyε Gly Tyr Ala Cyε Leu Lyε Gly Thr Ala Ala Met

350 355 360

Arg Aεn Thr Lyε Arg Gly Ser Trp Tyr lie Glu Ala Leu Ala Gin

365 370 375

Val Phe Ser Glu Arg Gly Cyε Aεp Met Hiε Val Ala Aεp Met Leu

380 385 390

Val Lyε Val Aεn Ala Leu lie Lyε Aεp Arg Glu Gly Tyr Ala Pro

395 400 405

Gly Thr Glu Phe Hiε Arg Cyε Lyε Glu Met Ser Glu Tyr Cyε Ser

410 415 420

Thr Leu Cyε Arg Hiε Leu Tyr Leu Phe Pro Gly Hiε Pro Pro Thr

425 430 435