AU751594B2 - Truncated platelet-activating factor acetylhydrolase - Google Patents

Description

WO 99/09147 PCT/US97/14212 TRUNCATED PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE FIELD OF THE INVENTION The present invention relates generally to platelet-activating factor acetylhydrolase and more specifically to novel purified and isolated polynucleotides encoding human plasma platelet-activating factor acetylhydrolase, to the plateletactivating factor acetylhydrolase products encoded by the polynucleotides, to materials and methods for the recombinant production of platelet-activating factor acetylhydrolase products and to antibody substances specific for platelet-activating factor acetylhydrolase.

BACKGROUND

Platelet-activating factor (PAF) is a biologically active phospholipid synthesized by various cell types. In vivo and at normal concentrations of 10 10 to 9 M, PAF activates target cells such as platelets and neutrophils by binding to specific G protein-coupled cell surface receptors [Venable et al., J. Lipid Res., 34: 691-701 (1993)]. PAF has the structure 1-Q-alkyl-2-acetyl-sn-glycero-3phosphocholine. For optimal biological activity, the sn-1 position of the PAF glycerol backbone must be in an ether linkage with a fatty alcohol and the sn-3 position must have a phosphocholine head group.

PAF functions in normal physiological processes inflammation, hemostasis and parturition) and is implicated in pathological inflammatory responses asthma, anaphylaxis, septic shock and arthritis) [Venable et al., supra, and Lindsberg et al., Ann. Neurol., 30: 117-129 (1991)]. The likelihood of PAF involvement in pathological responses has prompted attempts to modulate the activity of PAF and the major focus of these attempts has been the development of antagonists WO 99/09147 PCT/US97/14212 -2of PAF activity which interfere with binding of PAF to cell surface receptors. See, for example, Heuer et al., Clin. Exp. Allergy, 22: 980-983 (1992).

The synthesis and secretion of PAF as well as its degradation and clearance appear to be tightly controlled. To the extent that pathological inflammatory actions of PAF result from a failure of PAF regulatory mechanisms giving rise to excessive production, inappropriate production or lack of degradation, an alternative means of modulating the activity of PAF would involve mimicing or augmenting the natural process by which resolution of inflammation occurs.

Macrophages [Stafforini et al., J. Biol. Chem., 265(17): 9682-9687 (1990)], hepatocytes and the human hepatoma cell line HepG2 [Satoh et al., J. Clin. Invest., 87: 476-481 (1991) and Tarbet et al., J. Biol. Chem., 266(25): 16667-16673 (1991)] have been reported to release an enzymatic activity, PAF acetylhydrolase (PAF-AH), that inactivates PAF. In addition to inactivating PAF, PAF-AH also inactivates oxidatively fragmented phospholipids such as products of the arachidonic acid cascade that mediate inflammation. See, Stremler et al., J. Biol. Chem., 266(17): 11095- 11103 (1991). The inactivation of PAF by PAF-AH occurs primarily by hydrolysis of the PAF sn-2 acetyl group and PAF-AH metabolizes oxidatively fragmented phospholipids by removing sn-2 acyl groups. Two types of PAF-AH have been identified: cytoplasmic forms found in a variety of cell types and tissues such as endothelial cells and erythrocytes, and an extracellular form found in plasma and serum. Plasma PAF-AH does not hydrolyze intact phospholipids except for PAF and this substrate specificity allows the enzyme to circulate in vivo in a fully active state without adverse effects. The plasma PAF-AH appears to account for all of the PAF degradation in human blood ex vivo [Stafforini et al., J. Biol. Chem., 262(9): 4223- 4230 (1987)].

While the cytoplasmic and plasma forms of PAF-AH appear to have identical substrate specificity, plasma PAF-AH has biochemical characteristics which distinguish it from cytoplasmic PAF-AH and from other characterized lipases.

Specifically, plasma PAF-AH is associated with lipoprotein particles, is inhibited by diisopropyl fluorophosphate, is not affected by calcium ions, is relatively insensitive to proteolysis, and has an apparent molecular weight of 43,000 daltons. See, Stafforini et al. (1987), supra. The same Stafforini et al. article describes a WO 99/09147 PCTIUS97/1 4212 -3procedure for partial purification of PAF-AH from human plasma and the amino acid composition of the plasma material obtained by use of the procedure. Cytoplasmic PAF-AH has been purified from erythrocytes as reported in Stafforini et al., J. Biol.

Chem., 268(6): 3857-3865 (1993) and ten amino terminal residues of cytoplasmic PAF-AH are also described in the article. Hattori et al., J. Biol. Chem., 268(25): 18748-18753 (1993) describes the purification of cytoplasmic PAF-AH from bovine brain. Subsequent to filing of the parent application hereto the nucleotide sequence of bovine brain cytoplasmic PAF-AH was published in Hattori et al., J. Biol. Chem., 269(237): 23150-23155 (1994). On January 5, 1995, three months after the filing date of the parent application hereto, a nucleotide sequence for a lipoprotein associated phospholipase A 2 (Lp-PLA 2 was published in Smithkline Beecham PLC Patent Cooperation Treaty (PCT) International Publication No. WO 95/00649. The nucleotide sequence of the Lp-PLA 2 differs at one position when compared to the nucleotide sequence of the PAF-AH of the present invention. The nucleotide difference (corresponding to position 1297 of SEQ ID NO: 7) results in an amino acid difference between the enzymes encoded by the polynucleotides. The amino acid at position 379 of SEQ ID NO: 8 is a valine while the amino acid at the corresponding position in Lp-PLA 2 is an alanine. In addition, the nucleotide sequence of the PAF- AH of the present invention includes 124 bases at the 5' end and twenty bases at the 3' end not present in the Lp-PLA 2 sequence. Three months later, on April 10, 1995, a Lp-PLA 2 sequence was deposited in GenBank under Accession No. U24577 which differs at eleven positions when compared to the nucleotide sequence of the PAF-AH of the present invention. The nucleotide differences (corresponding to position 79, 81, 84, 85, 86, 121, 122, 904, 905, 911, 983 and 1327 of SEQ ID NO: 7) results in four amino acid differences between the enzymes encoded by the polynucleotides.

The amino acids at positions 249, 250, 274 and 389 of SEQ ID NO: 8 are lysine, aspartic acid, phenylalanine and leucine, respectively, while the respective amino acid at the corresponding positions in the GenBank sequence are isoleucine, arginine, leucine and serine.

The recombinant production of PAF-AH would make possible the use of exogenous PAF-AH to mimic or augment normal processes of resolution of inflammation in vivo. The administration of PAF-AH would provide a physiological WO 99/09147 PCT/US97/14212 -4advantage over administration of PAF receptor antagonists because PAF-AH is a product normally found in plasma. Moreover, because PAF receptor antagonists which are structurally related to PAF inhibit native PAF-AH activity, the desirable metabolism of PAF and of oxidatively fragmented phospholipids is thereby prevented.

Thus, the inhibition of PAF-AH activity by PAF receptor antagonists counteracts the competitive blockade of the PAF receptor by the antagonists. See, Stremler et al., supra. In addition, in locations of acute inflammation, for example, the release of oxidants results in inactivation of the native PAF-AH enzyme in turn resulting in elevated local levels of PAF and PAF-like compounds which would compete with any exogenously administed PAF receptor antagonist for binding to the PAF receptor.

In contrast, treatment with recombinant PAF-AH would augment endogenous PAF- AH activity and compensate for any inactivated endogenous enzyme.

There thus exists a need in the art to identify and isolate polynucleotide sequences encoding human plasma PAF-AH, to develop materials and methods useful for the recombinant production of PAF-AH and to generate reagents for the detection of PAF-AH in plasma.

SUMMARY OF THE INVENTION The present invention provides novel purified and isolated polynucleotides DNA and RNA both sense and antisense strands) encoding human plasma PAF-AH or enzymatically active fragments thereof. Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences. The DNA sequence encoding PAF-AH that is set out in SEQ ID NO: 7 and DNA sequences which hybridize to the noncoding strand thereof under standard stringent conditions or which would hybridize but for the redundancy of the genetic code, are contemplated by the invention. Also contemplated by the invention are biological replicas copies of isolated DNA sequences made in vivo or in vitro) of DNA sequences of the invention.

Autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating PAF-AH sequences and especially vectors wherein DNA encoding PAF-AH is operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided.

WO 99/09147 PCT/US97/14212 According to another aspect of the invention, procaryotic or eucaryotic host cells are stably transformed with DNA sequences of the invention in a manner allowing the desired PAF-AH to be expressed therein. Host cells expressing PAF- AH products can serve a variety of useful purposes. Such cells constitute a valuable source of immunogen for the development of antibody substances specifically immunoreactive with PAF-AH. Host cells of the invention are conspicuously useful in methods for the large scale production of PAF-AH wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by, for example, immunoaffinity purification.

A non-immunological method contemplated by the invention for purifying PAF-AH from plasma includes the following steps: isolating low density lipoprotein particles; solubilizing said low density lipoprotein particles in a buffer comprising 10mM CHAPS to generate a first PAF-AH enzyme solution; (c) applying said first PAF-AH enzyme solution to a DEAE anion exchange column; (d) washing said DEAE anion exchange column using an approximately pH 7.5 buffer comprising ImM CHAPS; eluting PAF-AH enzyme from said DEAE anion exchange column in fractions using approximately pH 7.5 buffers comprising a gradient of 0 to 0.5 M NaCI; pooling fractions eluted from said DEAE anion exchange column having PAF-AH enzymatic activity; adjusting said pooled, active fractions from said DEAE anion exchange column to 10mM CHAPS to generate a second PAF-AH enzyme solution; applying said second PAF-AH enzyme solution to a blue dye ligand affinity column; eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer comprising 10mM CHAPS and a chaotropic salt; applying the eluate from said blue dye ligand affinity column to a Cu ligand affinity column; eluting PAF-AH enzyme from said Cu ligand affinity column using a buffer comprising 10mM CHAPS and imidazole; subjecting the eluate from said Cu ligand affinity column to SDS-PAGE; and isolating the approximately 44 kDa PAF-AH enzyme from the SDS-polyacrylamide gel.

Preferably, the buffer of step is 25 mM Tris-HCI, 10mM CHAPS, pH 7.5; the buffer of step is 25 mM Tris-HCI, 1mM CHAPS; the column of step is a Blue Sepharose Fast Flow column; the buffer of step is 25mM Tris-HCl, WO 99/09147 PCTIUS9/14212 -6- CHAPS, 0.5M KSCN, pH 7.5; the column of step is a Cu Chelating Sepharose column; and the buffer of step is 25 mM Tris-HC1, 10mM CHAPS, 0.5M NaC1, imidazole at a pH in a range of about pH 7.5-8.0.

A method contemplated by the invention for purifying enzymaticallyactive PAF-AH from E. coli producing PAF-AH includes the steps of: preparing a centrifugation supernatant from lysed E. coli producing PAF-AH enzyme; (b) applying said centrifugation supernatant to a blue dye ligand affinity column; (c) eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer comprising 10mM CHAPS and a chaotropic salt; applying said eluate from said blue dye ligand affinity column to a Cu ligand affinity column; and eluting PAF- AH enzyme from said Cu ligand affinity column using a buffer comprising CHAPS and imidazole. Preferably, the column of step is a Blue Sepharose Fast Flow column; the buffer of step is 25mM Tris-HCI, 10mM CHAPS, KSCN, pH 7.5; the column of step is a Cu Chelating Sepharose column; and the buffer of step is 25mM Tris-HCl, 10mM CHAPS, 0.5M NaC1, 100mM imidazole, pH Another method contemplated by the invention for purifying enzymatically-active PAF-AH from E. coli producing PAF-AH includes the steps of: preparing a centrifugation supernatant from lysed E. coli producing PAF-AH enzyme; diluting said centrifugation supernatant in a low pH buffer comprising CHAPS; applying said diluted centrifugation supernatant to a cation exchange column equilibrated at about pH 7.5; eluting PAF-AH enzyme from said cation exchange column using 1M salt; raising the pH of said eluate from said cation exhange column and adjusting the salt concentration of said eluate to about 0.5M salt; applying said adjusted eluate from said cation exchange column to a blue dye ligand affinity column; eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer comprising about 2M to about 3M salt; and (h) dialyzing said eluate from said blue dye ligand affinity column using a buffer comprising about 0.1% Tween. Preferably, the buffer of step is 25mM MES, 10mM CHAPS, ImM EDTA, pH 4.9; the column of step is an S sepharose column equilibrated in 25mM MES, 10mM CHAPS, ImM EDTA, 50mM NaCI, pH PAF-AH is eluted in step using ImM NaCI; the pH of the eluate in step (e) WO 99/09147 PCT/US97/14212 -7is adjusted to pH 7.5 using 2M Tris base; the column in step is a sepharose column; the buffer in step is 25mM Tris, 10mM CHAPS, 3M NaCI, ImM EDTA, pH 7.5; and the buffer in step is 25mM Tris, 0.5M NaCI, 0.1 Tween pH Still another method contemplated by the invention for purifying enzymatically-active PAF-AH from E.coli includes the steps of: preparing an E.coli extract which yields solubilized PAF-AH supernatant after lysis in a buffer containing CHAPS; dilution of said supernatant and application to a anion exchange column equilibrated at about pH 8.0; eluting PAF-AH enzyme from said anion exchange column; applying said adjusted eluate from said anion exchange column to a blue dye ligand affinity column; eluting the said blue dye ligand affinity column using a buffer comprising 3.0M salt; dilution of the blue dye eluate into a suitable buffer for performing hydroxylapatite chromatography; (g) performing hydroxylapatite chromatography where washing and elution is accomplished using buffers (with or without CHAPS); diluting said hydroxylapatite eluate to an appropriate salt concentration for cation exchange chromatography; applying said diluted hydroxylapatite eluate to a cation exchange column at a pH ranging between approximately 6.0 to 7.0; elution of PAF-AH from said cation exchange column with a suitable formulation buffer; performing cation exchange chromatography in the cold; and formulation of PAF-AH in liquid or frozen form in the absence of CHAPS.

Preferably in step above the lysis buffer is 25mM Tris, 100mM NaCI, ImM EDTA, 20mM CHAPS, pH 8.0; in step the dilution of the supernatant for anion exchange chromatography is 3-4 fold into 25mM Tris, 1mM EDTA, 10mM CHAPS, pH 8.0 and the column is a Q-Sepharose column equilibrated with 25mM Tris, ImM EDTA, 50mM NaCI, 10mM CHAPS, pH 8.0; in step the anion exchange column is eluted using 25mM Tris, ImM EDTA, 350mM NaC1, lOmM CHAPS, pH 8.0; in step the eluate from step is applied directly onto a blue dye affinity column; in step the column is eluted with 3M NaCI, CHAPS, 25mM Tris, pH 8.0 buffer; in step dilution of the blue dye eluate for hydroxylapatite chromatography is accomplished by dilution into 10mM sodium phosphate, 100mM NaCI, 10mM CHAPS, pH 6.2; in step hydroxylapatite WO 99/09147 PCT/US97/14212 -8chromatography is accomplished using a hydroxylapatite column equilibrated with sodium phosphate, 100mM NaCI, 10mM CHAPS and elution is accomplished using 50mM sodium phosphate, 100mM NaCI (with or without) 10mM CHAPS, pH in step dilution of said hydroxylapatite eluate for cation exchange chromatography is accomplished by dilution into a buffer ranging in pH from approximately 6.0 to 7.0 comprising sodium phosphate (with or without CHAPS); in step a S Sepharose column is equilibrated with 50mM sodium phosphate, (with or without) 10mM CHAPS, pH 6.8; in step elution is accomplished with a suitable formulation buffer such as potassium phosphate 50mM, 12.5mM aspartic acid, 125mM NaCI, pH 7.5 containing 0.01% Tween-80; and in step cation exchange chromatrography is accomplished at 2-8"C. Examples of suitable formulation buffers for use in step which stabilize PAF-AH include 50mM potassium phosphate, 12.5mM Aspartic acid, 125mM NaCI pH 7.4 (approximately, with and without the addition of Tween-80 and or Pluronic F68) or 25mM potassium phosphate buffer containing (at least) 125mM NaCI, 25mM arginine and 0.01% Tween-80 (with or without Pluronic F68 at approximately 0.1 and Yet another method contemplated by the invention for purifying enzymatically active rPAF-AH products from E. coli includes the steps of: (a) preparing an E. coli extract which yields solubilized rPAF-AH product supernatant after lysis in a buffer containing Triton X-100, dilution of said supernatant and application to an immobilized metal affinity exchange column equilibrated at about pH 8.0; eluting rPAF-AH product from said immobilized metal affinity exchange column with a buffer comprising imidazole; adjusting the salt concentration and applying said eluate from said immobilized metal affinity column to an hydrophobic interaction column (HIC#1); eluting said HIC#1 by reducing the salt concentration and/or increasing the detergent concentration; titrating said HIC#1 eluate to a pH of about 6.4; applying said adjusted HIC#1 eluate to a cation exchange column (CEX#1) equilibrated at about pH 6.4; eluting said CEX#1 with concentration? sodium chloride; adjusting said CEX#1 eluate with sodium chloride to a concentration of about 2.0M; applying said adjusted CEX#1 eluate to a hydrophobic interaction column (HIC#2) equilibrated at about pH 8.0 and about sodium chloride; eluting said HIC#2 hy reducing the salt concentration and/or WO 99/09147 PCT/US97/14212 -9increasing the detergent concentration; diluting said HIC#2 eluate and adjusting to a pH of about 6.0; applying said adjusted HIC#2 eluate to a cation exchange column (CEX#2) equilibrated at about pH 6.0; eluting the rPAF-AH product from said CEX#2 with a suitable formulation buffer.

Preferably, in step above the lysis buffer is 90mM TRIS, 0.125% Triton X-100, 0.6M NaCI, pH 8.0, and lysis is carried out in a high pressure homogenizer; in step the supernatant is diluted into equilibration buffer TRIS, 0.5M NaCI, 0.1% Triton X-100, pH a zinc chelate column (Chelating Sepharose Fast Flow, Pharmacia, Uppsala, Sweden) is charged, equilibrated with equilibration buffer, loaded with the diluted supernatant, and washed with TRIS, 0.5M NaCI, 4M urea, 0.1 Triton X-100, pH 8.0, followed by washing with TRIS, 0.5M NaCI, 0.02% Triton X-100, pH 8.0; in step elution is accomplished with 20mM Tris, 50mM imidazole, 0.02% Triton X-100, pH 8.0; in step the eluate is adjusted to ImM EDTA and 2M NaCI, a Phenyl Sepharose 6 Fast Flow (Pharmacia) is equilibrated with equilibration buffer (2.0M NaCI, Tris, 0.02% Triton X-100, pH loaded with the adjusted eluate from step at room temperature, washed with equilibration buffer, and washed with 25mM NaPO 4 0.02% Triton X-100, pH6.5 at a flow rate of 30cm/hr; in step elution is accomplished with 25mM NaPO 4 3% Triton X-100, pH 6.5; in step a Macro- Prep High S Column (Bio-Rad Labs, Richmond, CA) is equilibrated with equilibration buffer (20mM NaPO 4 0.02% Triton X-100, pH loaded with the adjusted eluate from step washed with equilibration buffer, and washed with Tris, 0.02% Triton X-100, pH 8.0; in step elution is accomplished with Tris, 0.02 Triton X-100, 1.3M NaC1, pH 8.0; in step a Bakerbond Wide Pore Hi-Propyl C 3 (Baker, Phillipsburg, NJ) is equilibrated with equilibration buffer NaCI, 25mM Tris, 0.02% Triton X-100, pH loaded with adjusted eluate from step at room temperature, washed with equilibration buffer, and washed with Tris, 0.02% Triton X-100, pH 8.0 at 30 cm/hr; in step elution is accomplished with 10mM Tris, 3.0% Triton X-100, pH 8.0; in step dilution is into equilibration buffer (20mM succinate, 0.1 PLURONIC F68, pH in step (m) a SP Sepharose Fast Flow (Pharmacia) column is equilibrated with the equilibration buffer of step loaded with eluate from step and washed with equilibration WO 99/09147 PCT/US97/14212 buffer; and in step elution is accomplished with 50mM NaPO 4 0.7M NaCI, 0.1% PLURONIC F68, 0.02% TWEEN 80, pH PAF-AH products may be obtained as isolates from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving procaryotic or eucaryotic host cells of the invention. PAF-AH products having part or all of the amino acid sequence set out in SEQ ID NO: 8 are contemplated. Specifically contemplated are fragments lacking up to the first twelve N-terminal amino acids of the mature human PAF-AH amino acid sequence set out in SEQ ID NO: 8, particularly those having Met 4 6 Ala 47 or Ala 4 8 of SEQ ID NO: 8 as the initial N-terminal amino acid. Also contemplated are fragments thereof lacking up to thirty C-terminal amino acids of the amino acid sequence of SEQ ID NO: 8, particularly those having Ile 4 2 9 and Leu 4 3 1 as the C-terminal residue.

Further contemplated are variants of PAF-AH or PAF-AH or which have an amino acid replacement in the sequence of SEQ ID NO: 8 selected from the group consisting of S 108 A, S 273 A, D 286 A, D 286 N, D 296 A, D 304 A, D 338 A, H 351 A, H 395 A, H 399 A, C 67 S, C 229 S, C 291 S, C 334 S, C 407 S, D 286 A, D 286 N and D 304 A. As noted above, polynucleotides (including DNA) encoding such fragments or variant fragments are provided by the invention, as well as methods of recombinantly producing such fragments or variants by growing host cells comprising such DNA. Presently preferred PAF-AH products include the prokaryotic polypeptide expression products of DNA encoding amino acid residues Met 4 6 through Asn44 1 of SEQ ID NO: 8, designated rPH.2, and the prokaryotic polypeptide expression products of DNA encoding amino acid residues Met 4 6 through 11e 4 2 9 of SEQ ID NO: 8, designated rPH.9. Both the rPH.2 and rPH.9 products display less amino-terminal heterogeneity than, for example, the corresponding prokaryotic expression products of DNA encoding the full mature sequence of PAF-AH preceded by a translation initiation codon. Moreover, the rPH.9 product displays greater carboxy terminal homogeneity (consistency). The use of mammalian host cells is expected to provide for such post-translational modifications myristolation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. PAF-AH products of the invention may be full length WO 99/09147 PCT/US97/14212 -11polypeptides, fragments or variants. Variants may comprise PAF-AH analogs wherein one or more of the specified naturally encoded) amino acids is deleted or replaced or wherein one or more nonspecified amino acids are added: without loss of one or more of the enzymatic activities or immunological characteristics specific to PAF-AH; or with specific disablement of a particular biological activity of PAF-AH. Proteins or other molecules that bind to PAF-AH may be used to modulate its activity.

Also comprehended by the present invention are antibody substances monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like) and other binding proteins specific for PAF-AH. Specifically illustrating binding proteins of the invention are the monoclonal antibodies produced by hybridomas 90G11D and 90F2D which were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on September 30, 1994 and were respectively assigned Accession Nos. HB 11724 and HB 11725. Also illustrating binding proteins of the invention is the monoclonal antibody produced by hybridoma 143A which was deposited with the ATCC on June 1, 1995 and assigned Accession No. HB 11900.

Proteins or other molecules lipids or small molecules) which specifically bind to PAF-AH can be identified using PAF-AH isolated from plasma, recombinant PAF- AH, PAF-AH variants or cells expressing such products. Binding proteins are useful, in turn, in compositions for immunization as well as for purifying PAF-AH, and are useful for detection or quantification of PAF-AH in fluid and tissue samples by known immunological procedures. Anti-idiotypic antibodies specific for PAF-AHspecific antibody substances are also contemplated.

The scientific value of the information contributed through the disclosures of DNA and amino acid sequences of the present invention is manifest.

As one series of examples, knowledge of the sequence of a cDNA for PAF-AH makes possible the isolation by DNA/DNA hybridization of genomic DNA sequences encoding PAF-AH and specifying PAF-AH expression control regulatory sequences such as promoters, operators and the like. DNA/DNA hybridization procedures carried out with DNA sequences of the invention under conditions of stringency standard in the art are likewise expected to allow the isolation of DNAs encoding WO 99/09147 PCT/US97/14212 -12allelic variants of PAF-AH, other structurally related proteins sharing one or more of the biochemical and/or immunological properties of PAF-AH, and non-human species proteins homologous to PAF-AH. The DNA sequence information provided by the present invention also makes possible the development, by homologous recombination or "knockout" strategies [see, Kapecchi, Science, 244: 1288-1292 (1989)], of rodents that fail to express a functional PAF-AH enzyme or that express a variant PAF-AH enzyme. Polynucleotides of the invention when suitably labelled are useful in hybridization assays to detect the capacity of cells to synthesize PAF- AH. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in the PAF-AH locus that underlies a disease state or states. Also made available by the invention are anti-sense polynucleotides relevant to regulating expression of PAF-AH by those cells which ordinarily express the same.

Administration of PAF-AH preparations of the invention to mammalian subjects, especially humans, for the purpose of ameliorating pathological inflammatory conditions is contemplated. Based on implication of the involvement of PAF in pathological inflammatory conditions, the administration of PAF-AH is indicated, for example, in treatment of asthma [Miwa et al., J. Clin. Invest., 82: 1983-1991 (1988); Hsieh et al., J. Allergy Clin. Immunol., 91: 650-657 (1993); and Yamashita et al., Allergy, 49: 60-63 (1994)], anaphylaxis [Venable et al., supra], shock [Venable et al., supra], reperfusion injury and central nervous system ischemia [Lindsberg et al. (1991), supra], antigen-induced arthritis [Zarco et al., Clin. Exp.

Immunol., 88: 318-323 (1992)], atherogenesis [Handley et al., Drug Dev. Res., 7: 361-375 (1986)], Crohn's disease [Denizot et al., Digestive Diseases and Sciences, 37(3): 432-437 (1992)], ischemic bowel necrosis/necrotizing enterocolitis [Denizot et al., supra and Caplan et al., Acta Paediatr., Suppl. 396: 11-17 (1994)], ulcerative colitis (Denizot et al., supra), ischemic stroke [Satoh et al., Stroke, 23: 1090-1092 (1992)], ischemic brain injury [Lindsberg et al., Stroke, 21: 1452-1457 (1990) and Lindsberg et al. (1991), supra], systemic lupus erythematosus [Matsuzaki et al., Clinica Chimica Acta, 210: 139-144 (1992)], acute pancreatitis [Kald et al., Pancreas, 440-442 (1993)], septicemia (Kald et al., supra), acute post streptococcal glomerulonephritis [Mezzano et al., J. Am. Soc. Nephrol., 4: 235-242 WO 99/09147 PCT/US97/14212 -13- (1993)], pulmonary edema resulting from IL-2 therapy [Rabinovici et al., J. Clin.

Invest., 89: 1669-1673 (1992)], allergic inflammation [Watanabe et al., Br. J.

Pharmacol., 111: 123-130 (1994)], ischemic renal failure [Grino et al., Annals of Internal Medicine, 121(5): 345-347 (1994); preterm labor [Hoffman et al., Am. J.

Obstet. Gynecol., 162(2): 525-528 (1990) and Maki et al., Proc. Natl. Acad. Sci.

USA, 85: 728-732 (1988)]; adult respiratory distress syndrome [Rabinovici et al., J.

Appl. Physiol., 74(4): 1791-1802 (1993); Matsumoto et al., Clin. Exp. Pharmacol.

Physiol., 19 509-515 (1992); and Rodriguez-Roisin et al., J. Clin. Invest., 93: 188- 194 (1994)]. Also contemplated is the use of PAF-AH preparations to treat human immunodeficiency virus (HIV) infection of the central nervous system. "Treatment" as used herein includes both prophylactic and therapeutic treatment.

Animal models for many of the foregoing pathological conditions have been described in the art. For example, a mouse model for asthma and rhinitis is described in Example 16 herein; a rabbit model for arthritis is described in Zarco et at., supra; rat models for ischemic bowel necrosis/necrotizing enterocolitis are described in Furukawa et al., Ped. Res., 237-241 (1993) and Caplan et al., supra; a rabbit model for stroke is described in Lindsberg et al., (1990), supra; a mouse model for lupus is described in Matsuzaki et al., supra; a rat model for acute pancreatitis is described in Kald et al., supra: a rat model for pulmonary edema resulting from IL-2 therapy is described in Rabinovici et al., supra; a rat model of allergic inflammation is described in Watanabe et al., supra); a canine model of renal allograft is described in Watson et al., Transplantation, 56(4): 1047-1049 (1993); and rat and guinea pig models of adult respiratory distress syndrome are respectively described in Rabinovici et al., supra. and Lellouch-Tubiana, Am. Rev. Respir. Dis., 137: 948-954 (1988).

Specifically contemplated by the invention are PAF-AH compositions for use in methods for treating a mammal susceptible to or suffering from PAFmediated pathological conditions comprising administering PAF-AH to the mammal in an amount sufficient to supplement endogenous PAF-AH activity and to inactivate pathological amounts of PAF in the mammal.

Therapeutic/pharmaceutical compositions contemplated by the invention include PAF-AH products and a physiologically acceptable diluent or carrier and may WO 99/09147 PCT/US97/1 4212 -14also include other agents having anti-inflammatory effects. Dosage amounts indicated would be sufficient to supplement endogenous PAF-AH activity and to inactivate pathological amounts of PAF. For general dosage considerations see Remmington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA (1990).

Dosages will vary between about 0.1 to about 1000 1g PAF-AH/kg body weight.

Therapeutic compositions of the invention may be administered by various routes depending on the pathological condition to be treated. For example, administration may be by intraveneous, subcutaneous, oral, suppository, and/or pulmonary routes.

For pathological conditions of the lung, administration of PAF-AH by the pulmonary route is particularly indicated. Contemplated for use in pulmonary administration are a wide range of delivery devices including, for example, nebulizers, metered dose inhalers, and powder inhalers, which are standard in the art.

Delivery of various proteins to the lungs and circulatory system by inhalation of aerosol formulations has been described in Adjei et al., Pharm. Res., 565-569 (1990) (leuprolide acetate); Braquet et al., J. Cardio. Pharm., 13(Supp. s. 143- 146 (1989) (endothelin-1); Hubbard et al., Annals of Internal Medicine, 111(3), 206- 212 (1989) (al-antitrypsin); Smith et al., J. Clin. Invest., 84: 1145-1146 (1989) (a-1proteinase inhibitor); Debs et al., J. Immunol., 140: 3482-3488 (1933) (recombinant gamma interferon and tumor necrosis factor alpha); Patent Cooperation Treaty (PCT) International Publication No. WO 94/20069 published September 15, 1994 (recombinant pegylated granulocyte colony stimulating factor).

BRIEF DESCRIPTION OF THE DRAWING Numerous other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description thereof, reference being made to the drawing wherein: FIGURE 1 is a photograph of a PVDF membrane containing PAF-AH purified from human plasma; FIGURE 2 is a graph showing the enzymatic activity of recombinant human plasma PAF-AH; FIGURE 3 is a schematic drawing depicting recombinant PAF-AH fragments and their catalytic activity; WO 99/09147 PCT/US97/14212 FIGURE 4 depicts mass spectroscopy results for a recombinant PAF- AH product, rPH.2.

FIGURE 5 depicts mass spectroscopy results for a recombinant PAF- AH product, rPH.9.

FIGURE 6 is a bar graph illustrating blockage of PAF-induced rat foot edema by locally administered recombinant PAF-AH of the invention; FIGURE 7 is a bar graph illustrating blockage of PAF-induced rat foot edema by intravenously administered PAF-AH; FIGURE 8 is a bar graph showing that PAF-AH blocks PAF-induced edema but not zymosan A-induced edema; FIGURES 9A and 9B present dose response results of PAF-AH antiinflammatory activity in rat food edema; FIGURES 10A and 10B present results indicating the in vivo efficacy of a single dose of PAF-AH over time; FIGURE 11 is a line graph representing the pharmacokinetics of PAF- AH in rat circulation; and FIGURE 12 is a bar graph showing the anti-inflammatory effects of PAF-AH in comparison to the lesser effects of PAF antagonists in rat foot edema.

FIGURE 13 presents results indicating that PAF-AH neutralizes the apoptotic effects of conditioned media from HIV-1-infected and activated monocytes.

DETAILED DESCRIPTION The following examples illustrate the invention. Example 1 presents a novel method for the purification of PAF-AH from human plasma. Example 2 describes amino acid microsequencing of the purified human plasma PAF-AH. The cloning of a full length cDNA encoding human plasma PAF-AH is described in Example 3. Identification of a putative splice variant of the human plasma PAF-AH gene is described in Example 4. The cloning of genomic sequences encoding human plasma PAF-AH is described in Example 5. Example 6 desribes the cloning of canine, murine, bovine, chicken, rodent and macaque cDNAs homologous to the human plasma PAF-AH cDNA. Example 7 presents the results of an assay evidencing the enzymatic activity of recombinant PAF-AH transiently expressed in WO 99/09147 PCT/US97/14212 -16- COS 7 cells. Example 8 describes the expression of full length, truncated and chimeric human PAF-AH DNAs in E. coli, S. cerevisiae and mammalian cells.

Example 9 presents protocols for purification of recombinant PAF-AH from E. coli and assays confirming its enzymatic activity. Example 10 describes various recombinant PAF-AH products including amino acid substitution analogs and amino and carboxy-truncated products, and describes experiments demonstrating that native PAF-AH isolated from plasma is glycosylated. Results of a Northern blot assay for expression of human plasma PAF-AH RNA in various tissues and cell lines are presented in Example 11 while results of in situ hybridization are presented in Example 12. Example 13 describes the development of monoclonal and polyclonal antibodies specific for human plasma PAF-AH. Examples 14, 15, 16, 17, 18 and 19 respectively describe the in vivo therapeutic effect of administration of recombinant PAF-AH products of the invention on acute inflammation, pleurisy, asthma, necrotizing enterocolitis, adult respiratory distress syndrome and pancreatitis in animal models. Example 20 describes the in vitro effect of recombinant PAF-AH product on neurotoxicity associated with HIV infection. Example 21 presents the results of immunoassays of serum of human patients exhibiting a deficiency in PAF- AH activity and describes the identification of a genetic lesion in the patients which is apparently responsible for the deficiency.

Example 1 PAF-AH was purified from human plasma in order to provide material for amino acid sequencing.

A. Optimization of Purification Conditions Initially, low density lipoprotein (LDL) particles were precipitated from plasma with phosphotungstate and solubilized in 0.1% Tween 20 and subjected to chromatography on a DEAE column (Pharmacia, Uppsala, Sweden) according to the method of Stafforini et al. (1987), supra, but inconsistent elution of PAF-AH activity from the DEAE column required reevaluation of the solubilization and subsequent purification conditions.

WO 99/09147 PCT/US97/14212 -17- Tween 20, CHAPS (Pierce Chemical Co., Rockford, IL) and octyl glucoside were evaluated by centrifugation and gel filtration chromatography for their ability to solubilize LDL particles. CHAPS provided 25% greater recovery of solubilized activity than Tween 20 and 300% greater recovery than octyl glucoside.

LDL precipitate solubilized with 10mM CHAPS was then fractionated on a DEAE Sepharose Fast Flow column (an anion exchange column; Pharmacia) with buffer containing ImM CHAPS to provide a large pool of partially purified PAF-AH ("the DEAE pool") for evaluation of additional columns.

The DEAE pool was used as starting material to test a variety of chromatography columns for utility in further purifying the PAF-AH activity. The columns tested included: Blue Sepharose Fast Flow (Pharmacia), a dye ligand affinity column; S-Sepharose Fast Flow (Pharmacia), a cation exchange column; Cu Chelating Sepharose (Pharmacia), a metal ligand affinity column; Fractogel S (EM Separations, Gibbstown, NJ), a cation exchange column; and Sephacryl-200 (Pharmacia), a gel filtration column. These chromatographic procedures all yielded low, unsatisfactory levels of purification when operated in ImM CHAPS. Subsequent gel filtration chromatography on Sephacryl S-200 in ImM CHAPS generated an enzymatically active fraction which eluted over a broad size range rather than the expected 44 kDa approximate size. Taken together, these results indicated that the LDL proteins were aggregating in solution.

Different LDL samples were therefore evaluated by analytical gel filtration chromatography for aggregation of the PAF-AH activity. Samples from the DEAE pool and of freshly solubilized LDL precipitate were analyzed on Superose 12 (Pharmacia) equilibrated in buffer with ImM CHAPS. Both samples eluted over a very broad range of molecular weights with most of the activity eluting above 150 kDa. When the samples were then analyzed on Superose 12 equilibrated with CHAPS, the bulk of the activity eluted near 44 kDa as expected for PAF-AH activity.

However, the samples contained some PAF-AH activity in the high molecular weight region corresponding to aggregates.

Other samples eluted PAF-AH activity exclusively in the approximately 44 kDa range when they were subsequently tested by gel filtration. These samples were an LDL precipitate solubilized in 10mM CHAPS in the presence of 0.5M NaCI WO 99/09147 PCT/US97/14212 -18and a fresh DEAE pool that was adjusted to 10mM CHAPS after elution from the DEAE column. These data indicate that at least 10mM CHAPS is required to maintain non-aggregated PAF-AH. Increase of the CHAPS concentration from 1mM to 10mM after chromatography on DEAE but prior to subsequent chromatographic steps resulted in dramatic differences in purification. For example, the degree of PAF-AH purification on S-Sepharose Fast Flow was increased from 2-fold to PAF-AH activity bound the Blue Sepharose Fast Flow column irreversibly in ImM CHAPS, but the column provided the highest level of purification in 10mM CHAPS.

The DEAE chromatography was not improved with prior addition of 10mM CHAPS.

Chromatography on Cu Chelating Sepharose after the Blue Sepharose Fast Flow column concentrated PAF-AH activity 15-fold. It was also determined that PAF-AH activity could be recovered from a reduced SDS-polyacrylamide gel, as long as samples were not boiled. The activity of material eluted from the Cu Chelating Sepharose column when subjected to SDS-polyacrylamide gel electrophoresis coincided with a major protein band when the gel was silver stained.

B. PAF-AH Purification Protocol The novel protocol utilized to purify PAF-AH for amino acid sequencing therefore comprised the following steps which were performed at 4 C.

Human plasma was divided into 900 ml aliquots in 1 liter Nalgene bottles and adjusted to pH 8.6. LDL particles were then precipitated by adding 90 ml of 3.85 sodium phosphotungstate followed by 23 ml of 2M MgCl 2 The plasma was then centrifuged for 15 minutes at 3600 g. Pellets were resuspended in 800 ml of 0.2% sodium citrate. LDL was precipitated again by adding 10 g NaCI and 24 ml of 2M MgCI 2 LDL particles were pelleted by centrifugation for 15 minutes at 3600 g.

This wash was repeated twice. Pellets were then frozen at -20 0 C. LDL particles from 5L of plasma were resuspended in 5 L of buffer A (25mM Tris-HC1, CHAPS, pH 7.5) and stirred overnight. Solubilized LDL particles were centrifuged at 3600 g for 1.5 hours. Supernatants were combined and filtered with Whatman 113 filter paper to remove any remaining solids. Solubilized LDL supernatant was loaded on a DEAE Sepharose Fast Flow column (11 cm x 10 cm; 1 L resin volume; ml/minute) equilibrated in buffer B (25mM Tris-HC1, ImM CHAPS, pH The WO 99/09147 PCTIS97/14212 -19column was washed with buffer B until absorbance returned to baseline. Protein was eluted with an 8 L, 0 0.5M NaCI gradient and 480 ml fractions were collected.

This step was necessary to obtain binding to the Blue Sepharose Fast Flow column below. Fractions were assayed for acetylhydrolase activity essentially by the method described in Example 4.

Active fractions were pooled and sufficient CHAPS was added to make the pool about 10mM CHAPS. The DEAE pool was loaded overnight at 4 ml/minute onto a Blue Sepharose Fast Flow column (5 cm x 10 cm; 200 ml bed volume) equilibrated in buffer A containing 0.5M NaCI. The column was washed with the equilibration buffer at 16 ml/minute until absorbance returned to baseline. PAF-AH activity was step eluted with buffer A containing 0.5M KSCN (a chaotropic salt) at 16 ml/minute and collected in 50 ml fractions. This step resulted in greater than 1000-fold purification. Active fractions were pooled, and the pool was adjusted to pH with 1M Tris-HCl pH 8.0. The active pool from Blue Sepharose Fast Flow chromatography was loaded onto a Cu Chelating Sepharose column (2.5 cm x 2 cm; ml bed volume; 4 ml/minute) equilibrated in buffer C [25mM Tris-HCl, CHAPS, 0.5M NaCI, pH 8.0 (pH 7.5 also worked)], and the column was washed with 50 ml buffer C. PAF-AH activity was eluted with 100 ml 50mM imidazole in buffer C and collected in 10 ml fractions. Fractions containing PAF-AH activity were pooled and dialyzed against buffer A. In addition to providing a concentration of PAF-AH activity, the Cu Chelating Sepharose column gave a small purification. The Cu Chelating Sepharose pool was reduced in 50 mM DT for minutes at 37 0 C and loaded onto a 0.75 mm, 7.5% polyacrylamide gel. Gel slices were cut every 0.5 cm and placed in disposable microfuge tubes containing 200 zl 25mM Tris-HC1, 10mM CHAPS, 150mM NaCI. Slices were ground up and allowed to incubate overnight at 4 0 C. The supernatant of each gel slice was then assayed for PAF-AH activity to determine which protein band on SDS-PAGE contained PAF-AH activity. PAF-AH activity was found in an approximately 44 kDa band. Protein from a duplicate gel was electrotransferred to a PVDF membrane (Immobilon-P, Millipore) and stained with Coomassie Blue. A photograph of the PVDF membrane is presented in FIGURE 1.

WO 99/09147 PCT/US97/14212 As presented in Table 1 below, approximately 200 jg PAF-AH was purified 2 x 10 6 -fold from 5 L human plasma. In comparison, a 3 x 10 4 -fold purification of PAF-AH activity is described in Stafforini et al. (1987), supra.

Table 1 Sample Vol. Activity Total Prot. Specific Recovery Fold Purification (ml) (cpm x Activity Conc. Activity of Activity Step Cum.

106) (cpm x (mg/ (cpm x Step Cum.

9 ml) 106) Plasma 5000 23 116 62 0.37 100 100 1 1 LDL 4500 22 97 1.76 12 84 84 33 33 DEAE 4200 49 207 1.08 46 212 178 3.7 124 Blue 165 881 14 0.02 54200 70 126 1190 1.5 x 105 Cu 12 12700 152 0.15 82200 104 131 1.5 2.2 x 105 SDS-PAGE -10 2.2 x 106 In summary, the following steps were unique and critical for successful purification of plasma PAF-AH for microsequencing: solubilization and chromotography in 10mM CHAPS, chromatography on a blue ligand affinity column such as Blue Sepharose Fast Flow, chromatography on a Cu ligand affinity column such as Cu Chelating Sepharose, and elution of PAF-AH from

SDS-PAGE.

Example 2 For amino acid sequencing, the approximately 44 kDa protein band from the PAF-AH- containing PVDF membrane described in Example 1 was excised and sequenced using an Applied Biosystems 473A Protein sequencer. N-terminal sequence analysis of the approximately 44 kDa protein band corresponding to the PAF-AH activity indicated that the band contained two major sequences and two minor sequences. The ratio of the two major sequences was 1:1 and it was therefore difficult to interpret the sequence data.

WO 99/09147 PCT/US97/14212 -21- To distinguish the sequences of the two major proteins which had been resolved on the SDS gel, a duplicate PVDF membrane containing the approximately 44 kDa band was cut in half such that the upper part and the lower part of the membrane were separately subjected to sequencing.

The N-terminal sequence obtained for the lower half of the membrane was: SEQ ID NO: 1

FKDLGEENFKALVLIAF

A search of protein databases revealed this sequence to be a fragment of human serum albumin. The upper half of the same PVDF membrane was also sequenced and the N-terminal amino acid sequence determined was: SEQ ID NO: 2

IQVLMAAASFGQTKIP

This sequence did not match any protein in the databases searched and was different from the N-terminal amino acid sequence: SEQ ID NO: 3

MKPLVVFVLGG

which was reported for erythrocyte cytoplasmic PAF-AH in Stafforini et al. (1993), supra. The novel sequence (SEQ ID NO: 2) was utilized for cDNA cloning of human plasma PAF-AH as described below in Example 3.

Example 3 A full length clone encoding human plasma PAF-AH was isolated from a macrophage cDNA library.

A. Construction of a Macrophage cDNA Library Poly A+ RNA was harvested from peripheral blood monocyte-derived macrophages. Double-stranded, blunt-ended cDNA was generated using the Invitrogen Copy Kit (San Diego, CA) and BstXI adapters were ligated to the cDNA prior to insertion into the mammalian expression vector, pRc/CMV (Invitrogen). The resulting plasmids were introduced into E. coli strain XL-1 Blue by electroporation.

Transformed bacteria were plated at a density of approximately 3000 colonies per WO 99/09147 PCT/US97/14212 -22agarose plate on a total of 978 plates. Plasmid DNA prepared separately from each plate was retained in individual pools and was also combined into larger pools representing 300,000 clones each.

B. Library Screening by PCR The macrophage library was screened by the polymerase chain reaction utilizing a degenerate antisense oligonucleotide PCR primer based on the novel Nterminal amino acid sequence described in Example 2. The sequence of the primer is set out below in IUPAC nomenclature and where is an inosine.

SEQ ID NO: 4 5' ACATGAATTCGGIATCYTTIGTYTGICCRAA 3' The codon choice tables of Wada et al., Nuc. Acids Res., 19S: 1981-1986 (1991) were used to select nucleotides at the third position of each codon of the primer. The primer was used in combination with a primer specific for either the SP6 or T7 promoter sequences, both of which flank the cloning site of pRc/CMV, to screen the macrophage library pools of 300,000 clones. All PCR reactions contained 100 ng of template cDNA, 1 gg of each primer, 0.125mM of each dNTP, 10mM Tris-HCl pH 8.4, 50mM MgCI 2 and 2.5 units of Taq polymerase. An initial denaturation step of 94°C for four minutes was followed by 30 cycles of amplification of 1 minute at 94°C, 1 minute at 60 0 C and 2 minutes at 72 0 C. The resulting PCR product was cloned into pBluescript SK- (Stratagene, La Jolla, CA) and its nucleotide sequence determined by the dideoxy chain termination method. The PCR product contained the sequence predicted by the novel peptide sequence and corresponds to nucleotides 1 to 331 of SEQ ID NO: 7.

The PCR primers set out below, which are specific for the cloned PCR fragment described above, were then designed for identifying a full length clone.

Sense Primer (SEQ ID NO: TATITCTAGAAGTGTGGTGGAACTCGCTGG 3' Antisense Primer (SEQ ID NO: 6) CGATGAATTCAGCTTGCAGCAGCCATCAGTAC 3' PCR reactions utilizing the primers were performed as described above to first screen the cDNA pools of 300,000 clones and then the appropriate subset of the smaller WO 99/09147 PCT/US97/14212 -23pools of 3000 clones. Three pools of 3000 clones which produced a PCR product of the expected size were then used to transform bacteria.

C. Library Screening by Hybridization DNA from the transformed bacteria was subsequently screened by hybridization using the original cloned PCR fragment as a probe. Colonies were blotted onto nitrocellulose and prehybridized and hybridized in 50% formamide, 0.75M sodium chloride, 0.075M sodium citrate, 0.05M sodium phosphate pH 1% polyvinyl pyrolidine, 1% Ficoll, 1% bovine serum albumin and 50 ng/ml sonicated salmon sperm DNA. The hybridization probe was labeled by random hexamer priming. After overnight hybridization at 42 0 C, blots were washed extensively in 0.03M sodium chloride, 3mM sodium citrate, 0.1% SDS at 42°C. The nucleotide sequence of 10 hybridizing clones was determined. One of the clones, clone sAH 406-3, contained the sequence predicted by the original peptide sequence of the PAF-AH activity purified from human plasma. The DNA and deduced amino acid sequences of the human plasma PAF-AH are set out in SEQ ID NOs: 7 and 8, respectively.

Clone sAH 406-3 contains a 1.52 kb insert with an open reading frame that encodes a predicted protein of 441 amino acids. At the amino terminus, a relatively hydrophobic segment of 41 residues precedes the N-terminal amino acid (the isoleucine at position 42 of SEQ ID NO: 8) identified by protein microsequencing. The encoded protein may thus have either a long signal sequence or a signal sequence plus an additional peptide that is cleaved to yield the mature functional enzyme. The presence of a signal sequence is one characteristic of secreted proteins. In addition, the protein encoded by clone sAH 406-3 includes the consensus GxSxG motif (amino acids 271-275 of SEQ ID NO: 8) that is believed to contain the active site serine of all known mammalian lipases, microbial lipases and serine proteases. See Chapus er al., Biochimie, 70: 1223-1224 (1988) and Brenner, Nature, 334: 528-530 (1988).

Table 2 below is a comparison of the amino acid composition of the human plasma PAF-AH of the invention as predicted from SEQ ID NO: 8 and the WO 99/09147 PCTIUS97/1 4212 -24amino acid composition of the purportedly purified material described by Stafforini et al. (1987), supra.

Table 2 Ala Asp Asn Cys Glu Gin Phe Gly His Ile Lys Leu Met Pro Arg Ser Thr Val Trp Tyr Clone sAH 406-3 26 48 5 36 22 29 13 31 26 40 10 15 18 27 20 13 7 14 Stafforini et al.

24 37 14 42 12 58 24 17 26 7 11 16 36 14 Not determined 13 The amino acid composition of the mature form of the human plasma PAF-AH of the invention and the amino acid composition of the previously purified material that was purportedly the human plasma PAF-AH are clearly distinct.

When alignment of the Hattori et al., supra nucleotide and deduced amino acid sequences of bovine brain cytoplasmic PAF-AH with the nucleotide and amino acid sequences of the human plasma PAF-AH of the invention was attempted, no significant structural similarity in the sequences was observed.

WO 99/09147 PCTIUS97/1 4212 Example 4 A putative splice variant of the human PAF-AH gene was detected when PCR was performed on macrophage and stimulated PBMC cDNA using primers that hybridized to the 5' untranslated region (nucleotides 31 to 52 of SEQ ID NO: 7) and the region spanning the translation termination codon at the 3' end of the PAF- AH cDNA (nucleotides 1465 to 1487 of SEQ ID NO: The PCR reactions yielded two bands on a gel, one corresponding to the expected size of the PAF-AH cDNA of Example 3 and the other was about 100 bp shorter. Sequencing of both bands revealed that the larger band was the PAF-AH cDNA of Example 3 while the shorter band lacked exon 2 (Example 5 below) of the PAF-AH sequence which encodes the putative signal and pro-peptide sequences of plasma PAF-AH. The predicted catalytic triad and all cysteines were present in the shorter clone, therefore the biochemical activity of the protein encoded by the clone is likely to match that of the plasma enzyme.

To begin to assess the biological relevance of the PAF-AH splice variant that is predicted to encode a cytoplasmically active enzyme, the relative abundance of the two forms in blood monocyte-derived macrophages was assayed by RNase protection. Neither message was present in freshly isolated monocytes but both messages were found at day 2 of in vitro differentiation of the monocytes into macrophages and persisted through 6 days of culture. The quantity of the two messages was approximately equivalent throughout the differentiation period. In contrast, similar analyses of neural tissues revealed that only full length message predicted to encode the full length extracellular form of PAF-AH is expressed.

Example Genomic human plasma PAF-AH sequences were also isolated. The structure of the PAF-AH gene was determined by isolating lambda and P1 phage clones containing human genomic DNA by DNA hybridization under conditions of high stringency. Fragments of the phage clones were subcloned and sequenced using primers designed to anneal at regular intervals throughout the cDNA clone sAH 406- 3. In addition, new sequencing primers designed to anneal to the intron regions flanking the exons were used to sequence back across the exon-intron boundaries to WO 99/09147 PCT/US97/14212 -26confirm the sequences. Exon/intron boundaries were defined as the points where the genomic and cDNA sequences diverged. These analyses revealed that the human PAF-AH gene is comprised of 12 exons.

Exons 1, 2, 3, 4, 5, 6, and part of 7 were isolated from a male fetal placental library constructed in lamda FIX (Stratagene). Phage plaques were blotted onto nitrocellulose and prehybridized and hybridized in 50% formamide, 0.75M sodium chloride, 75mM sodium citrate, 50mM sodium phosphate (pH 1% polyvinyl pyrolidine, 1% Ficoll, 1% bovine serum albumin, and 50 ng/ml sonicated salmon sperm DNA. The hybridization probe used to identify a phage clone containing exons 2-6 and part of 7 consisted of the entire cDNA clone sAH 406-3.

A clone containing exon 1 was identified using a fragment derived from the 5' end of the cDNA clone (nucleotides 1 to 312 of SEQ ID NO: Both probes were labelled with 32 P by hexamer random priming. After overnight hybridization at 42 C, blots were washed extensively in 30mM sodium chloride, 3mM sodium citrate, 0.1% SDS at 42'C. The DNA sequences of exons 1, 2, 3, 4, 5, and 6 along with partial surrounding intron sequences are set out in SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively.

The remainder of exon 7 as well as exons 8, 9, 10, 11, and 12 were subcloned from a P1 clone isolated from a human PI genomic library. P1 phage plaques were blotted onto nitrocellulose and prehybridized and hybridized in 0.75M sodium chloride, 50mM sodium phosphate (pH 5mM EDTA, 1% polyvinyl pyrolidine, 1% Ficoll, 1% bovine serum albumin, 0.5% SDS, and 0.1 mg/ml total human DNA. The hybridization probe, labeled with 3 2 P by hexamer random priming, consisted of a 2.6 kb EcoRl fragment of genomic DNA derived from the 3' end of a lambda clone isolated above. This fragment contained exon 6 and the part of exon 7 present on the phage clone. After overnight hybridization at 65 blots were washed as described above. The DNA sequences of exons 7, 8, 9, 10, 11, and 12 along with partial surrounding intron sequences are set out in SEQ ID NOs: 16, 17, 18, 19, and 20, respectively.

WO 99/09147 PCT/US97/14212 -27- Example 6 Full length plasma PAF-AH cDNA clones were isolated from mouse, canine, bovine and chicken spleen cDNA libraries and a partial rodent clone was isolated from a rat thymus cDNA library. The clones were identified by low stringency hybridization to the human cDNA (hybridization conditions were the same as described for exons 1 through 6 in Example 5 above except that 20% formamide instead of 50% formamide was used). A 1 kb HindIII fragment of the human PAF- AH sAH 406-3 cDNA clone (nucleotides 309 to 1322 of SEQ ID NO: 7) was used as a probe. In addition, a partial monkey clone was isolated from macaque brain cDNA by PCR using primers based on nucleotides 285 to 303 and 851 to 867 of SEQ ID NO: 7. The nucleotide and deduced amino acid sequences of the mouse, canine, bovine, chicken, rat, and macaque cDNA clones are set out in SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively.

A comparison of the deduced amino acid sequences of the cDNA clones with the human cDNA clone results in the amino acid percentage identity values set out in Table 3 below.

Table 3 Human Dog Mouse Bovine Chicken Dog 80 100 64 82 Mouse 66 64 100 64 47 Monkey 92 82 69 80 52 Rat 74 69 82 69 Bovine 82 82 64 100 Chicken 50 50 47 50 100 About 38% of the residues are completely conserved in all the sequences. The most divergent regions are at the amino terminal end (containing the signal sequence) and the carboxyl terminal end which are shown in Example 10 as not critical for enzymatic activity. The Gly-Xaa-Ser-Xaa-Gly motif (SEQ ID NO: 27) found in neutral lipases and other esterases was conserved in the bovine, canine, mouse, rat and chicken PAF-AH. The central serine of this motif serves as the active WO 99/09147 PCT/S97/14212 -28site nucleophile for these enzymes. The predicted aspartate and histidine components of the active site (Example 10A) were also conserved. The human plasma PAF-AH of the invention therefore appears to utilize a catalytic triad and may assume the ca/e hydrolase conformation of the neutral lipases even though it does not exhibit other sequence homology to the lipases.

Moreover, human plasma PAF-AH is expected to have a region that mediates its specific interaction with the low density and high density lipoprotein particles of plasma. Interaction with these particles may be mediated by the Nterminal half of the molecule which has large stretches of amino acids highly conserved among species but does not contain the catalytic triad of the enzyme.

Example 7 To determine whether human plasma PAF-AH cDNA clone sAH 406-3 (Example 3) encodes a protein having PAF-AH activity, the pRc/CMV expression construct was transiently expressed in COS 7 cells. Three days following transfection by a DEAE Dextran method, COS cell media was assayed for PAF-AH activity.

Cells were seeded at a density of 300,000 cells per 60 mm tissue culture dish. The following day, the cells were incubated in DMEM containing mg/ml DEAE dextran, 0. ImM chloroquine and 5-10 tg of plasmid DNA for 2 hours.

Cells were then treated with 10% DMSO in phosphate-buffered saline for 1 minute, washed with media and incubated in DMEM containing 10% fetal calf serum previously treated with diisopropyl fluorophosphate (DFP) to inactivate endogenous bovine serum PAF-AH. After 3 days of incubation, media from transfected cells were assayed for PAF-AH activity. Assays were conducted in the presence and absence of either 10 mM EDTA or 1 mM DFP to determine whether the recombinant enzyme was calcium-independent and inhibited by the serine esterase inhibitor DFP as previously described for plasma PAF-AH by Stafforini et al. (1987), supra.

Negative controls included cells transfected with pRc/CMV either lacking an insert or having the sAH 406-3 insert in reverse orientation.

PAF-AH activity in transfectant supernatants was determined by the method of Stafforini et al. (1990), supra, with the following modifications. Briefly, PAF-AH activity was determined by measuring the hydrolysis of 3 H-acetate from WO 99/09147 PCT/US97/14212 -29- [acetyl- 3 H] PAF (New England Nuclear, Boston, MA). The aqueous free 3 H-acetate was separated from labeled substrate by reversed-phase column chromatography over octadecylsilica gel cartridges (Baker Research Products, Phillipsburg, PA). Assays were carried out using 10 zl transfectant supernatant in 0.1M Hepes buffer, pH 7.2, in a reaction volume of 50 A total of 50 pmoles of substrate were used per reaction with a ratio of 1:5 labeled: cold PAF. Reactions were incubated for minutes at 37°C and stopped by the addition of 40 1l of 10M acetic acid. The solution was then washed through the octadecylsilica gel cartridges which were then rinsed with 0.1M sodium acetate. The aqueous eluate from each sample was collected and counted in a liquid scintillation counter for one minute. Enzyme activity was expressed in counts per minute.

As shown in FIGURE 2, media from cells transfected with sAH 406-3 contained PAF-AH activity at levels 4-fold greater than background. This activity was unaffected by the presence of EDTA but was abolished by ImM DFP. These observations demonstrate that clone sAH 406-3 encodes an activity consistent with the human plasma enzyme PAF-AH.

Example 8 Full length and various truncated human plasma PAF-AH DNAs and a chimeric mouse-human PAF-AH DNA were expressed in E. coli and yeast and stably expressed in mammalian cells by recombinant methods.

A. Expression in E. coli PCR was used to generate a protein coding fragment of human plasma PAF-AH cDNA from clone sAH 406-3 which was readily amenable to subcloning into an E. coli expression vector. The subcloned segment began at the 5' end of the human gene with the codon that encodes Ile 4 2 (SEQ ID NO: the N-terminal residue of the enzyme purified from human plasma. The remainder of the gene through the native termination codon was included in the construct. The 5' sense PCR primer utilized was: WO 99/09147 PCT/US97/14212 SEQ ID NO: 28 3' and contained an XbaI cloning site as well as a translation initiation codon (underscored). The 3' antisense primer utilized was: SEQ ID NO: 29 ATTGATATCCTAATrGTATITCTCTATTCCTG 3' and encompassed the termination codon of sAH 406-3 and contained an EcoRV cloning site. PCR reactions were performed essentially as described in Example 3.

The resulting PCR product was digested with XbaI and EcoRV and subcloned into a pBR322 vector containing the Trp promoter [deBoer et al., PNAS, 80:21-25 (1983)] immediately upstream of the cloning site. E. coli strain XL-1 Blue was transformed with the expression construct, and cultured in L broth containing 100 jig/ml of carbenicillin. Transformants from overnight cultures were pelleted and resuspended in lysis buffer containing 50mM Tris-HCl pH 7.5, 50mM NaCI, 10mM CHAPS, ImM EDTA, 100 /g/ml lysozyme, and 0.05 trypsin-inhibiting units (TIU)/ml Aprotinin. Following a 1 hour incubation on ice and sonication for 2 minutes, the lysates were assayed for PAF-AH activity by the method described in Example 4.

E. coli transformed with the expression construct (designated trp AH) generated a product with PAF-AH activity. See Table 6 in Example 9.

Constructs including three additional promoters, the taclI promoter (deBoer, supra), the arabinose (ara) B promoter from Salmonella typhimurium [Horwitz et al., Gene, 14: 309-319 (1981)], and the bacteriophage T7 promoter, were also utilized to drive expression of human PAF-AH sequences in E. coli. Constructs comprising the Trp promoter (pUC trp AH), the taclI promoter (pUC tac AH), and the araB promoter (pUC ara AH) were assembled in plasmid pUC19 (New England Biolabs, MA) while the construct comprising the T7 promoter (pET AH) was assembled in plasmid pET15B (Novagen, Madison, WI). A construct containing a hybrid promoter, pHAB/PH, consisting of the araB promoter fused to the ribosome binding sites of the T7 promoter region was also assembled in pET15B. All E. coli constructs produced PAF-AH activity within a range of 20 to 50 U/ml/OD600. This activity corresponded to a total recombinant protein mass of 1 of the total cell protein.

WO 99/09147 PCT/US97/14212 -31- Several E. coli expression constructs were also evaluated which produce PAF-AH with extended amino termini. The N-terminus of natural plasma PAF-AH was identified as Ile 4 2 by amino acid sequencing (Example However, the sequence immediately upstream of Ile 42 does not conform to amino acids found at signal sequence cleavage sites the "-3-1-rule" is not followed, as lysine is not found at position see von Heijne, Nuc. Acids Res., 14:4683-4690 (1986)].

Presumably a more classical signal sequence (M 1

-A

17 or MI-P 2 1 is recognized by the cellular secretion system, followed by endoproteolytic cleavage. The entire coding sequence for PAF-AH beginning at the initiating methionine (nucleotides 162 to 1487 of SEQ ID NO: 7) was engineered for expression in E. coli using the trp promoter. As shown in Table 4, this construct made active PAF-AH, but expression was at about one fiftieth of the level of the original construct beginning at Ile 4 2 Another expression construct, beginning at Val 18 (nucleotides 213 to 1487 of SEQ ID NO: produced active PAF-AH at about one third the level of the original construct. These results suggest that amino terminal end extensions are not critical or necessary for activity of recombinant PAF-AH produced in E. coli.

Table 4 PAF-AH activity Construct Lysate Media pUC trp AH (Ile 4 2 N-terminus) 177.7 0.030 pUC trp AH Met 1 3.1 0.003 pUC trp AH Vall8 54.6 0.033 Truncated recombinant human PAF-AH products were also produced in E. coli using a low copy number plasmid and a promoter that can be induced by the addition of arabinose to the culture. One such N-terminally truncated PAF-AH product is the recombinant expression product of DNA encoding amino acid residues Met 4 6 through Asn441 of the polypeptide encoded by full length PAF-AH cDNA (SEQ ID NO: and is designated rPH.2. The plasmid used for production of rPH.2 in bacterial cells was pBAR2/PH.2, a pBR322-based plasmid that carries (1) nucleotides 297 to 1487 of SEQ ID NO: 7 encoding human PAF-AH beginning with WO 99/09147 PCT/US97/14212 -32the methionine codon at position 46, the araB-C promoters and araC gene from the arabinose operon of Salmonella typhimurium, a transcription termination sequence from the bacteriophage T7, and a replication origin from bacteriophage fl.

Specifically, pBAR2/PH.2 included the following segments of DNA: from the destroyed AatlI site at position 1994 to the EcoRI site at nucleotide 6274, vector sequence containing an origin of replication and genes encoding resistance to either ampicillin or tetracycline derived from the bacterial plasmid pBR322; from the EcoRI site at position 6274 to the XbaI site at position 131, DNA from the Salmonella typhimurium arabinose operon (Genbank accession numbers M11045, M11046, M 1047, J01797); from the XbaI site at position 131 to the NcoI site at position 170, DNA containing a ribosome binding site from pET- 21b (Novagen, Madison, WI); from the NcoI site at position 170 to the XhoI site at position 1363, human PAF-AH cDNA sequence; and from the XhoI site at position 1363 to the destroyed AatII site at position 1993, a DNA fragment from pET-21b (Novagen) that contains a transcription termination sequence from bacteriophae T7 and an origin of replication from bacteriophage fl.

Another PAF-AH product, designated rPH.9, is the recombinant expression product of DNA encoding amino acid residues Met 4 6 through Ile 4 2 9 of the polypeptide encoded by full length PAF-AH cDNA (SEQ ID NO: The DNA encoding rPH.9 was inserted into the same vector used for production of rPH.2 in bacterial cells. This plasmid was designated pBAR2/PH.9 and specifically included the following segments of DNA: from the destroyed AatII site at position 1958 to the EcoRI site at nucleotide 6239 of the vector sequence containing an origin of replication and genes encoding resistance to either ampicillin or tetracycline derived from the bacterial plasmid pBR322; from the EcoRI site at position 6239 to the XbaI site at position 131, DNA from the Salmonella typhimurium arabinose operon (Genbank accession numbers M11045, M11046, M 1047, J01797); from the Xbal site at position 131 to the NcoI site at position 170, DNA containing a ribosome binding site from pET-21b (Novagen, Madison, WI); from the NcoI site at position 170 to the XhoI site at position 1328, human PAF-AH DNA sequence; from the XhoI site at position 1328 to the destroyed Aatl site at position 1958, a WO 99/09147 PCTIUS97/14212 -33- DNA fragment from pET-21b (Novagen, Madison, WI) that contains a transcription termination sequence from bacteriophage T7 and a origin of replication from bacteriophage fl.

Expression of PAF-AH products in pBAR2/PH.2 and pBAR2/PH.9 is under the control of the araB promoter, which is tightly repressed in the presence of glucose and absence of arabinose, but functions as a strong promoter when Larabinose is added to cultures depleted of glucose. Selection for cells containing the plasmid can be accomplished through the addition of either ampicillin (or related antibiotics) or tetracycline to the culture medium. A variety of E. coli strains can be used as a host for recombinant expression of PAF-AH products, including but not limited to strains prototrophic for arabinose metabolism such as W3110, BL21, C600, JM101 and their derivatives, strains containing mutations reducing proteolysis such as CAG629, KY1429, and strains defective in their ability to degrade arabinose such as SB7219 and MC1061. The advantage of using a strain that is unable to break down arabinose is that the inducer (arabinose) for production of PAF- AH is not depleted from the medium during the induction period, resulting in higher levels of PAF-AH compared to that obtained with strains that are capable of metabolizing arabinose. Any suitable media and culturing conditions may be used to express active PAF-AH products in various E. coli strains. For example, either rich media formulations such LB, EDM295 (a M9 based minimum medium supplemented with yeast extract and acid hydrolysed casein), or "defined" media such as A675, an A based minimal medium set at pH 6.75 employing glycerol as a carbon source and supplemented with trace elements and vitamins, permit substantial production of rPAF-AH products. Tetracycline is included in the media to maintain selection of the plasmid.

The plasmid pBAR2/PH.2 was transformed into the E. coli strain MC1061 (ATCC 53338), which carries a deletion of the arabinose operon and thereby cannot metabolize arabinose. MC1061 is also a leucine auxotroph and was cultivated by batch-fed process using a defined media containing casamino acids that complement the leucine mutation.

The E. coli M1061 cells transformed with pBAR2/PH.2 were grown at 30 C in batch media containing 2 gm/L glucose. Glucose serves the dual purpose WO 99/09147 PCT/US97/14212 -34of carbon source for cell growth, and repressor of the arabinose promoter. When batch glucose levels were depleted 50 mg/L), a nutrient feed (containing 300 gm/L glucose) was started. The feed was increased linearly for 16 hours at a rate which limited acid bi-product formation. At this point, the nutrient feed was switched to media containing glycerol instead of glucose. Simultaneously, 500 gm/L L-arabinose was added to a final concentration of 5 gm/L. The glycerol feed was kept at a constant feed rate for 22 hours. Cells were harvested using hollow-fiber filtration to concentrate the suspension approximately 10-fold. Cell paste was stored at -70 C.

A final cell mass of about 80 gm/L was obtained (OD 6 0 0 50-60) with a PAF-AH activity of 65-70 U/OD/ml representing about 10% of total cell protein. The final culture volume of about 75 liters contained 50-60 gm PAF-AH.

High level production of rPAF-AH products can be achieved when pBAR2/PH.2 or PH.9 is expressed by strains SB7219 or MC1061. Other strains deficient in arabinose degradation are suitable for high cell density production.

Preferably, the cells are cultured under the following conditions. Exponentially growing SB7219;pBAR2/PH.2 and SB7219;pBAR2/PH.9 strains are seeded into fermentors containing batch medium containing 2 g/L glucose. Once glucose is consumed, the tanks are fed with a glycerol solution containing trace elements, vitamins, magnesium and ammonium salt to maintain healthy exponential growth.

The tanks are maintained at 300C, provided air to supply oxygen and agitated to maintain the dissolved oxygen level above about 15% saturation. When the cell density of the culture is above 110 g/L (wet cell mass), constant feed rate is imposed and a bolus addition of L-arabinose is added to the culture (about 0.5% final).

Product formation is observed for 16-22 hours. The cultures typically achieve 40-50 g/L (dry cell weight). Cells are harvested by centrifugation, stored at -70 0 C, and rPAF-AH product purified for analysis. Specific productivities in excess of 150 units/ml/OD 6 0 0 are routinely obtained.

B. Expression in Yeast Cells Recombinant human PAF-AH was also expressed in Saccharomyces cerevisiae. The yeast ADH2 promoter was used to drive rPAF-AH expression and produced 7 U/ml/OD 6 0 0 (Table 5 below).

WO 99/09147 PCT/US97/14212 Table Enzyme Activity Construct Promoter Strain (U/ml/OD) pUC tac AH tac E. coli W3110 pUC trp AH trp E. coli W3110 pUC ara AH araB E. coli W3110 pET AH T7 E. coli BL21 (DE3) (Novagen) pHAB/PH araB/T7 E. coli XL-1 34 pBAR2/PH.2 araB MC1061 pYep ADH2 AH ADH2 Yeast BJ2.28 7 C. Expression of PAF-AH in mammalian cells 1. Expression of Human PAF-AH cDNA Constructs Plasmids constructed for expression of PAF-AH, with the exception of pSFN/PAFAH.1, employ a strong viral promoter from cytomegalovirus, a polyadenylation site from the bovine growth hormone gene, and the SV40 origin of replication to permit high copy number replication of the plasmid in COS cells.

Plasmids were electroporated into cells.

A first set of plasmids was constructed in which the 5' flanking sequence (pDC1/PAFAH.1) or both the 5' or 3' flanking sequences (PDC1/PAFAH.2) of the human PAF-AH cDNA were replaced with flanking sequences from other genes known to be expressed at high levels in mammalian cells.

Transfection of these plasmids into COS, CHO or 293 cells led to production of PAF- AH at about the same level (0.01 units/ml or 2-4 fold above background) as that cited for clone sAH 406-3 in Example 7 after transient transfection of COS cells. Another plasmid was constructed which included a Friend spleen focus-forming virus promoter instead of the cytomegalovirus promoter. The human PAF-AH cDNA was inserted into plasmid pmH-neo [Hahn et al., Gene, 127: 267 (1993)] under control of the Friend spleen focus-forming virus promoter. Transfection of the myeloma cell line WO 99/09147 PCT/US97/14212 -36- NSO with the plasmid which was designated pSFN/PAFAH. 1 and screening of several hundred clones resulted in the isolation of two transfectants (4B11 and 1C11) that made 0.15-0.5 units/ml of PAF-AH activity. Assuming a specific activity of 5000 units/milligram, the productivity of these two NSO transfectants corresponds to about 0.1 mg/liter.

2. Expression of Mouse-Human Chimeric PAF-AH Gene Constructs A construct (pRc/MS9) containing the cDNA encoding mouse PAF-AH in the mammalian expression vector pRc/CMV resulted in production of secreted PAF-AH at the level of 5-10 units/ml (1000 fold above background) after transfection into COS cells. Assuming that the specific activity of the mouse PAF-AH is about the same as that of the human enzyme, the mouse cDNA is therefore expressed at a 500-1000 fold higher level than is the human PAF-AH cDNA.

To examine the difference between the expression levels of human and mouse PAF-AH in COS cells, two mouse-human chimeric genes were constructed and tested for expression in COS cells. The first of these constructs, pRc/PH.MHC1, contains the coding sequence for the N-terminal 97 amino acids of the mouse PAF- AH polypeptide (SEQ ID NO: 21) fused to the C-terminal 343 amino acids of human PAF-AH in the expression vector pRc/CMV (Invitrogen, San Diego, CA). The second chimeric gene, in plasmid pRc/PH.MHC2, contains the coding sequence for the N-terminal 40 amino acids of the mouse PAF-AH polypeptide fused to the Cterminal 400 residues of human PAF-AH in pRc/CMV. Transfection of COS cells with pRc/PH.MHC1 led to accumulation of 1-2 units/ml of PAF-AH activity in the media. Conditioned media derived from cells transfected with pRc/PH.MHC2 was found to contain only 0.01 units/ml of PAF-AH activity. From these experiments, it appears that the difference in expression level between mouse and human PAF-AH genes is attributable at least in part to the polypeptide segment between the residues and 97, or the corresponding RNA or DNA segment encoding this region of the PAF-AH protein.

WO 99/09147 PCT/US97/14212 -37- 3. Recoding of the First 290 bp of the PAF-AH Coding Sequence One hypothesis for the low level of human PAF-AH synthesized in transfected mammalian cells is that the codons utilized by the natural gene are suboptimal for efficient expression. However, it does not seem likely that codon usage can account for 500-1000 fold difference in expression levels between the mouse and human genes because optimizing codons generally has at most only a fold effect on expression. A second hypothesis to explain the difference between the mouse and human PAF-AH expression levels is that the human PAF-AH mRNA in the 5' coding region forms a secondary structure that leads to either relatively rapid degradation of the mRNA or causes inefficient translation initiation or elongation.

To test these hypotheses, a synthetic fragment encoding the authentic human PAF-AH protein from the amino-terminus to residue 96 but in which most of the codons have been substituted ("recoded") with a codon of a different sequence but encoding the same amino acid was constructed. Changing the second codon from GTG to GTA resulted in the creation of an Asp718 site, which was at one end of the synthetic fragment and which is present in the mouse cDNA. The other end of the fragment contained the BamHI site normally found at codon 97 of the human gene.

The approximately 290 bp Asp718/BamHI fragment was derived from a PCR fragment that was made using the dual asymmetric PCR approach for construction of synthetic genes described in Sandhu et al., Biotechniques, 12: 14-16 (1992). The synthetic Asp718/BamHI fragment was ligated with DNA fragments encoding the remainder of the human PAF-AH molecule beginning with nucleotide 453 of SEQ ID NO: 7 such that a sequence encoding authentic human PAF-AH enzyme was inserted into the mammalian expression vector pRc/CMV (Invitrogen, San Diego) to create plasmid pRc/HPH.4. The complete sequence of the recoded gene is set out in SEQ ID NO: 30. The 5' flanking sequence adjacent to the human PAF-AH coding sequence in pRc/HPH.4 is from that of a mouse cDNA encoding PAF-AH in pRc/MS9 (nucleotides 1 to 116 of SEQ ID NO: 21).

To test expression of human PAF-AH from pRc/HPH.4, COS cells were transiently transfected with pRc/HPH.4 (recoded human gene), pRc/MS9 (mouse PAF-AH), or pRc/PH.MHCl (mouse-human hybrid The conditioned media from the transfected cells were tested for PAF-AH activity and found to WO 99/09147 PCT/US97/14212 -38contain 5.7 units/ml (mouse gene), 0.9 units/ml (mouse-human hybrid or 2.6 units/ml (recoded human gene). Thus, the strategy of recoding the first 290 bp of coding sequence of human PAF-AH was successful in boosting expression levels of human PAF-AH from a few nanograms/ml to about 0.5 microgram/ml in a transient COS cell transfection. The recoded PAF-AH gene from pRc/HPH.4 will be inserted into a mammalian expression vector containing the dihydrofolate reductase (DHFR) gene and DHFR-negative chinese hamster ovary cells will be transfected with the vector. The transfected cells will be subjected to methotrexate selection to obtain clones making high levels of human PAF-AH due to gene amplification.

Example 9 Recombinant human plasma PAF-AH (beginning at Ile 4 2 expressed in E. coli was purified to a single Coomassie-stained SDS-PAGE band by various methods and assayed for activities exhibited by the native PAF-AH enzyme.

A. Purification of Recombinant PAF-AH The first purification procedure utilized is similar to that described in Example 1 for native PAF-AH. The following steps were performed at 4°C. Pellets from 50 ml PAF-AH producing E. coli (transformed with expression construct trp AH) were lysed as described in Example 8. Solids were removed by centrifugation at 10,000 g for 20 minutes. The supernatant was loaded at 0.8 ml/minute onto a Blue Sepharose Fast Flow column (2.5 cm x 4 cm; 20 ml bed volume) equilibrated in buffer D (25mM Tris-HC1, 10mM CHAPS, 0.5M NaCI, pH The column was washed with 100 ml buffer D and eluted with 100 ml buffer A containing KSCN at 3.2 ml/minute. A 15 ml active fraction was loaded onto a 1 ml Cu Chelating Sepharose column equilibrated in buffer D. The column was washed with 5 ml buffer D followed by elution with 5 ml of buffer D containing 100mM imidazole with gravity flow. Fractions containing PAF-AH activity were analyzed by SDS-

PAGE.

The results of the purification are shown in Table 6 wherein a unit equals Cimol PAF hydrolysis per hour. The purification product obtained at 4 0

C

appeared on SDS-PAGE as a single intense band below the 43 kDa marker with some WO 99/09147 PCTIUS97/142122 -39diffuse staining directly above and below it. The recombinant material is significantly more pure and exhibits greater specific activity when compared with PAF-AH preparations from plasma as described in Example 1.

Table 6 Sample Volume Activity Total Prot Cone Specific Recovery Fold (ml) (units/ Act. (mg/mL) Activity of Activity Purification ml) (units (units/ Step Cum. Step Cum.

x mg) 3 Lysate 4.5 989 4451 15.6 63 100 100 1 1 Blue 15 64 960 0.07 914 22 22 14.4 14.4 Cu 1 2128 2128 0.55 3869 220 48 4.2 61 When the same purification protocol was performed at ambient temperature, in addition to the band below the 43 kDa marker, a group of bands below the 29 kDa marker correlated with PAF-AH activity of assayed gel slices. These lower molecular weight bands may be proteolytic fragments of PAF-AH that retain enzymatic activity.

A different purification procedure was also performed at ambient temperature. Pellets (100 g) of PAF-AH-producing E. coli (transformed with the expression construct pUC trp AH) were resuspended in 200 ml of lysis buffer Tris, 20mM CHAPS, 50mM NaCI, ImM EDTA, 50 lg/ml benzamidine, pH and lysed by passing three times through a microfluidizer at 15,000 psi. Solids were removed by centrifugation at 14,300 x g for 1 hour. The supernatant was diluted fold in dilution buffer [25mM MES (2-[N-morpholino] ethanesulfonic acid), CHAPS, 1mM EDTA, pH 4.9] and loaded at 25 ml/minute onto an S Sepharose Fast Flow Column (200 ml) (a cation exchange column) equilibrated in Buffer E MES, 10mM CHAPS, ImM EDTA, 50mM NaC1, pH The column was washed with 1 liter of Buffer E, eluted with 1M NaCI, and the eluate was collected in 50 ml fractions adjusted to pH 7.5 with 0.5 ml of 2M Tris base. Fractions containing PAF- WO 99/09147 PCT/US97/14212 AH activity were pooled and adjusted to 0.5M NaCI. The S pool was loaded at 1 ml/minute onto a Blue Sepharose Fast Flow column (2.5 cm x 4 cm; 20 ml) equilibrated in Buffer F (25mM Tris, 10mM CHAPS, 0.5M NaCI, 1mM EDTA, pH The column was washed with 100 ml Buffer F and eluted with 100 ml Buffer F containing 3M NaCl at 4 ml/minute. The Blue Sepharose Fast Flow chromatography step was then repeated to reduce endotoxin levels in the sample.

Fractions containing PAF-AH activity were pooled and dialyzed against Buffer G Tris pH 7.5, 0.5M NaC1, 0.1 Tween 80, ImM EDTA).

The results of the purification are shown in Table 7 wherein a unit equals /mol PAF hydrolysis per hour.

Table 7 Sample Volume Activity Total Prot Cone Specific Recovery Fold (ml) (units/ Act. (mg/mL) Activity of Activity Purification ml) (units (units/ Step Cum. Step Cum.

x reg) 103) Lysate 200 5640 1128 57.46 98 100 100 1 1 S 111 5742 637 3.69 1557 57 56 16 16 Blue 100 3944 394 0.84 4676 35 62 3 48 The purification product obtained appeared on SDS-PAGE as a single intense band below the 43 kDa marker with some diffuse staining directly above and below it.

The recombinant material is significantly more pure and exhibits greater specific activity when compared with PAF-AH preparations from plasma as described in Example 1.

Yet another purification procedure contemplated by the present invention involves the following cell lysis, clarification, and first column steps. Cells are diluted 1:1 in lysis buffer (25mM Tris pH 7.5, 150mM NaCI, 1% Tween 2mM EDTA). Lysis is performed in a chilled microfluidizer at 15,000-20,000 psi with three passes of the material to yield 99% cell breakage. The lysate is diluted 1:20 in dilution buffer (25mM Tris pH 8.5, ImM EDTA) and applied to a column WO 99/09147 PCTIUS97/1 4212 -41packed with Q-Sepharose Big Bead chromatography media (Pharmacia) and equilibrated in 25mM Tris pH 8.5, ImM EDTA, 0.015% Tween 80. The eluate is diluted 1:10 in 25mM MES pH 5.5, 1.2M Ammonium sulfate, ImM EDTA and applied to Butyl Sepharose chromography media (Pharmacia) equilibrated in the same buffer. PAF-AH activity is eluted in 25mM MES pH 5.5, 0.1% Tween 80, ImM

EDTA.

Still another method contemplated by the invention for purifying enzymatically-active PAF-AH from E.coli includes the steps of: preparing an E.coli extract which yields solubilized PAF-AH supernatant after lysis in a buffer containing CHAPS; dilution of the said supernatant and application to a anion exchange column equilibrated at about pH 8.0; eluting PAF-AH enzyme from said anion exchange column; applying said adjusted eluate from said anion exchange column to a blue dye ligand affinity column; eluting the said blue dye ligand affinity column using a buffer comprising 3.0M salt; dilution of the blue dye eluate into a suitable buffer for performing hydroxylapatite chromatography; (g) performing hydroxylapatite chromatography where washing and elution is accomplished using buffers (with or without CHAPS); diluting said hydroxylapatite eluate to an appropriate salt concentration for cation exchange chromatography; applying said diluted hydroxylapatite eluate to a cation exchange column at a pH ranging between approximately 6.0 to 7.0; elution of PAF-AH from said cation exchange column with a suitable formulation buffer; performing cation exchange chromatography in the cold; and formulation of PAF-AH in liquid or frozen form in the absence of CHAPS.

Preferably in step above the lysis buffer is 25mM Tris, 100mM NaCI, ImM EDTA, 20mM CHAPS, pH 8.0; in step the dilution of the supernatant for anion exchange chromatography is 3-4 fold into 25mM Tris, ImM EDTA, 10mM CHAPS, pH 8.0 and the column is a Q-Sepharose column equilibrated with 25mM Tris, ImM EDTA, 50mM NaCI, 10mM CHAPS, pH 8.0; in step the anion exchange column is eluted using 25mM Tris, ImM EDTA, 350mM NaCI, 10mM CHAPS, pH 8.0; in step the eluate from step is applied directly onto a blue dye affinity column; in step the column is eluted with 3M NaCI, CHAPS, 25mM Tris, pH 8.0 buffer; in step dilution of the blue dye eluate for WO 99/09147 PCT/US97/1 4212 -42hydroxylapatite chromatography is accomplished by dilution into 10mM sodium phosphate, 100mM NaCI, 10mM CHAPS, pH 6.2; in step hydroxylapatite chromatography is accomplished using a hydroxylapatite column equilibrated with sodium phosphate, 100mM NaCI, 10mM CHAPS and elution is accomplished using 50mM sodium phosphate, 100mM NaCI (with or without) 10mM CHAPS, pH in step dilution of said hydroxylapatite eluate for cation exchange chromatography is accomplished by dilution into a buffer ranging in pH from approximately 6.0 to 7.0 comprising sodium phosphate (with or without CHAPS); in step a S Sepharose column is equilibrated with 50mM sodium phosphate, (with or without) 10mM CHAPS, pH 6.8; in step elution is accomplished with a suitable formulation buffer such as potassium phosphate 50mM, 12.5mM aspartic acid, 125mM NaCI, pH 7.5 containing 0.01% Tween-80; and in step cation exchange chromatrography is accomplished at 2-8 C. Examples of suitable formulation buffers for use in step which stabilize PAF-AH include 50mM potassium phosphate, 12.5mM Aspartic acid, 125mM NaCI pH 7.4 (approximately, with and without the addition of Tween-80 and or Pluronic F68) or 25mM potassium phosphate buffer containing (at least) 125mM NaCI, 25mM arginine and 0.01% Tween-80 (with or without Pluronic F68 at approximately 0.1 and B. Activity of Recombinant PAF-AH The most remarkable property of the PAF acetylhydrolase is its marked specificity for substrates with a short residue at the sn-2 position of the substrate.

This strict specificity distinguishes PAF acetylhydrolase from other forms of PLA 2 Thus, to determine if recombinant PAF-AH degrades phospholipids with long-chain fatty acids at the sn-2 position, hydrolysis of 1 -palmitoyl-2-arachidonoyl-sn-glycero-3phosphocholine (arachidonoylPC) was assayed since this is the preferred substrate for a well-characterized form of PLA 2 As predicted from previous studies with native PAF-AH, this phospholipid was not hydrolyzed when incubated with recombinant PAF-AH. In additional experiments, arachidonoylPC was included in a standard PAF hydrolysis assay at concentrations ranging from 0 to 125 pjM to determine whether it inhibited the hydrolysis of PAF by recombinant PAF-AH. There was no inhibition of PAF hydrolysis even at the highest concentration of PAF-AH, which was WO 99/09147 PCTIS97/1 4212 -43greater than the concentration of PAF. Thus, recombinant PAF-AH exhibits the same substrate selectivity as the native enzyme; long chain substrates are not recognized.

Moreover, recombinant PAF-AH enzyme rapidly degraded an oxidized phospholipid (glutaroylPC) which had undergone oxidative cleavage of the sn-2 fatty acid. Native plasma PAF-AH has several other properties that distinguish it from other phospholipases including calcium-independence and resistance to compounds that modify sulfhydryl groups or disrupt disulfides.

Both the native and recombinant plasma PAF-AH enzymes are sensitive to DFP, indicating that a serine comprises part of their active sites. An unusual feature of the native plasma PAF acetylhydrolase is that it is tightly associated with lipoproteins in circulation, and its catalytic efficiency is influenced by the lipoprotein environment. When recombinant PAF-AH of the invention was incubated with human plasma (previously treated with DFP to abolish the endogenous enzyme activity), it associated with low and high density lipoproteins in the same manner as the native activity. This result is significant because there is substantial evidence that modification of low density lipoproteins is essential for the cholesterol deposition observed in atheromas, and that oxidation of lipids is an initiating factor in this process. PAF-AH protects low density lipoproteins from modification under oxidizing conditions in vitro and may have such a role in vivo. Administration of PAF-AH is thus indicated for the suppression of the oxidation of lipoproteins in atherosclerotic plaques as well as to resolve inflammation.

These results all confirm that the cDNA clone sAH 406-3 encodes a protein with the activities of the the human plasma PAF acetylhydrolase.

Example Various other recombinant PAF-AH products were expressed in E.

coli. The products included PAF-AH analogs having single amino acid mutations and PAF-AH fragments.

A. PAF-AH Amino Acid Substitution Products PAF-AH is a lipase because it hydrolyses the phospholipid PAF.

While no obvious overall similarity exists between PAF-AH and other characterized WO 99/09147 PCTIUS97/1 4212 -44lipases, there are conserved residues found in comparisons of structurally characterized lipases. A serine has been identified as a member of the active site.

The serine, along with an aspartate residue and a histidine residue, form a catalytic triad which represents the active site of the lipase. The three residues are not adjacent in the primary protein sequence, but structural studies have demonstrated that the three residues are adjacent in three dimensional space. Comparisons of structures of mammalian lipases suggest that the aspartate residue is generally twenty-four amino acids C-terminal to the active site serine. In addition, the histidine is generally 109 to 111 amino acids C-terminal to the active site serine.

By site-directed mutagenesis and PCR, individual codons of the human PAF-AH coding sequence were modified to encode alanine residues and were expressed in E. coli. As shown in Table 8 below wherein, for example, the abbreviation "S108A" indicates that the serine residue at position 108 was changed to an alanine, point mutations of Ser 2 7 3 Asp 29 6 or His 3 5 1 completely destroy PAF- AH activity. The distances between active site residues is similar for PAF-AH (Ser to Asp, 23 amino acids; Ser to His, 78 amino acids) and other lipases. These experiments demonstrate that Ser 2 7 3 Asp 2 9 6 and His 3 5 1 are critical residues for activity and are therefore likely candidates for catalytic triad residues. Cysteines are often critical for the functional integrity of proteins because of their capacity to form disulfide bonds. The plasma PAF-AH enzyme contains five cysteines. To determine whether any of the five is critical for enzyme actvity, each cysteine was mutated individually to a serine and the resulting mutants were expressed in E. coli.

Preliminary activity results using partially purified preparations of these recombinantly produced mutants are shown below in the second column of Table 8, while results using more purified preparations are shown below in the third column of Table 8. The data show that all of the cysteine mutants had largely equivalent activity, so that none of the cysteines appears to be necessary for PAF-AH activity.

Other point mutations also had little or no effect on PAF-AH catalytic activity. In Table 8, represents wild type PAF-AH activity of about 40-60 U/ml/OD 6 0 0 represents about 20-40 U/ml/OD 60 0 activity, represents about 10-20 U/ml/OD 6 0 0 activity, represents 1-10 U/ml/OD600 activity, and indicates 1 U/ml/OD 6 0 0 activity.

WO 99/09147 PCT/US97/14212 Table 8 Mutation Wild type S108A S273A D286A D286N D296A D304A D338A H351A H395A, H399A C67S C229S C291S C334S C407S C67S, C334S, C407S PAF-AH activity Specific PAF-AH activity of purified preparations 6.9 mmol/mg/hr 5.7 mmol/mg/hr 6.5 mmol/mg/hr 5.9 mmol/mg/hr 6.8 mmol/mg/hr 6.4 mmol/mg/hr 6.8 mmol/mg/hr B. PAF-AH Fragment Products C-terminal deletions were prepared by digesting the 3' end of the PAF- AH coding sequence with exonuclease II for various amounts of time and then ligating the shortened coding sequence to plasmid DNA encoding stop codons in all three reading frames. Ten different deletion constructs were characterized by DNA sequence analysis, protein expression, and PAF-AH activity. Removal of twenty-one to thirty C-terminal amino acids greatly reduced catalytic activity and removal of fifty-two residues completely destroyed activity. See FIGURE 3.

Similar deletions were made at the amino terminal end of PAF-AH.

Fusions of PAF-AH with E. coli thioredoxin at the N-terminus were prepared to facilitate consistent high level expression PAF-AH activity [LaVallie et al., WO 99/09147 PCT/US97/14212 -46- Bio/technology, 11:187-193 (1993)]. Removal of nineteen amino acids from the naturally processed N-terminus (Ile 4 2 reduced activity by 99% while removal of twenty-six amino acids completely destroyed enzymatic activity in the fusion protein.

See FIGURE 3. Deletion of twelve amino acids appeared to enhance enzyme activity about four fold.

In subsequent purifications of PAF-AH from fresh human plasma by a method similar to that described in Example 1 (Microcon 30 filter from Amicon were utilized to concentrate Blue sepharose eluate instead of a Cu column), two Ntermini in addition to Ile 4 2 were identified, Ser 3 5 and Lys 5 5 The heterogeneity may be the natural state of the enzyme in plasma or may occur during purification.

The purified material described above was also subject to analysis for glycosylation. Purified native PAF-AH was incubated in the presence or absence of N-Glycanase, an enzyme that removes N-linked carbohydrates from glycoproteins.

The treated PAF-AH samples were electrophoresed through a 12% SDS polyacrylamide gel then visualized by Western blotting using rabbit polyclonal antisera. Protein not treated with N-Glycanase migrated as a diffuse band of 45-50 kDa whereas the protein treated with the glycanase migrated as a tight band of about 44 kDa, demonstrating that native PAF-AH is glycosylated.

N-terminal heterogeneity was also observed in purified preparations of recombinant PAF-AH (Ile 4 2 N-terminus). These preparations were a mixture of polypeptides with N-termini beginning at Ala 4 7 Ile 4 2 or the artificial initiating Met_ 1 adjacent to Ile 4 2 1. Preliminary comparison of PAF-AH fragments with PAF-AH In view of the observed heterogeneity of recombinantly produced PAF- AH, other recombinant products were prepared and tested for homogeneity after recombinant expression and purification. The composition of the recombinant expression products of pBAR2/PH.2 and pBAR2/PH.9 in E. coli strain MC1061 was analyzed at different time points during the production phase of cell fermentation.

Partially purified samples of the recombinant PH.2 and PH.9 from cells collected at time points ranging between 5 and 22 hours after induction of protein expression were WO 99/09147 PCT/US97/14212 -47analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-

MS).

When the PH.2 expression vector was utilized, two peaks were observed in the spectrum of the partially purified protein at a mass value expected for rPAF-AH protein. Two peaks were observed at all time points, with greater heterogeneity being observed at time points when fermentation is stressed as indicated by an accumulation of acetate and/or a depletion of oxygen in the media. The accuracy of the MALDI-MS technique in this mass range was approximately about the mass of one amino acid. The higher mass peak observed was consistent with the presence of the expected full length translation product for the PH.2 vector, minus the translation initiating methionine which is expected to be posttranslationally removed. The lower mass peak was approximately 1200 atomic mass units less.

When the PH.9 expression vector was utilized, a single peak predominated in the spectrum of the partially purified protein at a mass value expected for rPAF-AH protein. This single peak was observed at all time points, with no increase in heterogeneity seen at different time points. The observed mass of this protein was consistent with the presence of the expected full length translation product for the PH.9 vector, minus the initiating methionine.

2. Purification of PAF-AH fragments Recombinantly expressed rPH.2 (the expression product of DNA encoding Met 4 6 -Asn441) and rPH.9 (the expression product of DNA encoding Met 4 6 -Ile 4 2 9 preparations were purified for further comparison with purified rPAF- AH (expression product of DNA encoding Ile 4 2 -Asn441). rPH.9 was produced by E. coli strain SB7219 and purified generally according to the zinc chelate purification procedure described above, while rPH.2 was produced by E. coli strain MC1061 and purified as described below. The transformed cells were lysed by dilution of the cell paste with lysis buffer (100 mM succinate, 100 mM NaCI, 20 mM CHAPS, pH The slurry was mixed and lysed by high pressure disruption. The lysed cells were centrifuged and the supernatant containing rPH.2 was retained. The clarified supernatant was diluted 5-fold in 25 mM sodium phosphate buffer containing, 1 mM WO 99/09147 PCTIUS97/1 4212 -48- EDTA, 10 mM CHAPS, pH 7.0. The diluted supernatant was then applied to the Q Sepharose column. The column was washed first with 3 column volumes of 25 mM sodium phosphate buffer containing 1 mM EDTA, 50 mM NaCI, 10 mM CHAPS, pH 7.0 (Wash then washed with 10 column volumes of 25 mM Tris buffer containing 1 mM EDTA, 10 mM CHAPS, pH 8.0 (Wash 2) and with 10 column volumes of 25 mM Tris buffer containing 1 mM EDTA, 100 mM NaC1, 10 mM CHAPS, pH 8.0 (Wash Elution was accomplished with 25 mM Tris buffer containing 1 mM EDTA, 350 mM NaCI, 10 mM CHAPS, pH 8.0. The Q Sepharose eluate was diluted 3-fold in 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 then applied to a Blue Sepharose column. The column was washed first with 10 column volumes of 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0. The column was then washed with 3 column volumes of 25 mM Tris, 0.5 M NaCI, 10 mM CHAPS, pH 8.0. Elution was accomplished with 25 mM Tris, 3.0 M NaCI, 10 mM CHAPs, pH 8.0. The Blue Sepharose eluate was diluted 5-fold in 10 mM sodium phosphate, 10 mM CHAPS, pH 6.2 then applied to the chromatography column. The column was washed with 10 column volumes of 10 mM sodium phosphate, 100 mM NaCI, 0.1 Pluronic F68, pH 6.2. rPH.2 was eluted with 120 mM sodium phosphate, 100 mM NaCI, 0.1% Pluronic F-68, pH 7.5. The hydroxyapatite eluate was diluted 6fold with 10 mM sodium phosphate, 0.1 Pluronic F68, pH 6.8. The diluted hydroxyapatite eluate was adjusted to pH 6.8 using 0.5 N succinic acid and then applied to a SP Sepharose column. The SP Sepharose column was washed with column volumes 50 mM sodium phosphate, 0.1% Fluronic F68, pH 6.8 and eluted with 50 mM sodium phosphate, 125 mM NaCI, 0.1 Pluronic F68, pH 7.5. The eluted rPH.2 was formulated by diluting to a final concentration of 4 mg/ml in mM sodium phosphate, 125 mM NaCI, 0.15% Pluronic F68, pH 7.5, and Tween was added to a final concentration of 0.02 Tween 80. The formulated product was then filtered through a 0.2A membrane and stored prior to use.

3. Comparison of PAF-AH fragments with PAF-AH by sequencing The purified rPH.2 and rPH.9 preparations were compared with purified rPAF-AH preparations by N-terminal sequencing using an Applied Biosystems Model 473A Protein Sequencer (Applied Biosystems, Foster City, CA) WO 99/09147 PCTIIJS97/14212 -49and by C-terminal sequencing using a Hewlett-Packard Model G1009A C-terminal Protein Sequencer. The rPH.2 preparation had less N-terminal heterogeneity compared to rPAF-AH. The N-terminus analysis of the rPH.9 preparation was similar to that of rPH.2, but less C-terminal heterogeneity was observed for the rPH.9 preparation relative to rPH.2.

The purified rPH.2 preparation contained a major sequence with an Nterminus of Ala 4 7 (about 86-89 and a minor sequence with an N-terminus of Ala 4 8 (about 11-14%), with the ratio of the two N-termini being fairly consistent under different fermentation conditions. The purified rPH.9 preparation also contained a major sequence with an N-terminus of Ala 4 7 (about 83-90%) and a minor sequence with an N-terminus of Ala 4 8 (about 10-17%). In contrast, attempts to produce in bacteria the polypeptide beginning at Ile 4 2 (rPAF-AH) resulted in a varying mixture of polypeptides with N-termini beginning at Ala 4 7 (20-53 Ile 4 2 or at the artificial initiating Met_l methionine (37-72%) adjacent to Ile 4 2 For rPH.2 and rPH.9, the initiating methionine is efficiently removed by an amino-terminal peptidase after bacterial synthesis of the polypeptide, leaving the alanine at position 47 (or the alanine at position 48) as the N-terminal residue.

C-terminal sequencing was carried out on one lot of rPH.2, which was observed to have a C-terminus of HOOC-Asn-Tyr as the major sequence (about consistent with the predicted HOOC-Asn44 1 -Tyr 44 0 C-terminus of the translation product, while about 20% was HOOC-Leu. After the rPH.2 preparation had been fractionated by SDS-PAGE, additional sequencing of the primary and secondary bands yielded a C-terminal sequence of HOOC-Leu-Met from a lower secondary band (AHL, described below in section consistent with a product that is 10 amino acids shorter than the full length translation product, as well as low levels of HOOC-His. Further peptide mapping has shown that additional C-termini are present in some lots of PH.2 protein. The C-terminus of rPH.9 was primarily HOOC-Ile-His (about 78 to 91%, depending on the lot) by direct sequencing, consistent with the predicted HOOC-Ile 42 9 -His 4 2 8 C-terminus of the translation product. There appears to be some background ("noise") in this technique, so low levels of other sequences could not be ruled out.

WO 99/09147 PCT/US97/14212 4. Comparison of PAF-AH fragments with PAF-AH by MALDI-MS MALDI-MS was performed on purified rPH.2 and rPH.9 preparations.

The rPH.2 spectrum exhibited two peaks in the spectrum at a mass value expected for the rPAF-AH product (see FIGURE similar to the pattern observed with the partially purified protein in section B.1. above. The secondary, lower molecular weight peak was typically present at approximately 20% to 30% of the total. The rPH.9 spectrum showed a predominant peak at a mass consistent with that expected for the full length translation product for the PH.9 vector, minus the translation initiating methionine (see FIGURE A small slightly lower molecular weight shoulder peak was also observed for rPH.9 that represented approximately 5 of the total.

Comparison of PAF-AH fragments with PAF-AH by SDS-PAGE Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) was performed on purified rPAF-AH, rPH.2 and rPH.9 preparations. A complicated banding pattern was observed for rPH.2 around the electrophoretic migration range expected for the rPAF-AH product, based on protein molecular weight standards.

One, or in some gels, two predominant bands were seen, with readily observed secondary bands above and below the primary band. These upper secondary, middle primary and lower secondary bands, respectively, were termed AHU, AHM and AHL. All of these bands reacted with an anti-rPAF-AH monoclonal antibody on Western blot and have thus been identified as rPAF-AH related products. The upper secondary band AH U increased in intensity over time with storage of the protein and presumably represents a modified form of the rPAF-AH product. The SDS-PAGE of the rPAF-AH preparation is similar to that of rPH.2. There are two major bands that migrate near the expected molecular weight for rPAF-AH, as well as a minor band above and a shadow below the major bands. In contrast, rPH.9 displayed a single predominant band on SDS-PAGE with no apparent splitting. Faint bands at a slightly lower molecular weight and at an expected dimer position were also seen.

No AHU-like band was observed.

The composition of the purified rPH.2 and rPH.9 preparations was also analyzed on 2D gels (isoelectric focusing (IEF) in urea followed by SDS-PAGE in the WO 99/09147 PCT/US97/14212 -51second dimension). For rPH.9, the 2D gels showed five main spots separated in the IEF direction. The charge heterogeneity appeared consistent between lots of rPH.9.

In contrast, the 2D gel pattern of rPH.2 was more complicated as it contained approximately 15 spots separated in the IEF and SDS-PAGE dimensions.

6. Comparison of activity of PAF-AH fragments with PAF-AH Purified rPH.2 and rPH.9 have enzymatic activity indistinguishable from that of endogenous PAF-AH purified from serum, and rPH.2 and rPH.9 bind to lipropotein in a similar manner as purified endogenous PAF-AH.

Example 11 A preliminary analysis of expression patterns of human plasma PAF- AH mRNA in human tissues was conducted by Northern blot hybridization.

RNA was prepared from human cerebral cortex, heart, kidney, placenta, thymus and tonsil using RNA Stat 60 (Tel-Test Friendswood, TX).

Additionally, RNA was prepared from the human hematopoietic precursor-like cell line, THP-1 (ATCC TIB 202), which was induced to differentiate to a macrophagelike phenotype using the phorbol ester phorbolmyristylacetate (PMA). Tissue RNA and RNA prepared from the premyelocytic THP-1 cell line prior to and 1 to 3 days after induction were electrophoresed through a 1.2% agarose formaldehyde gel and subsequently transferred to a nitrocellulose membrane. The full length human plasma PAF-AH cDNA, sAH 406-3, was labelled by random priming and hybridized to the membrane under conditions identical to those described in Example 3 for library screening. Initial results indicate that the PAF-AH probe hybridized to a 1.8 kb band in the thymus, tonsil, and to a lesser extent, the placental RNA.

PAF is synthesized in the brain under normal physiological as well as pathophysiological conditions. Given the known pro-inflammatory and potential neurotoxic properties of the molecule, a mechanism for localization of PAF synthesis or for its rapid catabolism would be expected to be critical for the health of neural tissue. The presence of PAF acetylhydrolase in neural tissues is consistent with it playing such a protective role. Interestingly, both a bovine heterotrimeric intracellular PAF-AH [the cloning of which is described in Hattori et al., J. Biol.

WO 99/09147 PCT/US97/14212 -52- Chem., 269(37): 23150-23155 (1994)] and PAF-AH of the invention have been identified in the brain. To determine whether the two enzymes are expressed in similar or different compartments of the brain, the human homologue of the bovine brain intracellular PAF-AH cDNA was cloned, and its mRNA expression pattern in the brain was compared by Northern blotting to the mRNA expression pattern of the PAF-AH of the invention by essentially the same methods as described in the foregoing paragraph. The regions of the brain examined by Northern blotting were the cerebellum, medulla, spinal cord, putamen, amygdala, caudate nucleus, thalamus, and the occipital pole, frontal lobe and temporal lobe of the cerebral cortex. Message of both enzymes was detected in each of these tissues although the heterotrimeric intracellular form appeared in greater abundance than the secreted form. Northern blot analysis of additional tissues further revealed that the heterotrimeric intracellular form is expressed in a broad variety of tissues and cells, including thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, macrophages, brain, liver, skeletal muscle, kidney, pancreas and adrenal gland. This ubiquitous expression suggests that the heterotrimeric intracellular PAF-AH has a general housekeeping function within cells.

The expression of PAF-AH RNA in monocytes isolated from human blood and during their spontaneous differentiation into macrophages in culture was also examined. Little or no RNA was detected in fresh monocytes, but expression was induced and maintained during differentiation into macrophages. There was a concomitant accumulation of PAF-AH activity in the culture medium of the differentiating cells. Expression of the human plasma PAF-AH transcript was also observed in the THP-1 cell RNA at 1 day but not 3 days following induction. THP-1 cells did not express mRNA for PAF-AH in the basal state.

Example 12 PAF-AH expression in human and mouse tissues was examined by in situ hybridization.

Human tissues were obtained from National Disease Research Interchange and the Cooperative Human Tissue Network. Normal mouse brain and spinal cord, and EAE stage 3 mouse spinal cords were harvested from S/JLJ mice.

WO 99/09147 PCT/US97/14212 -53- Normal S/JLJ mouse embryos were harvested from eleven to eighteen days after fertilization.

The tissue sections were placed in Tissue Tek II cryomolds (Miles Laboratories, Inc., Naperville, IL) with a small amount of OCT compound (Miles, Inc., Elkhart, IN). They were centered in the cryomold, the cryomold filled with OCT compound, then placed in a container with 2-methylbutane [C 2

H

5

CH(CH

3 2 Aldrich Chemical Company, Inc., Milwaukee, WI] and the container placed in liquid nitrogen. Once the tissue and OCT compound in the cryomold were frozen, the blocks were stored at -80 C until sectioning. The tissue blocks were sectioned at 6 /m thickness and adhered to Vectabond (Vector Laboratories, Inc., Burlingame, CA) coated slides and stored at -70 C and placed at 50 °C for approximately 5 minutes to warm them and remove condensation and were then fixed in 4% paraformaldehyde for 20 minutes at 4 C, dehydrated 95%, 100% ethanol) for 1 minute at 4°C in each grade, then allowed to air dry for 30 minutes at room temperature. Sections were denatured for 2 minutes at 70 0 C in 70% formamide/2X SSC, rinsed twice in 2X SSC, dehydrated and then air dried for 30 minutes. The tissues were hybridized in situ with radiolabeled single-stranded mRNA generated from DNA derived from an internal 1 Kb HindIm fragment of the PAF-AH gene (nucleotides 308 to 1323 of SEQ ID NO: 7) by in vitro RNA transcription incorporation 35S-UTP (Amersham) or from DNA derived from the heterotrimeric intracellular PAF-AH cDNA identified by Hattori et al. The probes were used at varying lengths from 250-500 bp.

Hybridization was carried out overnight (12-16 hours) at 500C; the 3 5 S-labeled riboprobes (6 x 105 cpm/section), tRNA (0.5 ag/section) and diethylpyrocarbonate (depc)-treated water were added to hybridization buffer to bring it a final concentration of 50% formamide, 0.3M NaCI, 20 mM Tris pH 7.5, 10% dextran sulfate, 1X Denhardt's solution, 100 mM dithiothretol (DTT) and 5 mM EDTA.

After hybridization, sections were washed for 1 hour at room temperature in 4X mM DTT, then for 40 minutes at 60 C in 50% formamide/IX SSC/10 mM DTT, 30 minutes at room temperature in 2X SSC, and 30 minutes at room temperature in 0.1X SSC. The sections were dehydrated, air dried for 2 hours, coated with Kodak NTB2 photographic emulsion, air dried for 2 hours, developed WO 99/09147 PCT/US97/14212 -54- (after storage at 4"C in complete darkness) and counterstained with hematoxylin/eosin.

A. Brain Cerebellum. In both the mouse and the human brains, strong signal was seen in the Purkinje cell layer of the cerebellum, in basket cells, and individual neuronal cell bodies in the dentate nucleus (one of the four deep nuclei in the cerebellum). Message for the heterotrimeric intracellular PAF-AH was also observed in these cell types. Additionally, plasma PAF-AH signal was seen on individual cells in the granular and molecular layers of the grey matter.

Hippocampus. In the human hippocampus section, individual cells throughout the section, which appear to be neuronal cell bodies, showed strong signal. These were identified as polymorphic cell bodies and granule cells. Message for the heterotrimeric intracellular PAF-AH was also observed in hippocampus.

Brain stem. On both human and mouse brain stem sections, there was strong signal on individual cells in the grey matter.

Cortex. On human cortex sections taken from the cerebral, occipital, and temporal cortexes, and on mouse whole brain sections, individual cells throughout the cortex showed strong signal. These cells were identified as pyramidal, stellate and polymorphic cell bodies. There does not appear to be differentiation in the expression pattern in the different layers of the cortex. These in situ hybridization results are different from the results for cerebral cortex obtained by Northern blotting.

The difference is likely to result from the greater sensitivity of in situ hybridization compared to that of Northern blotting. As in the cerebellum and hippocampus, a similar pattern of expression of the heterotrimeric intracellular PAF-AH was observed.

Pituitary. Somewhat weak signal was seen on scattered individual cells in the pars distalis of the human tissue section.

B. Human colon Both normal and Crohn's disease colons displayed signal in the lymphatic aggregations present in the mucosa of the sections, with the level of signal WO 99/09147 PCT/US97/14212 being slightly higher in the section from the Crohn's disease patient. The Crohn's disease colon also had strong signal in the lamina propria. Similarly, a high level of signal was observed in a diseased appendix section while the normal appendix exhibited a lower but still detectable signal. The sections from the ulcerative colitis patient showed no evident signal in either the lymphatic aggregations or the lamina propria.

C. Human tonsil and thymus Strong signal was seen on scattered groups of individual cells within the germinal centers of the tonsil and within the thymus.

D. Human lymph node Strong signal was observed on the lymph node section taken from a normal donor, while somewhat weak signal was observed in the lymph nodules of the section from a donor with septic shock.

E. Human small intestine Both normal and Crohn's disease small intestine had weak signal in the Peyer's patches and lamina propria in the sections, with the signal on the diseased tissue slightly higher.

F. Human spleen and lung Signal was not observed on any of the spleen (normal and splenic abcess sections) or lung (normal and emphysema sections) tissues.

G. Mouse spinal cord In both the normal and EAE stage 3 spinal cords, there was strong signal in the grey matter of the spinal cord, with the expression being slightly higher in the EAE stage 3 spinal cord. In the EAE stage 3 spinal cord, cells in the white matter and perivascular cuffs, probably infiltrating macrophages and/or other leukocytes, showed signal which was absent in the normal spinal cord.

WO 99/09147 PCT/US97/14212 -56- F. Mouse embryos In the day 11 embryo signal was apparent in the central nervous system in the fourth ventricle, which remained constant throughout the embryo time course as it developed into the cerebellum and brain stem. As the embryos matured, signal became apparent in central nervous system in the spinal cord (day 12), primary cortex and ganglion Gasseri (day 14), and hypophysis (day 16). Signal was observed in the peripheral nervous system (beginning on day 14 or 15) on nerves leaving the spinal cord, and, on day 17, strong signal appeared around the whiskers of the embryo.

Expression was also seen in the liver and lung at day 14, the gut (beginning on day 15), and in the posterior portion of the mouth/throat (beginning on day 16). By day 18, the expression pattern had differentiated into signal in the cortex, hindbrain (cerebellum and brain stem), nerves leaving the lumbar region of the spinal cord, the posterior portion of the mouth/throat, the liver, the kidney, and possible weak signal in the lung and gut.

G. Summary PAF-AH mRNA expression in the tonsil, thymus, lymph node, Peyer's patches, appendix, and colon lymphatic aggregates is consistent with the conclusions that the probable predominant in vivo source of PAF-AH is the macrophage because these tisues all are populated with tissue macrophages that serve as phagocytic and antigen-processing cells.

Expression of PAF-AH in inflamed tissues would be consistent with the hypothesis that a role of monocyte-derived macrophages is to resolve inflammation. PAF-AH would be expected to inactivate PAF and the proinflammatory phospholipids, thus down-regulating the inflammatory cascade of events initiated by these mediators.

PAF has been detected in whole brain tissue and is secreted by rat cerebellar granule cells in culture, In vitro and in vivo experiments have demonstrated that PAF binds a specific receptor in neural tissues and induces functional and phenotypic changes such as calcium mobilization, upregulation of transcription activating genes, and differentiation of the neural precursor cell line, PC12. These observations suggested a physiologic role for PAF in the brain, and WO 99/09147 PCT/US97/14212 -57consistent with this, recent experiments using hippocampal tissue section cultures and PAF analogs and antagonists have implicated PAF as an important retrograde messenger in hippocampal long term potentiation. Therefore, in addition to its pathological effect in inflammation, PAF appears to participate in routine neuronal signalling processes. Expression of the extracellular PAF-AH in the brain may serve to regulate the duration and magnitude of PAF-mediated signalling.

Example 13 Monoclonal antibodies specific for recombinant human plasma PAF-AH were generated using E. coli produced PAF-AH as an immunogen.

Mouse #1342 was injected on day 0, day 19, and day 40 with recombinant PAF-AH. For the prefusion boost, the mouse was injected with the immunogen in PBS, four days later the mouse was sacrificed and its spleen removed sterilely and placed in 10ml serum free RPMI 1640. A single-cell suspension was formed by grinding the spleen between the frosted ends of two glass microscope slides submerged in serum free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 1g/ml streptomycin (RPMI) (Gibco, Canada). The cell suspension was filtered through sterile 70-mesh Nitex cell strainer (Becton Dickinson, Parsippany, New Jersey), and washed twice by centrifuging at 200 g for 5 minutes and resuspending the pellet in 20 ml serum free RPMI. Thymocytes taken from 3 naive Balb/c mice were prepared in a similar manner. NS-1 myeloma cells, kept in log phase in RPMI with 11% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion, were centrifuged at 200 g for 5 minutes, and the pellet was washed twice as described in the foregoing paragraph.

One x 108 spleen cells were combined with 2.0 x 107 NS-1 cells, centrifuged and the supernatant was aspirated. The cell pellet was dislodged by tapping the tube and 1 ml of 37 C PEG 1500 (50% in 75mM Hepes, pH (Boehringer Mannheim) was added with stirring over the course of 1 minute, followed by adding 7 ml of serum free RPMI over 7 minutes. An additional 8 ml RPMI was added and the cells were centrifuged at 200 g for 10 minutes. After discarding the supernatant, the pellet was resuspended in 200 ml RPMI containing WO 99/09147 PCT/US97/14212 -58- FBS, 100 gM sodium hypoxanthine, 0.4 uM aminopterin, 16 /M thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 x 106 thymocytes/ml and plated into 10 Coming flat bottom 96 well tissue culture plates (Coming, Coming New York).

On days 2, 4, and 6, after the fusion, 100 gl of medium was removed from the wells of the fusion plates and replaced with fresh medium. On day 8, the fusion was screened by ELISA, testing for the presence of mouse IgG binding to recombinant PAF-AH. Immulon 4 plates (Dynatech, Cambridge, MA) were coated for 2 hours at 37" C with 100 ng/well recombinant PAF-AH diluted in 25mM TRIS, pH 7.5. The coating solution was aspirated and 200ul/well of blocking solution fish skin gelatin (Sigma) diluted in CMF-PBS] was added and incubated for minutes at 37 C. Plates were washed three times with PBS with 0.05% Tween (PBST) and 50 1 l culture supernatant was added. After incubation at 37' C for minutes, and washing as above, 50 Il of horseradish peroxidase conjugated goat antimouse IgG(fc) (Jackson ImmunoResearch, West Grove, Pennsylvania) diluted 1:3500 in PBST was added. Plates were incubated as above, washed four times with PBST and 100 gL substrate, consisting of 1 mg/ml o-phenylene diamine (Sigma) and 0.1 pl/ml 30% H 2 0 2 in 100 mM Citrate, pH 4.5, was added. The color reaction was stopped in 5 minutes with the addition of 50 g1 of 15 H 2

SO

4

A

4 9 0 was read onn a plate reader (Dynatech).

Selected fusion wells were cloned twice by dilution into 96 well plates and visually scoring the number of colonies/well after 5 days. Hybridomas cloned were 90D1E, 90E3A, 90E6C, 90G11D (ATCC HB 11724), and 90F2D (ATCC HB 11725).

The monoclonal antibodies produced by hybridomas were isotyped using the Isostrip system (Boehringer Mannheim, Indianapolis, IN). Results showed that the monoclonal antibodies produced by hybridomas from fusion 90 were all IgGi.

All of the monoclonal antibodies produced by hybridomas from fusion 90 functioned well in ELISA assays but were unable to bind PAF-AH on Western blots. To generate antibodies that could recognize PAF-AH by Western, mouse #1958 was immunized with recombinant enzyme. Hybridomas were generated as WO 99/09147 PCT/US97/14212 -59described for fusion 90 but were screened by Western blotting rather than ELISA to identify Western-competent clones.

For Western analyses, recombinant PAF-AH was mixed with an equal volume of sample buffer containing 125mM Tris, pH 6.8, 4% SDS, 100mM dithiothreitol and 0.05 bromphenol blue and boiled for five minutes prior to loading onto a 12% SDS polyacrylamide gel (Novex). Following electrophoresis at mAmps, proteins were electrotransferred onto a polyvinylidene fluoride membrane (Pierce) for 1 hour at 125 V in 192mM glycine, 25mM Tris base, 20% methanol, and 0.01% SDS. The membrane was incubated in 20mM Tris, 100mM NaCI (TBS) containing 5% bovine serum albumin (BSA, Sigma) overnight at 4 C. The blot was incubated 1 hour at room temperature with rabbit polyclonal antisera diluted 1/8000 in TBS containing 5 BSA, and then washed with TBS and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG in TBS containing 5 BSA for 1 hour at room temperature. The blot was again washed with TBS then incubated with 0.02% 5-bromo-4-chloro-3-indolyl phosphate and 0.03% nitroblue tetrazolium in 100mM Tris-HC1, pH 9.5, 100mM NaCI, and 5mM MgCl 2 The reaction was stopped with repeated water rinses.

Selected fusion wells, the supernatants of which were positive in Western analyses, were processed as described above. Hybridoma 143A reacted with PAF-AH in Western blots and was cloned (ATCC HB 11900).

Polyclonal antisera specific for human plasma PAF-AH was raised in rabbits by three monthly immunizations with 100 tAg of purified recombinant enzyme in Fruend's adjuvant.

Example 14 Experimental studies were performed to evaluate the in vivo therapeutic effects of recombinant PAF-AH of the invention on acute inflammation using a rat foot edema model [Henriques et al., Br. J. Pharmacol., 106: 579-582 (1992)]. The results of these studies demonstrated that rPAF-AH blocks PAF-induced edema.

Parallel studies were done to compare the effectiveness of PAF-AH with two commercially available PAF antagonists.

WO 99/09147 PCTIS97/1 4212 A. Preparation of PAF-AH E. coli transformed with the PAF-AH expression vector puc trp AH were lysed in a microfluidizer, solids were centrifuged out and the cell supernatants were loaded onto a S-Sepharose column (Pharmacia). The column was washed extensively with buffer consisting of 50mM NaC1, 10mM CHAPS, 25mM MES and ImM EDTA, pH 5.5. PAF-AH was eluted by increasing the NaCI concentration of the buffer to IM. Affinity chromatography using a Blue Sepharose column (Pharmacia) was then used as an additional purification step. Prior to loading the PAF-AH preparation on the Blue Sepharose column, the sample was diluted 1:2 to reduce the NaCI concentration to 0.5M and the pH was adjusted to 7.5. After washing the Blue Sepharose column extensively with buffer consisting of 0.5M NaCI, tris, 10mM CHAPS and ImM EDTA, pH 7.5 the PAF-AH was eluted by increasing the NaCI concentration to Purity of PAF-AH isolated in this manner was generally 95% as assessed by SDS-PAGE with activity in the range of 5000-10,000 U/ml. Additional quality controls done on each PAF-AH preparation included determining endotoxin levels and hemolysis activity on freshly obtained rat erythrocytes. A buffer containing 25mM Tris, 10mM CHAPS, 0.5M NaCI, pH 7.5 functioned as storage media of the enzyme as well as carrier for administration. Dosages used in experiments were based on enzyme activity assays conducted immediately prior to experiments.

B. Induction of Edema Six to eight-week-old female Long Evans rats (Charles River, Wilmington, MA), weighing 180-200 grams, were used for all experiments. Prior to experimental manipulations, animals were anesthetized with a mixture of the anesthetics Ketaset (Fort Dodge Laboratories, Fort Dodge, IA), Rompun (Miles, Shawnee Mission, KS), and Ace Promazine (Aveco, Fort Dodge, IA) administered subcutaneously at approximately 2.5 mg Ketaset, 1.6 mg Rompun, 0.2 mg Ace Promazine per animal per dose. Edema was induced in the foot by administration of either PAF or zymosan as follows. PAF (Sigma #P-1402) was freshly prepared for each experiment from a 19. 1mM stock solution stored in chloroform/methanol (9:1) WO 99/09147 PCT/US97/14212 -61at -20* C. Required volumes were dried down under N 2 diluted 1:1000 in a buffer containing 150mM NaCI, 10mM Tris pH 7.5, and 0.25 BSA, and sonicated for five minutes. Animals received 50 p1 PAF (final dose of 0.96 nmoles) subcutaneously between the hind foot pads, and edema was assessed after 1 hour and again after 2 hours in some experiments. Zymosan A (Sigma #A-8800) was freshly prepared for each experiment as a suspension of 10 mg/ml in PBS. Animals received 50 l of zymosan (final dose of 500 tg) subcutaneously between the hind foot pads and edema was assessed after 2 hours.

Edema was quantitated by measuring the foot volume immediately prior to administration of PAF or zymosan and at indicated time point post-challenge with PAF or zymosan. Edema is expressed as the increase in foot volume in milliliters.

Volume displacement measurements were made on anesthetized animals using a plethysmometer (UGO Basile, model #7150) which measures the displaced water volume of the immersed foot. In order to insure that foot immersion was comparable from one time point to the next, the hind feet were marked in indelible ink where the hairline meets the heel. Repeated measurements of the same foot using this technique indicate the precision to be within 5 C. PAF-AH Administration Routes and Dosages PAF-AH was injected locally between the foot pads, or systematically by IV injection in the tail vein. For local administration rats received 100 1 l PAF- AH (4000-6000 U/ml) delivered subcutaneously between the right hind foot pads.

Left feet served as controls by administration of 100 p1 carrier (buffered salt solution). For systemic administration of PAF-AH, rats received the indicated units of PAF-AH in 300 1 l of carrier administered IV in the tail vein. Controls received the appropriate volume of carrier IV in the tail vein.

D. Local Administration of PAF-AH Rats were injected with 100 1 l of PAF-AH (4000-6000 U/ml) subcutaneously between the right foot pads. Left feet were injected with 100 p1 carrier (buffered salt solution). Four other rats were injected only with carrier. All rats were immediately challenged with PAF via subcutaneous foot injection and foot WO 99/09147 PCT/US97/14212 -62volumes assessed 1 hour post-challenge. FIGURE 6, wherein edema is expressed as average increase in foot volume (ml) SEM for each treatment group, illustrates that PAF-induced foot edema is blocked by local administration of PAF-AH. The group which received local PAF-AH treatment prior to PAF challenge showed reduced inflammation compared to the control injected group. An increase in foot volume of 0.08 ml 0.08 (SEM) was seen in the PAF-AH group as compared to 0.63 0.14 (SEM) for the carrier treated controls. The increase in foot volume was a direct result of PAF injection as animals injected in the foot only with carrier did not exhibit an increase in foot volume.

E. Intravenous Administration of PAF-AH Rats (N=4 per group) were pretreated IV with either PAF-AH (2000 U in 300 gl carrier) or carrier alone, 15 minutes prior to PAF challenge. Edema was assessed 1 and 2 hours after PAF challenge. FIGURE 7, wherein edema is expressed as average increase in volume (ml) SEM for each treatment group, illustrates that IV administration of PAF-AH blocked PAF induced foot edema at one and two hours post challenge. The group which received 2000 U of PAF-AH given by the IV route showed a reduction in inflammation over the two hour time course. Mean volume increase for the PAF-AH treated group at two hours was 0.10 ml 0.08 (SEM), versus 0.56 ml 0.11 for carrier treated controls.

F. Comparison of PAF-AH Protection in Edema Induced by PAF or Zymosan Rats (N=4 per group) were pretreated IV with either PAF-AH (2000 U in 300 gl carrier) or carrier alone. Fifteen minutes after pretreatment, groups received either PAF or zymosan A, and foot volume was assessed after 1 and 2 hours, respectively. As shown in FIGURE 8, wherein edema is expressed as average increase in volume (ml) SEM for each treatment group, systemic administration of PAF-AH (2000 U) was effective in reducing PAF-induced foot edema, but failed to block zymosan induced edema. A mean increase in volume of 0.08 0.02 was seen in the PAF-AH treated group versus 0.49 0.03 for the control group.

WO 99/09147 PCTIUS97/14212 -63- G. Effective Dose Titration of PAF-AH Protection In two separate experiments, groups of rats (N=3 to 4 per group) were pretreated IV with either serial dilutions of PAF-AH or carrier control in a 300 volume, 15 minutes prior to PAF challenge. Both feet were challenged with PAF (as described above) and edema was assessed after 1 hour. FIGURE 9 wherein edema is expressed as average increase in volume (ml) SEM for each treatment group, illustrates the increase in protection from PAF-induced edema in rats injected with increasing dosages of PAF-AH. In the experiments, the ID 5 0 of PAF-AH given by the IV route was found to be between 40 and 80 U per rat.

H. In Vivo Efficacy of PAF-AH as a Function of Time After Administration In two separate experiments, two groups of rats (N=3 to 4 per group) were pretreated IV with either PAF-AH (2000 U in 300 A1 carrier) or carrier alone.

After administration, groups received PAF at time points ranging from 15 minutes to 47 hours post PAF-AH administration. Edema was then assessed 1 hour after PAF challenge. As shown in FIGURE 10, wherein edema is expressed as average increase in volume (ml) SEM for each treatment group, administration of 2000 U of PAF- AH protects rats from PAF induced edema for at least 24 hours.

I. Pharmacokinetics of PAF-AH Four rats received 2000 U of PAF-AH by IV injection in a 300 il volume. Plasma was collected at various time points and stored at 4 C and plasma concentrations of PAF-AH were determined by ELISA using a double mAb capture assay. In brief, monoclonal antibody 90GllD (Example 13) was diluted in carbonate buffer pH 9.6 at 100 ng/ml and immobilized on Immulon 4 ELISA plates overnight at 4°C. After extensive washing with PBS containing 0.05% Tween the plates were blocked for 1 hour at room temperature with 0.5% fish skin gelatin (Sigma) diluted in PBS. Serum samples diluted in PBS with 15mM CHAPS were added in duplicate to the washed ELISA plate and incubated for 1 hour at room temperature. After washing, a biotin conjugate of monoclonal antibody 90F2D (Example 13) was added to the wells at a concentration of 5 /g/ml diluted in PBS and incubated for 1 hour at room temperature. After washing, 50 1 l of a 1:1000 dilution WO 99/09147 PCT/US97/14212 -64of ExtraAvidin (Sigma) was added to the wells and incubated for 1 hour at room temperature. After washing, wells were developed using OPD as a substrate and quantitated. Enzyme activity was then calculated from a standard curve. FIGURE 11, wherein data points represent means SEM, shows that at one hour plasma enzyme levels approached the predicted concentration based on a 5-6 ml plasma volume for 180-200 gram rats, mean 374 U/ml 58.2. Beyond one hour plasma levels steadily declined, reaching a mean plasma concentration of 19.3 U/ml 3.4 at 24 hours, which is still considerably higher than endogenous rat PAF-AH levels which have been found to be approximately 4 U/ml by enzymatic assays.

J. Effectiveness of PAF-AH Versus PAF Antagonists Groups of rats (N=4 per group) were pretreated with one of three potential antiinflammatories: the PAF antagonist CV3988 (Biomol #L-103) administered IP (2 mg in 200 jl EtOH), the PAF antagonist Alprazolam (Sigma #A- 8800) administered IP (2 mg in 200 Il EtOH), or PAF-AH (2000 U) administered IV. Control rats were injected IV with a 300 Cl volume of carrier. The PAF antagonists were administered IP because they are solubilized in ethanol. Rats injected with either CV3988 or Alprazolam were challenged with PAF 30 minutes after administration of the PAF antagonist to allow the PAF antagonist to enter circulation, while PAF-AH and carrier-treated rats were challenged 15 minutes after enzyme administration. Rats injected with PAF-AH exhibited a reduction in PAFinduced edema beyond that afforded by the established PAF antagonists CV3988 and Alprazolam. See FIGURE 12 wherein edema is expressed as average increase in volume (ml) SEM for each treatment group.

In summary, rPAF-AH is effective in blocking edema mediated by PAF in vivo. Administration of PAF-AH products can be either local or systemic by IV injection. In dosing studies, IV injections in the range of 160-2000 U/rat were found to dramatically reduce PAF mediated inflammation, while the ID 5 0 dosage appears to be in the range of 40-80 U/rat. Calculations based on the plasma volume for 180-200 gram rats predicts that a plasma concentration in the range of 25-40 U/ml should block PAF-elicited edema. These predictions are supported by preliminary pharmacokinetic studies. A dosage of 2000 U of PAF-AH was found to be effective WO 99/09147 PCT/US97/14212 in blocking PAF mediated edema for at least 24 hours. At 24 hours following administration of PAF-AH plasma concentrations of the enzyme were found to be approximately 25 U/ml. PAF-AH was found to block PAF-induced edema more effectively than the two known PAF antagonists tested.

Collectively, these results demonstrate that PAF-AH effectively blocks PAF induced inflammation and may be of therapeutic value in diseases where PAF is the primary mediator.

Example Recombinant PAF-AH of the invention was tested in a second in vivo model, PAF-induced pleurisy. PAF has previously been shown to induce vascular leakage when introduced into the pleural space [Henriques et al., supra]. Female rats (Charles River, 180-200 g) were injected in the tail vein with 200 /l of 1% Evans blue dye in 0.9% with 300 /l recombinant PAF-AH (1500 Amol/ml/hour, prepared as described in Example 14) or with an equivalent volume of control buffer. Fifteen minutes later the rats received an 100 1 injection of PAF (2.0 nmol) into the pleural space. One hour following PAF challenge, rats were sacrificed and the pleural fluid was collected by rinsing the cavity with 3 ml heparinized phosphate buffered saline.

The degree of vascular leak was determined by the quantity of Evans blue dye in the pleural space which was quantitated by absorbance at 620 nm. Rats pretreated with PAF-AH were found to have much less vascular leakage than control animals (representing more than an 80% reduction in inflammation).

The foregoing results support the treatment of subjects suffering from pleurisy with recombinant PAF-AH enzyme of the invention.

1- Example 16 Recombinant PAF-AH enzyme of the invention was also tested for efficacy in a model of antigen-induced eosinophil recruitment. The accumulation of eosinophils in the airway is a characteristic feature of late phase immune responses which occur in asthma, rhinitis and eczema. BALB/c mice (Charles River) were sensitized by two intraperitoneal injections consisting of 1 Ig of ovalbumin (OVA) in 4 mg of aluminum hydroxide (Imject alum, Pierce Laboratories, Rockford, IL) WO 99/09147 PCT/US97/14212 -66given at a 2 week interval. Fourteen days following the second immunization, the sensitized mice were challenged with either aerosolized OVA or saline as a control.

Prior to challenge mice were randomly placed into four groups, with four mice/group. Mice in groups 1 and 3 were pretreated with 140 A1 of control buffer consisting of 25mM tris, 0.5M NaCI, ImM EDTA and 0.1% Tween 80 given by intravenous injection. Mice in groups 2 and 4 were pretreated with 750 units of PAF-AH (activity of 5,500 units/ml given in 140 Al of PAF-AH buffer). Thirty minutes following administration of PAF-AH or buffer, mice in groups 1 and 2 were exposed to aerosolized PBS as described below, while mice in groups 3 and 4 were exposed to aerosolized OVA. Twenty-four hours later mice were treated a second time with either 140 /l of buffer (groups 1 and 3) or 750 units of PAF-AH in 140 il of buffer (groups 2 and 4) given by intravenous injection.

Eosinophil infiltration of the trachea was induced in the sensitized mice by exposing the animals to aerosolized OVA. Sensitized mice were placed in 50 ml conical centrifuge tubes (Corning) and forced to breath aerosolized OVA (50 mg/ml) dissolved in 0.9% saline for 20 minutes using a nebulizer (Model 646, DeVilbiss Corp., Somerset, PA). Control mice were treated in a similar manner with the exception that 0.9% saline was used in the nebulizer. Forty-eight hours following the exposure to aerosolized OVA or saline, mice were sacrificed and the tracheas were excised. Tracheas from each group were inbeded in OCT and stored at -70° until sections were cut.

To evaluate eosinophil infiltration of the trachea, tissue sections from the four groups of mice were stained with either Luna solution and hematoxylin-eosin solution or with peroxidase. Twelve 6 im thick sections were cut from each group of mice and numbered accordingly. Odd numbered sections were stained with Luna stain as follows. Sections were fixed in formal-alcohol for 5 minutes at room temperature, rinsed across three changes of tap water for 2 minutes at room temperature then rinsed in two changed of dH20 for 1 minute at room temperature.

Tissue sections were stained with Luna stain 5 minutes at room temperature (Luna stain consisting of 90 ml Weigert's Iron hematoxylin and 10 ml of 1% Biebrich Scarlet). Stained slides were dipped in 1% acid alcohol six times, rinsed in tap water for 1 minute at room temperature, dipped in 0.5% lithium carbonate solution five WO 99/09147 PCT/US97/14212 -67times and rinsed in running tap water for 2 minutes at room temperature. Slides were dehydrated across 70%-95 %-100% ethanol 1 minute each, at room temperature, then cleared in two changes of xylene for 1 minute at room temperature and mounted in Cytoseal For the peroxidase stain, even numbered sections were fixed in 4°C acetone for 10 minutes and allowed to air dry. Two hundred 1l of DAB solution was added to each section and allowed to sit 5 minutes at room temperature. Slides were rinsed in tap water for 5 minutes at room temperature and 2 drops of 1% osmic acid was applied to each section for 3-5 seconds. Slides were rinsed in tap water for minutes at room temperature and counterstained with Mayers hematoxylin at 25 C at room temperature. Slides were then rinsed in running tap water for 5 minutes and dehydrated across 70%-95%-100% ethanol 1 minute each at room temperature.

Slides were cleared through two changes of xylene for 1 minute each at room temperature and mounted in Cytoseal The number of eosinophils in the submucosal tissue of the trachea was evaluated. Trachea from mice from groups 1 and 2 were found to have very few eosinophils scattered throughout the submucosal tissue. As expected tracheas from mice in group 3, which were pretreated with buffer and exposed to nebulized OVA, were found to have large numbers of eosinophils throughout the submucosal tissue.

In contrast, the tracheas from mice in group 4, which were pretreated with PAF-AH and exposed to nebulized OVA were found to have very few eosinophils in the submucosal tissue comparable to what was seen in the two control groups, groups 1 and 2.

Thus, therapeutic treatment with PAF-AH of subjects exhibiting a late phase immune response involving the accumulation of eosinophils in the airway, such as that which occurs in asthma and rhinitis is indicated.

Example 17 A PAF-AH product of the invention was also tested in two different rat models for treatment of necrotizing enterocolitis (NEC), an acute hemorrhagic necrosis of the bowel which occurs in low birth weight infants and causes a significant morbidity and mortality. Previous experiments have demonstrated that WO 99/09147 PCT/US97/14212 -68treatment with glucocorticoids decreases the incidence of NEC in animals and in premature infants, and the activity of glucocorticoids has been suggested to occur via an increase in the activity of plasma PAF-AH.

A. Activity in Rats With NEC Induced by PAF Challenge 1. Prevention of NEC A recombinant PAF-AH product, rPH.2 (25,500 units in 0.3 ml, groups 2 and or vehicle/buffer alone (25mM tris, 0.5M NaCI, ImM EDTA and 0.1% Tween 80) (groups 1 and 3) was administered into the tail veins of female Wistar rats weighing 180-220 grams. Either BSA (0.25%)-saline (groups 1 and 2) or PAF (0.2 ixg/100 gm) suspended in BSA saline (groups 3 and 4) was injected into the abdominal aorta at the level of the superior mesenteric artery minutes after rPH.2 or vehicle injection as previously described by Furukawa, et al.

[J.Pediatr.Res. 34:237-241 (1993)]. The small intestines were removed after 2 hours from the ligament of Trietz to the cecum, thoroughly washed with cold saline and examined grossly. Samples were obtained from microscopic examination from the upper, middle and lower portions of the small intestine. The tissues were fixed in buffered formalin and the sample processed for microscopic examination by staining with hematoxylin and eosin. The experiment was repeated three times.

Gross findings indicated a normal appearing bowel in groups treated with the vehicle of BSA saline. Similarly, rPH.2 injected in the absence of PAF had no effect on the gross findings. In contrast, the injection of PAF into the descending aorta resulted in rapid, severe discoloration and hemorrhage of the serosal surface of the bowel. A similar hemorrhage was noted when a section of the small bowel was examined on the mucosal side and the intestine appeared to be quite necrotic. When rPH.2 was injected via the tail vein 15 minutes prior to the administration of PAF into the aorta, the bowel appeared to be normal.

Upon microscopic examination, the intestine obtained from groups 1, 2 and 4 demonstrated a normal villous architecture and a normal population of cells within the lamina propria. In contrast, the group treated with PAF alone showed a full thickness necrosis and hemorrhage throughout the entire mucosa.

WO 99/09147 PCT/US97/14212 -69- The plasma PAF-AH activities were also determined in the rats utilized in the experiment described above. PAF-AH activity was determined as follows.

Prior to the tail vein injection, blood samples were obtained. Subsequently blood samples were obtained from the vena cava just prior to the injection of PAF and at the time of sacrifice. Approximately 50 C 1 of blood was collected in heparinized capillary tubes. The plasma was obtained following centrifugation (980 x g for minutes). The enzyme was assayed as previously described by Yasuda and Johnston, Endocrinology, 130:708-716 (1992).

The mean plasma PAF-AH activity of all rats prior to injection was found to be 75.5 2.5 units (1 unit equals 1 nmoles x min 1 x ml- 1 plasma). The mean plasma PAF-AH activities 15 minutes following the injection of the vehicle were 75.2 2.6 units for group 1 and 76.7 3.5 units for group 3. After minutes, the plasma PAF-AH activity of the animals injected with 25,500 units rPH.2 was 2249 341 units for group 2 and 2494 623 units for group 4. The activity of groups 2 and 4 remained elevated (1855 257 units) until the time of sacrifice (2 1/4 hours after rPH.2 injection) (Group 2 1771 308; Group 4 1939 478). These results indicate that plasma PAF-AH activity of the rats which were injected with the vehicle alone (groups 1 and 3) did not change during the course of the experiment. All the animals receiving the PAF injection alone developed NEC while all rats that were injected with rPH.2 followed by PAF injection were completely protected.

2. Dose-Dependency of Prevention of NEC In order to determine if the protection against NEC in rats was dose dependent, animals were treated with increasing doses of rPH.2 15 minutes prior to PAF administration. Initially, rPH.2, ranging from 25.5 to 25,500 units were administered into the tail vein of rats. PAF (0.4 Ig in 0.2 ml of BSA-saline) was subsequently injected into the abdominal aorta 15 minutes after the administration of rPH.2. The small intestine was removed and examined for NEC development 2 hours after PAF administration. Plasma PAF-AH activity was determined prior to the exogenous administration of the enzyme, and 15 minutes and 2 1/4 hours after rPH.2 administration. The results are the mean of 2-5 animals in each group.

WO 99/09147 PCT/US97/14212 Gross findings indicated that all rats receiving less than 2,000 units of the enzyme developed NEC. Plasma PAF-AH activity in animals receiving the lowest protective amount of enzyme (2040 units) was 363 units per ml of plasma after minutes, representing a five-fold increase over basal levels. When rPH.2 was administered at less than 1,020 total units, resultant plasma enzyme activity averaged approximately 160 or less, and all animals developed NEC.

3. Duration of Protection Against NEC In order to determine the length of time exogenous PAF-AH product afforded protection against development of NEC, rats were injected once with a fixed amount of the enzyme via the tail vein and subsequently challenged with PAF at various time points. rPH.2 (8,500 units in 0.3 ml) or vehicle alone was administered into the tail vein of rats, and PAF (0.36 Ig in 0.2 ml of BSA-saline) was injected into the abdominal aorta at various times after the enzyme administration. The small intestines were removed 2 hours after the PAF injection for gross and histological examinations in order to evaluate for NEC development. Plasma PAF-AH activities were determined at various times after enzyme administration and two hours after PAF administration. The mean value standard error for enzyme activity was determined for each group.

Results indicated that none of the rats developed NEC within the first eight hours after injection of rPH.2, however 100% of the animals challenged with PAF at 24 and 48 hours following injection of the enzyme developed NEC.

4. Reversal of NEC In order to determine if administration of PAF-AH product was capable of reversing development of NEC induced by PAF injection, 25,500 units of enzyme was administered via injection into the vena cava two minutes following PAF administration (0.4 Ag). None of the animals developed NEC. However, when rPH.2 was administered via this route 15 minutes after the PAF injection, all animals developed NEC, consistent with the rapid time course of NEC development as induced by the administration of PAF previously reported Furukawa et al. [supra].

WO 99/09147 PCT/US97/14212 -71- The sum of these observations indicate that a relatively small (five-fold) increase in the plasma PAF-AH activity is capable of preventing NEC. These observations combined with previous reports that plasma PAF-AH activity in fetal rabbits [Maki, et al., Proc.Natl.Acad.Sci. (USA) 85:728-732 (1988)] and premature infants [Caplan, et al., J.Pediatr. 116:908-964 (1990)] has been demonstrated to be relatively low suggests that prophylactic administration of human recombinant PAF- AH products to low birth weight infants may be useful in treatment of NEC.

B. Activity in a Neonatal Model of NEC The efficacy of a PAF-AH product, rPH.2, was evaluated as follows in an NEC model in which newborn rats are stressed by formula feeding and asphyxia, two common risk factors for the disease in humans. In this model, approximately 70-80% of the animals develop gross and microscopic intestinal injury similar to neonatal NEC by the third day of life. Newborn rats were obtained from pregnant Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) that were anesthetized with CO 2 and delivered via abdominal incision. Newborn animals were collected, dried, and maintained in a neonatal incubator during the entire experiment.

First, separate groups of animals were used to assess the dosing and absorption characteristics of rPH.2. Normal newborn rat pups were given one of three different enteral or intraperitoneal doses of rPH.2 (3X, 15X, or 75X) at time 0, and blood was collected at 1 hour, 6 hours, or 24 hours later for assessment of plasma PAF-AH activity. PAF-AH activity was measured using a substrate incubation assay [Gray et al., Nature, 374:549 (1995)] and an ELISA utilizing an anti-human rPAF-AH monoclonal antibody for each sample (90F2D and 90G11D, described in Example 13). For selected samples, immunohistochemical analysis was performed using two different monoclonal antibodies developed against human rPAF- AH (90F2D and 90G11D, described in Example 13). Immunohistochemistry was done with standard techniques using a 1:100 dilution of the antibody and overnight incubations.

Following enteral dosing of rPH.2 in normal newborn rats, there was no measurable plasma PAF-AH activity at any time point using either the substrate incubation assay or the ELISA technique. With intraperitoneal administration of WO 99/09147 PCT/US97/14212 -72rPH.2, significant circulating PAF-AH activity was measurable using both methods by 1 hour after dosing, and this activity peaked at 6 hours. Higher doses of rPH.2 (from 3 to 75X, 10 to 250 U) resulted in higher plasma PAF-AH activity.

Immunohistochemical analysis revealed the presence of rPAF-AH product in the epithelial cells of the intestinal mucosa following enteral administration. The reactivity clustered mostly in the intestinal villi with minimal staining present in the crypt cells. There was more staining in the ileum than jejunum, and some rPAF-AH product was immunochemically identified in portions of colon. There was no demonstrable staining in any control samples or in specimens recovered from animals dosed via the intraperitoneal route. Thus, enteral administration of rPAF-AH product resulted in local mucosal epithelial accumulation of the enzyme without any measurable systemic absorption, while, in contrast, intraperitoneal administration of rPAF-AH product resulted in high circulating enzyme levels but no local mucosal accumulation.

In the NEC model, NEC was induced in newborn rats according to Caplan et al., Pediatr. Pathol., 14:1017-1028 (1994). Briefly, animals were fed with newborn puppy formula reconstituted from powder (Esbiliac, Borden Inc) every three hours via a feeding tube. The feeding volume began at 0.1 ml/feed initially and advanced as tolerated to 0.4 ml/feed by the 4th day of the protocol. All animals were challenged with asphyxial insults twice daily by breathing 100% nitrogen for seconds in a closed plastic chamber followed by exposure to cold (40C) for minutes. Bowel and bladder function was stimulated with gentle manipulation after every feeding. Animals were maintained for 96 hours or until they showed signs of distress. Morbid animals had abdominal distention, bloody stools, respiratory distress, cyanosis, and lethargy, and were euthanized via decapitation. After sacrifice, the intestine of each rat was examined grossly for signs of necrosis and then formalin-fixed for later histological analysis. Specimens were paraffin-embedded, sectioned with a microtome, stained with hematoxylin and eosin, and examined in a blinded fashion by two observers. Intestinal injury was scored as 1+ for epithelial cell lifting or separation, 2 for sloughing of epithelial cells to mid villous level, 3 for necrosis of entire villi, and 4+ for transmural necrosis.

WO 99/09147 PCT/US97/1 4212 -73- To assess the efficacy of rPH.2, three different groups of rats were treated with the compound via enteral delivery, intraperitoneal delivery or both. The rPH.2 preparation had 0.8 mg/ml protein and approximately 4000 Units/mg PAF-AH activity, with a <0.5 EU/mg endotoxin/protein ratio. Enterally dosed animals were given 25X (80 U) of rPH.2 via the orogastric tube diluted into each feeding (every three hours). Intraperitoneally dosed animals were given 75X by intraperitoneal injection twice daily. Control animals received appropriate volumes of buffer mM NaPO 4 pH 7.4) without the rPH.2 and were studied simultaneously with each experimental group. Mortality and signs of NEC were evaluated for each treatment group, and differences were analyzed statistically using Fischer's Exact test. A pvalue of <0.05 was considered significant. Results are shown in Table 9 below.

Table 9 NEC Death Control admin.) 7/10 8/10 rPH.2 (240 U i.p. twice daily) 6/11 8/11 Control (enteral admin.) 19/26 21/26 rPH.2 (80 U enterally every 3 hours) 6/26 7/26 Control +enteral admin.) 10/17 12/17 rPH.2 (240 U i.p. twice daily and 3/14 7/14 U enterally every 3 hours) Data represent cumulative results from four different experiments for i.p. dosing, four experiments for enteral dosing, and three experiments for i.p. +enteral dosing.

Enteral rPH.2 administration significantly reduced the incidence of both NEC and death compared to control animals. Results from four different enterallydosed experiments showed that pretreatment with rPH.2 decreased NEC from 19/26 (control) to 6/26 (p <0.001). Intestinal injury was variable among treated and control animals, but in most cases was characterized by midvillous necrosis in some segments, total villous necrosis in other areas, occasional areas of transmural necrosis, and remaining portions of normal intestinal histology. The worst degree of NEC in treated animals and control animals with intestinal injury was similar (median score 2.8 in controls vs. 2.4 in rPH.2-treated rats, p 0.05).

WO 99/09147 PCT/US97/14212 -74- Intraperitoneal dosing with rPH.2 had no significant impact on NEC or death in this model. The onset of symptoms was similar between this group and controls (40 5 hours in controls vs 36 7 hours in rPH.2-treated rats) and the degree of NEC in both groups was similar (median score 2.6 in controls vs. 2.5 in rPH.2-treated rats).

Additional experiments were done in which rats were dosed both enterally and intraperitoneally with rPH.2 at the same doses as the single treatment groups (25X of rPH.2 in each feeding every three hours, plus 75X by intraperitoneal injection twice daily). Results are shown above in Table 9. Although there were no significant differences between treated and control groups in the incidence of death, the rPH.2 treatment significantly reduced the incidence of NEC (10/17 in controls vs.

3/14 in rPH.2-treated rats, p 0.04). Of note, 6 out of the 7 animals who died in the rPH.2-treated group had positive blood cultures for E. coli obtained just prior to death.

These results further support the protective role of PAF-AH products in a neonatal model of non-PAF-induced NEC. Enteral treatment with rPAF-AH product prevented NEC while intraperitoneal treatment at these doses had no demonstrable effect. These findings suggest that PAF-AH product supplementation for formula-fed premature newborns at risk for NEC may reduce the incidence of this disease.

Example 18 The efficacy of PAF-AH product in a guinea pig model of acute respiratory distress syndrome (ARDS) was examined.

Platelet-activating factor (PAF) injected intravenously into guinea pigs produces a profound lung inflammation reminiscent of early ARDS in humans.

Within minutes after intravenous administration of PAF, the lung parenchyma becomes congested with constricted bronchi and bronchioles [Lellouch-Tubiana et al., supra. Platelets and polymorphonuclear neutrophils begin to marginate and cellular aggregates are easily identified along arterioles of the lung [Lellouch-Tubiana, Br. J.

Exp Path., 66:345-355 (1985)]. PAF infusion also damages bronchial epithelial cells which dissociate from the airway walls and accumulate in the airway lumens. This WO 99/09147 PCT/US97/14212 damage to airway epithelial cells is consistent with hyaline membrane formation that occurs in humans during the development of ARDS. Margination of the neutrophils and platelets is quickly followed by diapedesis of these cells into the alveolar septa and alveolar spaces of the lung. Cellular infiltrates elicited by PAF are accompanied by significant vascular leakage resulting in airway edema [Kirsch, Exp. Lung Res., 18:447-459 (1992)]. Evidence of edema is further supported by in vitro studies where PAF induces a dose-dependent (10-1000 ng/ml) extravasation of 125I labeled fibrinogen in perfused guinea pig lungs [Basran, Br. J. Pharmacol., 77:437 (1982)].

Based on the above observations, an ARDS model in guinea pigs was developed. A cannula is placed into the jugular vein of anaesthetized male Hartly guinea pigs (approximately 350-400 grams) and PAF diluted in a 500 zl volume of phosphate buffered saline with 0.25 bovine serum albumin as a carrier (PBS-BSA) is infused over a 15 minute period of time at a total dosage ranging from 100-400 ng/kg. At various intervals following PAF infusion, animals are sacrificed and lung tissue is collected. In guinea pigs infused with PAF, dose dependent lung damage and inflammation is clearly evident by 15 minutes and continues to be present at minutes. Neutrophils and red blood cells are present in the alveolar spaces of PAF treated guinea pigs but absent in control or sham infused animals. Evidence of epithelial cell damage is also evident and reminiscent of hyaline membrane formation in human ARDS patients. Protein determinations done on bronchoalveolar lavage (BAL) samples taken from guinea pigs infused with PAF shows a dramatic accumulation of protein in the inflamed lung, clear evidence of vascular leakage.

rPH.2 was found to completely protect against PAF mediated lung injury in the guinea pig model of ARDS. Groups of guinea pigs were pretreated with either rPH.2 (2000 units in 500 pl) or 500 1l of the PAF-AH buffer only. Fifteen minutes later these guinea pigs were infused with 400 ng/kg PAF in a 500 $l volume, infused over a 15 minute period. In addition, a sham group of guinea pigs was infused with 500 /l of PBS-BSA. At the completion of the PAF infusion the animals were sacrificed and BAL fluid was collected by lavaging the lungs 2X with 10ml of saline containing 2 A/ml heparin to prevent clotting. To determine protein concentration in the BAL, samples were diluted 1:10 in saline and the OD 280 was determined. BAL fluid from sham guinea pigs was found to have a protein WO 99/09147 PCT/US97/14212 -76concentration of 2.10 1.3 mg/ml. In sharp contrast, BAL fluid from animals infused with PAF was found to have a protein concentration of 12.55 1.65 mg/ml.

In guinea pigs pretreated with rPH.2, BAL fluid was found to have a protein concentration of 1.13 0.25 mg/ml which is comparable to the sham controls and demonstrates that PAF-AH product completely blocks lung edema in response to

PAF.

Example 19 The efficacy of a PAF-AH product, rPH.2, was evaluated in two different models of acute pancreatitis.

A. Activity in a Rat Pancreatitis Model Male Wistar rats (200-250 g) were purchased from Charles River Laboratories (Wilmington, MA). They were housed in a climate controlled room at 23±2 °C with a 12 hour light/dark cycle and fed standard laboratory chow with water ad libitum. Animals were randomly assigned to either control or experimental groups. Rats were anesthetized with 50 mg/kg pentobarbital sodium intraperitoneally, and a polyvinyl catheter (size V3, Biolab products, Lake Havasu, AZ) was placed by cutdown into the jugular vein. The catheter was tunneled subcutaneously to exit in the dorsal cervical area, and the animals were allowed to recover from anesthesia.

The rats were given free access to water but were fasted overnight. Experiments were performed the next day on conscious animals. During the interim, catheter patency was maintained by constant infusion of saline (0.2 ml/h). On the day of the experiment, the animals were intravenously injected with rPH.2 or vehicle control, followed by an infusion of either 5 L/g/kg per hour of caerulein for 3.5 hours, or 10 /tg/kg per hour of caerulein for 5 hours, (Research Plus, Bayonne, NJ).

Immediately after completion of the infusion, the animals were anesthetized with pentobarbital sodium, their abdomens were opened, and 5 ml of blood aspirated from the inferior vena cava for subsequent assays. They were then sacrificed by exsanguination. Serum amylase, serum lipase and serum bilirubin were measured, and the pancreas was harvested. Pieces of pancreas were either fixed in a 4% phosphate buffered formaldehyde solution for histological examination or immediately WO 99/09147 PCT/US97/14212 -77deep frozen at -80 0 C for measurements of myeloperoxidase activity. Additional pieces of pancreas were assessed for pancreatic water content and pancreatic amylase and trypsin as described below. Myeloperoxidase activity, a measure of neutrophil sequestration, was assessed in the pancreas and lung as described below. Pulmonary vascular permeability was also assessed as described below. Statistical analysis of the data was accomplished using unpaired Student's t-test. The data reported represent means S.E.M. of at least three different experiments. Differences in the results were considered significant when p<0.05.

1. Pancreatic water content Pancreas pieces were blotted dry and weighed (wet weight), and were then desiccated for 34 hrs at 1200C and reweighed (dry weight). Pancreatic water content was calculated as the difference between wet and dry weight and expressed as a percentage of the pancreatic wet weight. A rise in pancreatic water content was considered to indicate the development of edema.

2. Serum and Pancreatic Amylase Amylase activity in serum was measured using 4,6-ethylidene (G 7 nitrophenyl (G 1 )-cqD-maltoplaside (ET-G 7 PNP) (Sigma Chemical Co., St. Louis, MO) as substrate according to Pierre et al., Clin. Chem., 22:1219 (1976). Amylase activity in pancreatic tissue homogenized in 10 mM phosphate buffer, pH 7.4, was measured using the same method.

3. Pancreatic Trypsin Trypsin activity was measured fluorimetrically using Boc-Gin-Ala-Arg- MCA as the substrate. Briefly, 200 /l of the sample and 2.7ml of 50 mM Tris-buffer (pH 8.0) containing 150 mM NaCI, ImM CaCl 2 and 0.1% bovine serum albumin were mixed in a cuvette. One hundred /l of substrate was added to the sample after seconds of preincubation to start the reaction. The fluorescence reading was taken (excitation 380 nm, emission 440 nm) and expressed as slope. To allow pooling of data from different experiments trypsin activity in the fractions was expressed as percent of total trypsin activity.

WO 99/09147 PCT/US97/14212 -78- 4. Histology and Morphometry For light microscopy, complete random cross-sections of the head, body and tail of the pancreas were fixed in 10% neutral phosphate-buffered formalin.

Paraffin embedded-5 um sections were stained with hematoxylin-eosin and examined in a blinded fashion by an experienced morphologist. Acinar cell injury/necrosis was defined as either the presence of acinar cell ghosts or (b) vacuolization and swelling of acinar cells and destruction of the histo-architecture of whole or parts of the acini, both of which had to be associated with an inflammatory reaction. The amount of acinar cell injury/necrosis and the total area occupied by acinar tissue were each quantitated morphometrically using computerized planimetric image analysis video unit (model CCD-72, Dage-MT1, Michigan city, IN) equipped with NIH-1200 image analysis software. Ten randomly chosen microscopic fields (125x) were examined for each tissue sample. The extent of acinar cell injury/necrosis was expressed as the percent of total acinar tissue which was occupied by areas which met the criteria for injury/necrosis.

Pancreas and Lung Myleoperoxidase (MPO) Activity Measurement Neutrophil sequestration in pancreas and lung was evaluated by measurement of tissue myeloperoxidase activity. Tissue samples harvested at the time of sacrifice were stored at -70 OC until the time of assay. Samples (50 mg) were thawed and homogenized in 1 mL of 20 mM phosphate buffer (pH 7.4) and centrifuged (10,000 x g, 10 min 4 OC). The resulting pellet was resuspended in mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma, St. Louis, MO) and subjected to four cycles of freezing-thawing.

The suspension was then further disrupted by sonication for 40 sec. and centrifuged (10,000 x g, 5 min. at 4 OC). A reaction mixture consisting of the extracted enzyme, 1.6 mM tetramethylbenzidine (Sigma Chemical Co., St. Louis, MO), 80 mM sodium phosphate buffer (pH 5.4) and 0.3 mM hydrogen peroxide was incubated at 370C for 110 sec, and the absorbance was measured at 655 nm in a CobasBio autoanalyzer.

This absorbance was then corrected for the fraction dry weight of the tissue sample.

WO 99/09147 PCT/US97/14212 -79- 6. Measurement of Pulmonary Vascular Permeability Obstruction of the common biliopancreatic duct also typically results in severe pancreatitis-associated lung injury quantifiable by lung vascular permeability and histological examination.

Two hours before the animals were killed, an intravenous bolus injection of 5 mg/kg fluorescein isothiocyanate albumin (FITC-albumin, Sigma Chemical Co., St. Louis, MO) was given. Pulmonary microvascular permeability was evaluated by quantifying the leakage of FITC-albumin from the vascular compartment into the bronchoalveolar space. Briefly, just after sacrifice, the right bronchus was blocked using a clamp and the trachea exposed. Subsequently, the right lung was lavaged by using a cannula inserted into the trachea. Three washes of saline ml lavage) were pooled and the FITC fluorescence in serum and lavage was measured at excitation 494 nm and emission 520 nm. The fluorescence ratio of lavage fluid to blood was calculated and taken as a measure of microvascular permeability in the lung. The lung was also stained with H&E and examined histologically.

7. Effect of Caerulein and rPH.2 administration Infusion of caerulein alone at 5 1 /g/kg/h for 3.5 hours resulted in a typical mild secretagogue-induced pancreatitis in the rats, which was characterized by hyperamylasemia, pancreatic edema as measured by pancreatic water content, and histological changes including marked acinar cell vacuolization and pancreatic edema.

Saline infusion in control animals did not result in any of these biochemical or histological changes. Administration of rPH.2 intravenously at doses of 5, 10 or mg/kg 30 min. before the start of caerulein infusion did not significantly alter the magnitude of the changes in pancreatic edema (water content) and histology that were induced by infusion of caerulein alone. Administration of rPH.2 also had no effect on caerulein-induced activation of pancreatic trypsinogen or amylase content.

Infusion of a higher dose of caerulein, 10 /g/kg/h for 5 hours, to rats resulted in a more severe pancreatitis, characterized relative to the controls by a more pronounced increase in serum amylase activity and pancreatic edema, a marked increase in pancreatic MPO activity, and a significant increase in trypsinogen WO 99/09147 PCTIS97/14212 activation and amylase activity in the pancreas. Pancreatic histology indicated not only pancreatic edema and acinar cell vacuolization but also some patchy necrosis and a few infiltrating cells.

Administration of rPH.2 (5 or 10 mg/kg intravenously) 30 min. before the start of caerulein (10 ag/kg/h) infusion ameliorated the magnitude of many of the pancreatic changes induced by the infusion of caerulein alone. Results are shown in Table 10 below. rPH.2 treatment at a dose of 5mg/kg resulted in decrease of serum amylase activity (from 10984+1412 to 6763±1256). The higher 10 mg/kg dose of rPH.2 did not result in further improvement of hyperamylasemia. Treatment with either 5 or 10 mg/kg rPH.2 also resulted in some decrease in caerulein-induced development of pancreatic edema as measured by water content (90.61 ±0.27 for caerulein alone vs. 88.21 0.61 for caerulein 5 mg/kg rPH.2). The 5 mg/kg dose of rPH.2 provided a significant amelioration of pancreatic MPO activity (2.92+0.32 fold increase over controls for caerulein alone vs. 1.19+0.21 for caerulein with rPH.2, p<0.05). Higher doses of rPH.2 did not result in further improvement of MPO activity. Neither dose of rPH.2 significantly altered the extent of trypsinogen activation or the amylase content in the pancreas. Pancreatic histology indicated some improvement in microscopic necrosis and infiltration after rPH.2 pretreatment.

Pancreatitis associated lung injury has been observed both clinically and in several models of pancreatitis. Infusion of caerulein at 5 ag/kg/h for 3.5 h, which resulted in a mild form of pancreatitis, did not result in significant injury to the lungs.

However, infusion of caerulein at 10 Ag/kg/h for 5 hours, which resulted in more severe pancreatitis, also resulted in lung injury quantified by increased lung vascular permeability (0.31+0.04 to 0.79±0.09), lung MPO activity (indicating neutrophil sequestration) and neutrophil infiltration on histological examination.

Administration of rPH.2 at a dose of 5 mg/kg 30 min prior to caerulein infusion significantly ameliorated the rise in lung MPO activity induced by the infusion of caerulein alone (3.55 0.93 for caerulein alone vs. 1.51 0.26 for caerulein with rPH.2). rPH.2 treatment significantly decreased the severity of microscopic changes observed in the lung tissue after caerulein infusion. The caerulein-induced increase in lung vascular permeability was reduced by rPH.2 treatment, although not statistically significant. The higher 10 mg/kg dose of rPH.2 WO 99/09147 WO 9909147PCT/US97/1 4212 -81was no more effective than the lower dose in decreasing the severity of caemuleininduced lung injury.

Table Control (no CER) Serum Amylase

(U/I)

Pancreas Water Content (%wet weight) 961± 174 72.71 +0.64 Caerulein

(CER)

l0jtg/kglh 10984±1412 90.61 ±0.27 CER 5 mg/kg rPH. 2 6763±1256 88.21±+0.61 CER 10 mg/kg rPH.2 8576± 1024 89.00 ±0.94 Pancreas MPG (fold increase over control) 2.92+0.32 1. 19 ±0.21 1.42 19 Pancreas Trypsin Activity (1 O00xslope/ ttg DNA Pancreas Amylase Content (U/Izg DNA) 0. 12+0.06 9.70 ±2.50 8.33±1.75 9. 15± 1.28 0.28+0.06 0.42 +0.07 0.45 +0.04 0.46+0.044 Lung Vascular Permeability (Lavage! Serum Lung MPO (fold increase over control) 0.31+0.04 0.79±+0.09 0. 70 +0.09 0.70±+0.07 3.55±+0.93 1.51 +0.26 1.64+0.22 13 Artivitv in an Qposm Pncreatitis Model Healthy, randomly trapped American opossums (Dideiphis irginiana) WO 99/09147 PCT/S97/1 4212 -82of either sex (2.0 kg to 4.0 kg) were obtained from Scott-Haas and housed in climate controlled rooms at 23±2 0 C with a 12 hour light/dark cycle and fed a standard laboratory chow with water ad libitum. After an overnight fast, the animals were anesthetized with 50mg/kg sodium-pentobarbital i.p. (Veterinary Laboratories Inc., Lenexa, KS). A celiotomy was performed through a midline incision under sterile conditions and the common bile pancreatic duct was ligated in all animals to induce acute necrotizing pancreatitis. Additionally, the cystic duct was ligated to prevent the gallbladder from serving as a bile reservoir. The animals were randomly assigned to either control or experimental groups. Starting at Day 2 after ligation of the pancreatic duct, the experimental group received 5 mg/kg body weight per day of rPH.2 (supplied in a 4mg/ml solution) intravenously via the tail vein, while the control group received an intravenous injection of the same volume of placebo vehicle only. After 1 and 2 days of treatment (at Day 3 and Day 4 after ligation of the pancreatic duct) the animals were euthanized by a sodium-pentobarbital overdose.

Blood samples were drawn from the heart for measurements of serum amylase, serum lipase and serum bilirubin, and the pancreas was harvested. Pieces of pancreas were either fixed in a 4% phosphate buffered formaldehyde solution for histological examination or immediately deep frozen at -80 0 C for measurements of myeloperoxidase activity. Additional pieces of pancreas were assessed for pancreatic water content and pancreatic amylase as described above in section A of this example.

Myeloperoxidase activity, a measure of neutrophil sequestration, was assessed in the pancreas as described above. Pulmonary vascular permeability was also assessed as described above.

The results reported represent mean standard error of the mean (SEM) values obtained from multiple determinations in 3 or more separate experiments. The significance of changes was evaluated using Student's t-test when the data consisted of only two groups or by analysis of variance (ANOVA) when comparing three or more groups. If ANOVA indicated a significant difference, the data were analyzed using Tukey's method as a post hoc test for the difference between groups. A p-value of 0.05 was considered to indicate a significant difference.

WO 99/09147 PCT/US97/14212 -83- Results are shown in Table 11. Obstruction of the common biliopancreatic duct resulted in severe necrotizing pancreatitis characterized by hyperamylasemia, hyperlipasemia and extensive necrosis of the pancreas.

Furthermore, obstruction of the common biliopancreatic duct was associated with an marked increase in serum bilirubin levels. Intravenous administration of rPH.2 mg/kg/day) starting at Day 2 after ligation of the pancreatic duct ameliorated the magnitude of many of the pancreatic changes induced by duct obstruction and placebo treatment alone. One day of rPH.2 treatment reduced serum amylase levels in comparison to placebo treated animals, although the difference was not statistically significant, and two days of rPH.2 treatment (at Day 4 after ligation of the pancreatic duct) significantly reduced serum amylase levels compared to placebo. One or two days of rPH.2 treatment reduced serum lipase levels relative to controls, although the difference was not statistically significant. Two days of rPH.2 treatment reduced pancreatic amylase content relative to controls, although one day of treatment resulted in an increase in pancreatic amylase. Treatment with rPH.2 was not observed to affect serum bilirubin levels, pancreas myeloperoxidase activity or pancreas water content.

The major characteristic histological changes induced by obstruction of the biliopancreatic duct included marked necrosis, infiltration of inflammatory cells, acinar cell vacuolization, and marked distention of the acinar lumina.

Morphometrical examination of the pancreas for acinar cell injury showed a major protective effect of rPH.2 on the pancreas after one and two days of rPH.2 treatment.

After one day of rPH.2 treatment, the acinar cell injury was reduced to about 23 of total acinar cell tissue, compared to 48 injury for the placebo-treated animals.

This reduction of acinar cell injury was even more pronounced after two days of treatment, at which time rPH.2 treatment resulted in about 35% injury of the total acinar cell tissue, compared to about 60% injury for the placebo-treated animals.

Lung vascular permeability, quantified by FITC injection showed a highly significant difference after one and two days of rPH.2 treatment compared to placebo group. Histological examination of the lung showed severe lung injury in all placebo-treated animals. Lung injury was characterized by an extensive inflammatory response with interstitial and intraalveolar infiltration of mainly macrophages, WO 99/09147 PCT/US97/14212 -84lymphocytes and neutrophils, and by a patchy but marked interstitial edema and thickening of the alveolar membranes. Administration of rPH.2 resulted in a marked decrease of infiltration of inflammatory cells and a reduction of interstitial edema at all times.

In summary, these results showed that administration of rPH.2 intravenously at a dose of 5 mg/kg/day beginning at 48 hours after ligation of the pancreatic duct resulted in significant amelioration of the increase in blood levels of amylase and lipase and acinar cell injury as quantitated by morphometric analysis of H&E stained sections, and a significant decrease in the severity of pancreatitisinduced lung injury. Administration of rPAF-AH product in this clinically relevant model of pancreatitis showed beneficial effects in decreasing the severity of pancreatitis.

WO 99/09147 PCT/US97/14212 Table 11 After 1 day of treatment (Sacrifice at Day 3) After 2 days of treatment (Sacrifice at Day 4) Placebo Serum bilirubin 5.49 (mg/dl) Serum amylase 5618 (U/l) Serum lipase 2226 (U/1) Pancreas Water 81.1 Content Pancreas MPO 1345 (OD/fraction dry weight) Pancreatic Amylase 706-4 (U/pg DNA) Lung Vascular Permeability 0.76 (FITC Lavage/ Serum Acinar Cell Injury of 48% Total Acinar Tissue) *p=0.02 vs. placebo 0.001 vs. placebo ±0.96 ±899 ±554 )±0.56 ±286 rPH.2 5mg/kg 7.10+0.60 4288 675 1241 ±263 81.52+0.79 1142±83 1101 ±105 0.21 +0.04** Placebo 6.54±0.55 6538±1355 1424±257 80.05 ±1.07 1149±232 950±85 0.57±0.13 60% rPH.2 4.91±0.79 3106±467* 1023 ±295 79.32+0.49 1033±130 712±131 0.23 ±0.04* -92 +0.09 23% Example A study was conducted to evaluate the effect of a PAF-AH product, rPH.2, on neurotoxicity associated with HIV infection. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system results in neuronal loss by apoptosis. HIV-1-infected monocytes activated by a variety of antigenic stimuli, including contact with neural cells, secrete high levels of neurotoxic pro-inflammatory WO 99/09147 PCT/US97/14212 -86cytokines, including PAF. The effect of rPH.2 on the neurotoxicity of conditioned media from HIV-infected and activated monocytes was assessed.

Monocytes were infected with HIV and activated as follows.

Monocytes were recovered from peripheral bone marrow cells (PBMC) of HIV- and hepatitis B-seronegative donors after leukopheresis and purified by countercurrent centrifugal elutriation as described in Genis et al., J. Exp. Med., 176:1703-1718 (1992). Cells were cultured as adherent monolayers (1 x 104 cells/m in T-75 culture flasks) in DMEM (Sigma, St. Louis, MO) with recombinant human macrophage colony stimulatory factor (MSCF) (Genetics Institute, Inc. Cambridge, MA). Under these conditions, monocytes differentiate into macrophages. After 7-10 days of culture, macrophages were exposed to HIV-lADA (accession number M60472) at a multiplicity of infection (MOI) of 0.01 infectious virions/target cell.

Under these conditions, 20-50 of the monocytes were infected at 7 days after HIV-1 inoculation, as determined by immunofluorescent and in situ hybridization techniques [Kalter et al., J. Immunol., 146:298-306 (1991)]. All cultures were refed with fresh medium every 2 to 3 days. Five to seven days after HIV-1 infection and during the peak of reverse transcriptase activity (107 cpm/ml), assessed according to Kalter et al., supra, cultures of HIV-1-infected and parallel cultures of uninfected monocytes were stimulated with LPS (10 ng/ml) or vehicle for 30 min. at 37 0 C, then snapfrozen at -800C until used in the neurotoxicity assay.

Human cerebral cortical neuron cell cultures were established as follows. Human fetal brain tissue was obtained from the telencephalon of second trimester (13-16 weeks gestation) human fetal brain tissue according to a modified procedure of Banker and Cowan, Brain Res., 126:397-425 (1977). Briefly, brain tissue was collected, washed in 30 ml of cold Hank's BSS (containing Ca+ 2 and Mg 2 25 mM HEPES, and 5X gentamicin), separated from adherent meninges and blood, and cut into 2 mm 3 pieces. The tissue was forced through a 230 1M Nitex bag and gently triturated through a flame-polished Pasteur pipet 10-15 times.

The tissue was centrifuged at 550 rpm, 5 minutes, 4 0 C, and the pellet was resuspended in 5-10 ml of MEM-hipp (D-glucose, 5 grams/liter; L-glutamine, 2 mM; HEPES, 10 mM; Na pyruvate, 1 mM; KC1, 20 mM) containing N1 components (insulin, 5 mg/l; transferrin, 5 mg/l; selenite, 5 ug/l, progesterone 20 nM; putrescine, WO 99/09147 PCT/US97/14212 -87- 100 fJM), as well as 10% fetal calf serum (FCS), PSN antibiotic mix (penicillin, mg/l; streptomycin, 50 mg/l; neomycin, 100 mg/1), and fungizone (2.5 mg/1). The cell count and viability were determined by diluting Hank's BSS with 0.4% trypan blue (1:1 v/v) and counting with a hemocytometer. Cells were tently triturated times with a 10 ml pipet and plated at a density of 105 cells/12 mm glass coverslip pre-coated with poly-L-lysine (70K-150K MW, Sigma, St. Louis, MO) placed in 24 well culture dishes. One ml of media was pipetted into each culture well. Cells were cultured for 10-28 days at 37 0 C in a humidified atmosphere of 5% C0 2 /95% air, changing media every 3 days. Under these conditions, cultures were >60-70% homogeneous for neurons, with 20-30% astrocytes, microglia and macrophage and microglia staining. After 14-28 days of culture, neuronal cultures express sufficient levels of N-methyl-D-aspartate (NMDA) or non-NMDA receptors to die after excitotoxic doses of NMDA or alpha-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid (AMPA).

The neurotoxicity assay was conducted as follows. The test samples, which were conditioned media from LPS-stimulated HIV-1 infected monocytes, control media, conditioned media with added rPH.2 at 51 gg/ml or (d) conditioned media with added vehicle for rPH.2, were applied to the neuronal cell cultures at a 1:10 v/v concentration for 24 hours. Neurotoxicity was measured by identifying apoptotic nuclei in situ on neuronal coverslips fixed in 4% paraformaldehyde, employing a commercial kit (Apop Tag; ONCOR, Gaithersburg, MD) that uses terminal deoxynucleotidyl transferase (TdT) to bind digoxigenin-dUPT to free 3'-OH ends of newly cleaved DNA (TUNEL staining). Digitized images of TUNEL-stained neurons in 15 randomly selected microscopic fields were analyzed for number of TUNEL-stained nuclei/number of total neurons per 50X field using computerized morphometry (MCID, Imaging Research, St. Catherine, Ontario, Canada). Data were expressed at neuronal nuclei positive for TUNEL staining SEM and are shown in FIGURE 13. Tests of statistical significance between control and experimental treatments were determined by ANOVA or paired t-tests, with significance at p.<0.05. Quantitation of these cultures confirmed that conditioned media from HIV-infected and activated monocytes induced neuronal cell death in nearly 25 of the total population of cerebral cortical neurons, and rPH.2 was able WO 99/09147 PCT/US97/14212 -88to reduce this toxicity to less than 5 of the total neurons. The rPH.2 by itself was not neurotoxic, since 50 /g/ml rPH.2 had no effect on neuronal cell death relative to cultures treated with control media. These results clearly indicate that a major component of the neurotoxicity induced by application of conditioned media from activated HIV-1 infected monocytes must be due to PAF, since neurotoxity can be almost completely abrogated by co-incubation with PAF-AH product, the enzyme responsible for metabolism of PAF in the central nervous system. These findings suggest potential therapeutic interventions in the treatment of the CNS neurologic disease associated with HIV-1 infection.

Example 21 Nearly four percent of the Japanese population has low or undetectable levels of PAF-AH activity in their plasma. This deficiency has been correlated with severe respiratory symptoms in asthmatic children [Miwa et al., J. Clin. Invest,. 82: 1983-1991 (1988)] who appear to have inherited the deficiency in an autosomal recessive manner.

To determine if the deficiency arises from an inactive but present enzyme or from an inability to synthesize PAF-AH, plasma from multiple patients deficient in PAF-AH activity was assayed both for PAF-AH activity (by the method described in Example 10 for transfectants) and for the presence of PAF-AH using the monoclonal antibodies 90G11D and 90F2D (Example 13) in a sandwich ELISA as follows. Immulon 4 flat bottom plates (Dynatech, Chantilly, VA) were coated with 100 ng/well of monoclonal antibody 90G1 ID and stored overnight. The plates were blocked for 1 hour at room temperature with 0.5 fish skin gelatin (Sigma) diluted in CMF-PBS and then washed three times. Patient plasma was diluted in PBS containing 15mM CHAPS and added to each well of the plates (50 Il/well). The plates were incubated for 1 hour at room temperature and washed four times. Fifty ,l of 5 tg/ml monoclonal antibody 90F2D, which was biotinylated by standard methods and diluted in PBST, was added to each well, and the plates were incubated for 1 hour at room temperature and then washed three times. Fifty /l of ExtraAvidin (Sigma) diluted 1/1000 in CMF-PBST was subsequently added to each well and plates were incubated for 1 hour at room temperature before development.

WO 99/09147 PCTIUS97/1 4212 -89- A direct correlation between PAF-AH activity and enzyme levels was observed. An absence of activity in a patient's serum was reflected by an absence of detectable enzyme. Similarly, plasma samples with half the normal activity contained half the normal levels of PAF-AH. These observations suggested that the deficiency of PAF-AH activity was due to an inability to synthesize the enzyme or due to an inactive enzyme which the monoclonal antibodies did not recognize.

Further experiments revealed that the deficiency was due to a genetic lesion in the human plasma PAF-AH gene. Genomic DNA from PAF-AH deficient individuals was isolated and used as template for PCR reactions with PAF-AH gene specific primers. Each of the coding sequence exons were initially amplified and sequenced from one individual. A single nucleotide change within exon 9 was observed (a G to T at position 996 of SEQ ID NO: The nucleotide change results in an amino acid substitution of a phenylalanine for a valine at position 279 of the PAF-AH sequence (V279F). Exon 9 was amplified from genomic DNA from an additional eleven PAF-AH deficient individuals who were found to have the same point mutation.

To test whether this mutation crippled the enzyme, an E. coli expression construct containing the mutation was generated by methods similar to that described in Example 10. When introduced into E. coli, the expression construct generated no PAF-AH activity while a control construct lacking the mutation was fully active. This amino acid substitution presumably results in a structural modification which causes the observed deficiency of activity and lack of immunoreactivity with the PAF-AH antibodies of the invention.

PAF-AH specific antibodies of the invention may thus be used in diagnostic methods to detect abnormal levels of PAF-AH in serum (normal levels are about 1 to 5 U/ml) and to follow the progression of treatment of pathological conditions with PAF-AH. Moreover, identification of a genetic lesion in the PAF- AH gene allows for genetic screening for the PAF-AH deficiency exhibited by the Japanese patients. The mutation causes the gain of a restriction endonuclease site (Mae II) and thus allows for the simple method of Restriction Fragment Length Polymorphism (RFLP) analysis to differentiate between active and mutant alleles.

See Lewin, pp. 136-141 in Genes V, Oxford University Press, New York, New York (1994).

Screening of genomic DNA from twelve PAF-AH deficient patients was carried out by digestion of the DNA with Maell Southern blotting, and hybridisation with an exon 9 probe (nucleotides 1-396 of SEQ ID NO: 17). All patients were found to have RFLPs consistent with the mutant allele.

While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the appended claims should be placed on the invention.

Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

WO 99/09147 PCT/US97/14212 -91- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: ICOS CORPORATION (ii) TITLE OF INVENTION: Platelet-Activating Factor Acetylhydrolase (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Marshall, O'Toole, Gerstein, Murray Borun STREET: 6300 Sears Tower, 233 South Wacker Drive CITY: Chicago STATE: Illinois COUNTRY: United States of America ZIP: 60606-6402 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Rin-Laures, Li-Hsien REGISTRATION NUMBER: 33,547 REFERENCE/DOCKET NUMBER: 27866/34026 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (312) 474-6300 TELEFAX: (312) 474-0448 TELEX: 25-3658 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 99/09147 PCT/US97/14212 -92- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala 1 5 10 Phe INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Ile Gin Val Leu Met Ala Ala Ala Ser Phe Gly Gin Thr Lys Ile Pro 1 5 10 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Lys Pro Leu Val Val Phe Val Leu Gly Gly 1 5 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: Modified-site LOCATION: group(13, 21, 27) OTHER INFORMATION: /note= "The nucleotide at each of these positions is an inosine." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ACATGAATTC GGNATCYTTG NGTYTGNCCR AA 32 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 99/09147 PCTIUS97/14212 -93- (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID TATTTCTAGA AGTGTGGTGG AACTCGCTGG INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGATGAATTC AGCTTGCAGC AGCCATCAGT AC 32 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 1520 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 162..1484 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GCTGGTCGGA GGCTCGCAGT GCTGTCGGCG AGAAGCAGTC GCGTTGGTGC GCGGTGGAAC GCGCCCAGGG ACCCCAGTTC CGCCTGAGAG ACTAAGCTGA AACTGCTGCT CAGCTCCCAA GGGTTTGGAG CGCTTGGGTC CCGCGAGCAG CTCCGCGCCG G ATG GTG CCA CCC Met Val Pro Pro 1 120 173

AAA

Lys

CCT

Pro TTG CAT GTG CTT Leu His Val Leu TGC CTC TGC GGC Cys Leu Cys Gly CTG GCT GTG GTT Leu Ala Val Val TTT GAC TGG Phe Asp Trp

CAA

Gln

AAA

Lys ATA AAT CCT Ile Asn Pro GCC CAT ATG Ala His Met GCA TGG GTC Ala Trp Val CAA ACT AAA Gln Thr Lys GAC TTA ATG Asp Leu Met TAT CCA TCC ATA CAA GTA Ile Gin Val

CTG

Leu ATG GCT GCT GCA Met Ala Ala Ala AAA TCA TCA Lys Ser Ser AGC TTT GGC Ser Phe Gly GGT TGT ACA Gly Cys Thr CGT TTA TAT Arg Leu Tyr 269 317 ATC CCC CGG Ile Pro Arg TTT GAT CAC Phe Asp His GGA AAT Gly Asn 60 ACT AAT Thr Asn 75 GGG CCT TAT Gly Pro Tyr AAG GGC ACC Lys Gly Thr TCC GTT Ser Val TTC TTG Phe Leu 413 461 CAA GAT AAT GAT CGC CTT GAC ACC CTT TGG ATC CCA AAT WO 99/09147 PCT/US97/1 4212 Tyr Pro Ser Gin Asp Asn 90 Asp Arg Leu Asp Thr 95 Leu Trp Ile Pro Asn 100 AAA GAA TAT TT Lys Glu Tyr Phe GGT CIT AGC AAA TTT CTT GGA ACA CAC Gly Leu Ser Lys Phe Leu Gly Thr His 110 TGG CTT Trp Leu 115 ATG GGC AAC Met Gly Asn AAC TGG AAT Asn Trp Asn 135

AT

Ile 120 TTG AGG TTA CTC Leu Arg Leu Leu

TTT

Phe 125 GGT TCA ATG ACA Gly Ser Met Thr ACT CCT GCA Thr Pro Ala 130 CTT GTT GTT Leu Val Val TCC CCT CTG AGG Ser Pro Leu Arg

CCT

Pro 140 GGT GAA AAA TAT Gly Glu Lys Tyr

CCA

Pro 145 TT TCT Phe Ser 150 CAT GGT CIT GGG His Gly Leu Gly

GCA

Ala 155 TTC AGG ACA CTT Phe Arg Thr Leu

TAT

Tyr 160 TCT GCT ATT GGC Ser Ala Ile Gly

ATT

Ile 165 GAC CTG GCA TCT Asp Leu Ala Ser

CAT

His 170 GGG =rr ATA GTT Gly Phe Ile Val

GCT

Ala 175 GCT GTA GAA CAC Ala Val Giu His

AGA

Arg 180 GAT AGA TCT GCA Asp Arg Ser Ala

TCT

Ser 185 GCA ACT TAC TAT Ala Thr Tyr Tyr AAG GAC CAA Lys Asp Gin GAA ATA GGG Glu Ile Gly GAG GAG ACA Glu Glu Thr 215

GAC

Asp 200 AAG TCT TGG CTC Lys Ser Tip Leu

TAC

Tyr 205 CTT AGA ACC CTG Leu Arg Thr Leu TCT GCT GCA Ser Ala Ala 195 AAA CAA GAG Lys Gin Glu 210 GCA AAA GAA Ala Lys Glu CAT ATA CGA AAT His Ile Arg Asn

GAG

Glu 220 CAG GTA CGG CAA Gin Val Arg Gin

AGA

Arg 225 TGT TCC Cys Ser 230 CAA GCT CTC AGT Gin Ala Leu Ser

CTG

Leu 235 ATT CIT GAC ATT Ile Leu Asp Ile

GAT

Asp 240 CAT GGA AAG CCA His Gly Lys Pro

GTG

Val 245 AAG AAT GCA TTA Lys Asn Ala Leu

GAT

Asp 250 TTA AAG TTT GAT Leu Lys Phe Asp

ATG

Met 255 GAA CAA CTG AAG Glu Gin Leu Lys

GAC

Asp 260 TCT ATT GAT AGG Ser Ile Asp Arg

GAA

Glu 265 AAA ATA GCA GTA Lys Ile Ala Val

ATT

Ile 270 GGA CAT TCT TTT Gly His Ser Phe GGT GGA Gly Gly 275 GCA ACG GTT Ala Thr Val ATT GCC CTG Ile Aia Leu 295 CAG ACT CTT AGT Gin Thr Leu Ser GAT CAG AGA TTC Asp Gin Arg Phe AGA TGT GGT Arg Cys Gly 290 GTA TAT TCC Val Tyr Ser GAT GCA TGG ATG TTT CCA CTG GGT GAT Asp Ala Tip Met Phe Pro Leu Gly Asp

GAA

Glu 305 AGA ATT Arg Ile 310 CCT CAG CCC CTC Pro Gin Pro Leu

TTT

Phe 315 TTT ATC AAC TCT Phe Ile Asn Ser

GAA

Glu 320 TAT TTC CAA TAT Tyr Phe Gin Tyr 1037 1085 1133 1181 1229

CCT

Pro 325 GCT AAT ATC ATA Ala Asn Ile Ile

AAA

Lys 330 ATG AAA AAA TGC Met Lys Lys Cys TCA CCT GAT AAA Ser Pro Asp Lys

GAA

Glu 340 AGA AAG ATG ATT Arg Lys Met Ile

ACA

Thr 345 ATC AGG GGT TCA Ile Arg Gly Ser

GTC

Val 350 CAC CAG AAT TTT His Gin Asn Phe GCT GAC Ala Asp 355 WO 99/09147PCUS/142 PCTIUS97/14212 TIC ACT TTT GCA ACT GGC AAA ATA ATT GGA CAC ATG CTC AAA TTA Phe Thr Phe Ala Thr Gly Lys Ile Ile Gly His Met Leu Lys Leu 360 365 370 GGA GAC ATA GAT TCA AAT GTA GCT ATT GAT CTT AGC AAC AAA GCT Gly Asp Ile Asp Ser Asn Val Ala Ile Asp Leu Ser Asn Lys Ala 375 380 385 TTA GCA TTC TTA CAA AAG, CAT TIA GGA CTT CAT AAA GAT IT GAT Leu Ala Phe Leu Gln Lys His Leu Gly Leu His Lys Asp Phe Asp 390 395 400 TGG GAC TGC TTG ATT GAA GGA GAT GAT GAG AAT CTT ATT CCA GGG Trp Asp Cys Leu Ile Giu Gly Asp Asp Glu Asn Leu Ile Pro Gly 405 410 415 AAC ATI AAC ACA ACC AAT CAA CAC ATC ATG TTA CAG AAC TCT TCA Asn Ile Asn Thr Thr Asn Gin His Ile Met Leu Gin Asn Ser Ser 425 430 435 ATA GAG AAA TAC AAT TAGGATTAAA ATAGGrrITT TAAAAAAAAA AAAAAA Ile Giu Lys Tyr Asn 440 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 44i amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein

AAG

Lys

TCA

Ser

CAG

Gin

ACC

Thr 420

GGA

Gly 1277 1325 1373 1421 1469 1520 (xi) SEQUENCE DESCRIPTION: SEQ Met Vai Pro Pro Lys Leu His Val Leu P Ala Met Al a Vai Leu Trp Thr Thr Pro 145 Ser Val Lys Ser Gly Arg Ile His Thr 130 Leu Al a Val Ser Phe Cys Leu Pro Trp 115 Pro Val Ile Tyr Ser Gly Thr Tyr Asn 100 Leu Ala Val Gly Pro Al a Gin Asp Tyr Lys Met Asn Phe Ile Phe Trp Thr Leu 70 Pro Glu Gly Trp Ser 150 Asp Asp Val Lys 55 Met Ser Tyr Asn Asn 135 His Leu Gin 25 Lys Pro Asp Asp Trp 105 Leu Pro Leu Ser

I

A

H

A

G

A

L-

G

H

ID NO: 8: he Cys Leu yr Ile Asn le Gin Val .rg Gly Asn is Thr Asn sn Asp Arg ly Leu Ser rg Leu Leu eu Arg Pro 140 ly Ala Phe 155 is Gly Phe Cys Pro Leu Gly Lys Leu Lys Phe 125 Gly Arg Ile Gly Val Met Pro Gly Asp Phe 110 Gly Giu Thr Val WO 99/09147PCUS7142 PCTIUS97/14212 Val Giu His Gin Leu Arg 225 His Gin Ser Phe Giu 305 Tyr Pro Asn Leu Asn 385 Asp Ile Asn Ser Lys 210 Aia Giy Leu Phe Arg 290 Val Phe Asp Phe Lys 370 Lys Phe Pro Ser Aila 195 Gin Lys Lys Lys Giy 275 Cys Tyr Gin Lys Ala 355 Leu Ala Asp Gly Ser 435 Arg 180 Ala Giu Giu Pro Asp 260 Giy Gly Ser Tyr Glu 340 Asp Lys Ser Gin Thr 420 Gly Glu Glu Cys Vai 245 Ser Ala Ile Arg Pro 325 Arg Phe Gly Leu Trp, 405 Asn Ile Asp Arg Ser Ala Ser Gly Thr 215 Gin Asn Asp Val Leu 295 Pro Asn Met Phe Ile 375 Phe Cys Asn Lys Asp 200 His Ala Ala Arg Ile 280 Asp Gin Ile Ile Al a 360 Asp Leu Leu Thr Tyr 440 185 Lys Ile Leu Leu Glu 265 Gin Ala Pro Ile Thr 345 Thr Ser Gin Ile Thr 425 Asn Ala Ser Arg Ser Asp 250 Lys Thr Trp, Leu Lys 330 Ile Gly Asn Lys Giu 410 Asn Thr Trp Asn Leu 235 Leu Ile Leu Met Phe 315 Met Arg Lys Val His 395 Gly Gin Tyr Leu Giu 220 Ile Lys Ala Ser Phe 300 Phe Lys Gly Ile Al a 380 Leu Asp His Tyr Tyr 205 Gln Leu Phe Val Glu 285 Pro Ile Lys Ser Ile 365 Ile Gly Asp Ile Phe 190 Leu Val Asp Asp Ile 270 Asp Leu Asn Cys Val 350 Gly Asp Leu Giu Met 430 Lys Arg Arg Ile Met 255 Gly Gin Giy Ser Tyr 335 His His Leu His Asn 415 Leu INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 1123 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: Not Determined WO 99/09147PCIS7112 PCTIUS97/14212 -97- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

AAATATAAAT

TGAAGTT'FrA

GAAGATGAAG

AAACGGCTCC

ATTGTCA'rIT

GGTCCACACA

CAGGGCATTG

ACAAGGAAAA

AGGTCTGCGG

GGCGGGCTAA

GCGCTCGAGC

CCTCCCCGGT

AGGCGGACCC

GCCCGCAGCC

TCGGGTrTGG

CCCGCGAGCA

GGGAGGAAGG

GTGGGCTCGC

CCTCGGAAAC

TTrTAATAACA

AATGAGTGTG

GAAGGCGTTT

TTCT.AGCTCC

CCAGGGAGAA

ACTTCCGAAT

CCAACTATAG

CCGGCCTGAC

AAAGGAGCTG

GTTAACCTCG

AGGCTACGTC

ACCTCCTCCA

AGACACAGCC

AGGGGGACAG

AGCGCTTGGG

GCTCCGCGCC

GCACGGTCGC

TGAGTCGCAC

AGCGCTAGGA

CCACACATAA

TTTTTAATTT

CAGTTAAACC

XTCTCCTC

ATGACACCAG

TGGTGTTCAG

ATGCTCGGAG

TGGGGGGTGA

GTGAGCACGA

GGTCCAGGTG

GGGAGCCGCC

GCATCACCAG

GCGCGCAGCC

CGGCTGGTCG

TCGCGTrGGT

GCGCCTGAGT

CGCGCTGGAG

CCGCTCTGCT

TCC'ITCGGGA

ATTTCAAACT

ATTAGAAAGT

CCAAATAACT

AGACCTAAGT

CACAGTGGCA

TGTAAAGTGT

TAATTCAGTG

ATTCAGCAGG

CACCACCAGG

CGGGCC.ATGG

GCTGCTAGTG

GGGAGGAGAG

CACCCGCCCG

GAGGCTCGCA

GCGCGGTGGA

AC TrCCCTA

GGATTGAAGA

CTGTGTrAC.A GCTATrCCTG

GGCCTTCCAA

ATCGGAGTGC

TATTCAGAGA

GAGTAAATCT

CATTGCCTGG,

TCTTGGGGAG

AGAGCCGGGC

GGTCGGGCAC

CCGCCTGCCA.

GTGCTGTCGG

AC CCC CCAGG

AGT'ITCTAGC

GAAAACATTG

CTGAGCTATG

ATTGTCCTrC

TCTGGAGCAC

GGAAAATGCG

ACACGGTGAA

GATCGGCATC

CTCTCTCCGC

GGTGCTGGGT

CACACACGCT

AAGGCGCGCT

GAGCTGCTCG

CGAGAAGCAG

GACCCCAGTT

AGGCGGGAGT

ACCGGCCGGG

TCCCTGCAGC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1123 GAGGAGGGGC CCC( GTCGGGACCC CGG GGCCGGTCCT GGG( GAGGAGAGAT GAC 3GGGGCG

%GCGGCG

ZTCACAG

INFORMATION FOR SEQ ID NO:l0: SEQUENCE CHARACTERISTICS: LENGTH: 417 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: 145. .287 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0: GTACCAATCT AAAACCCAGC ACAGAAAAAT ACATGTTTrA TTTTTTCCAA TACCTCAGCC TTTCTTGATT TGTCAGCTTA TTTAAGGCCT CTTCATTGCA 1-rCTT'rrAAT CATCTGC'rrC GAAGGAGACT AAGCTGAAAC TGCTGCTCAG GGTGCCACCC AAATTGC.ATG TGCTTITCTG CCTCTGCGGC TGCCTGGCTG TTTTGACTGG CAATACATAA ATCCTGTTGC CCATATGAAA TCATCAGGTA

GTGTTACTAG

TACTTCTTT

CTCCCAAGAT

TGGTTTATCC

AGAGGTGTAT

120 180 240 300 WO 99/09147 WO 9909147PCTIUS97/1 4212 -98- TTGTTCAAGG TCTTGAGCAA CTGATCTGTC GCCATACTTC AAGTGGGCCC CAAGAAGTTG CACATCTGCA CATCTAAACA AGTCCTATrr AAAGGCTrAT GGAGATCCTG TAITCTC INFORMATION FOR SEQ ID NO:ll: SEQUENCE CHARACTERISTICS: LENGTH: 498 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: 251. .372 360 417 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: CATI'AGGAGG TAACAGTCCA AGGCAGCTGA GAGAAAGGCT ATGTCTACT ACCCTCCAAA ACCCCTACAC AGTGTTTC.AA ACAGAGAGAC CCTCAATAAT ACTTGTTAGG TTGAGAAAGA AAGAAGGCCA GAAACTATGG GAAGTAACTT GAATTCTTTT GCATAATAAA ATCTGATATG TAATGGATGA CAAATGAGAT TGTrTrTCAG, CATGGGTCAA CAAAATACAA GTACTGATGG CTGCTGCAAC ACTAAAATCC CCCGGGGAAA TGGGCCTTAT TCCGTTGGTT GTAC.AGACTT CACACTAATA AGGTAATGCT TTGA TTTATA CAACTTATCC TGATACTCTA TCGCT.ATGGA CCACTAGAAG GTG ITCAAAT GTGACCTTGC CCTCACCTGA TTTTCGAATT TGTATTGT

TCATCTCTTT

TGCATATCTr

GATTCCG'ITG

AATATITACC

GTTTGGCCAA

AATGTTTGAT

ATATTGTCTG

GAATGACTCA

120 180 240 300 360 420 480 498 INFORMATION FOR SEQ ID NO;12: SEQUENCE CHARACTERISTICS: LENGTH: 433 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: 130. .274 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CAGCAGCCTA AAGTCTTAGA C TTrGTGAAC ACAGAGGTAT CGAAAATAGC TGCTGGAATA TGTTTGAGAC ACAACTTCTC TCTTAACAGG GCACCTTCTT GCGTTTATAT TATCCATCCC ACCCTrTGGA TCCCAAATAA AGAATA'TTT TGGGGTCTTA TGGCTTATGG GCAACA'TITT GAGGTTACTC TTTGGTAAG.A TGAGTCCCAC TAATTAATAT

TAAAAGTGCA

AAGATAATGA

GCAAATITCT

T'rrCTGTTGA C ITCAAGTAG TTAATTrCTT

TCACCITGAC

TGGAACACAC

TCCTTCTTTG

TTAAGACC.AA

TAGGCTC'ITG

ACAAC.AAGAA

CATGTATGAA AACCTTGAAA WO 99/09147PCUS/142 PCTIUS97/14212 -99- AGTAGAT~rr TCTTC.AGTCC AAATAGCTCC TAAAATGATA AGGAAAGTAT TTTrAAAG CCCAGGCAAC TAC 420 433 INFORMATION FOR SEQ ID NO:13: Wi SEQUENCE CHARACTERISTICS: LENGTH: 486 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: 164. .257 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TrGGTGGGTA TCTAGTAGCA GTCTTrAA TGAATCTACT.

ATATAAATCA GATGGGTCTG CATITI'ATGC TAATGAGATA CACTCAGAGA AAACCTTAAC TATAACCTTC CATTGTrGTC GCAAACTGGA ATI'CCCCTCT GAGGCCTGGT GAAAAATATC

GGTCTTGGGG

TGACAATTTT

CTGGAGGAGT

TCTGGTCAAG

GAAAGG

CATTCAGGTA ATG'FITGAGA TTATTCAAG AAAGAAATAG TGGGGTTCCT CAATAA'ITGG TAGTTTrTTTC CCTACTATCA

GGTTGAACAA

CAGAGTTTGG

CTGTGGGTCT

GCTCATTGGG

ATTCATCCAT

TGAATrAAAT

TAGGTTCAAT

CACTTGTTGT

TTTTGGCTTC

A.ATGTCATGC

ATTGATCAGT

ATTAGCCTCA

AAAAAAGTAG

TCACTAGCAA

GACAACTCCT

TTITrCTCAT

CAGGAATAAA

AGGCCCTrGT

CCTAGACCTG

CAGCAGAGAA

120 180 240 300 360 420 480 486 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 363 base pairs TYPE: nucleic acid STRAN'DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: 113. .181 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CCCCAGGCTC TACTACAGGG, TGTAATGGCC TCCATGTTCC CAGTTTATT CCTTGTAA'rr CATGACTGGT AG'ITGTAATT CTTCCCTCTT TTTGTTTTGA ATTCTGCTAT TGGCATTGAC CTGGCATCTC ATGGGTTTAT AGTTGCTGCT GGTATG'rrAC CTGATATAAT TGGGCTC'rrT GGCCAACTAC AGGGAATGTC ACTATG'rrTC TAAT=TTCAT AAAAGTTITAT TTAA.AATGTT GATGGAACT TAACATCATG AGCAAAAAAG GAGATTGAGT TTTATCGACT TAAAAGACTT

AGTGACTCAG

AGGACACTTT

GTAGAACACA

AATGCTC.ATA

TCAAGTATGG

AAAAGCACCT

120 180 240 300 360 WO 99/09147 PTU9/41 PCTIUS97/14212 -100-

AAC

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 441 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: 68. .191 (xi) SEQUENCE DESCRIPTION: SEQ ID GAACTGAGAA ACATGGTCAG ATGAGGAAGG GAAGGAGCAT ATTATAGAGA TAGATCTGCA TCTGCAACTT ACTA? TCAA TAGGGGACAA GTCTTGGCTC TACCTIAGAA CCCTGAAACA GAAATGAGCA GGTACATTGC AGTGAAAGGA GAGGTGGTTG

GCATAAATAA

GGACC.AATCT

AGAGGAGGAG

GTGACCTAAA

TrATGCTCCT

AGTAAAATTC

TTGGGAGATG

TTITGCTrGT

GCTGCAGAAA

ACACATATAC

AGCATGTACA

AGCTCTCCTA

CCTT.AATGGA

ACAGTGAATA

AAAGGATGAC

'rTTCCCATTC

ATATCTAGTT

TTCAGAATTC

AT'FrGTT.AAT

CCAAAAGATC

CATAGTAAAA

CTCGAGCCGG

TTAATITAC

TGTCAATAGA

ACCTGGCAAG

TI'CCTGGAGC

120 180 240 300 360 420 441 ACAAAGGCAA ATACAAAAAT INFORMATION FOR SEQ ID NO:l6: SEQUENCE CHARACTERISTICS: LENGTH: 577 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: 245. .358 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GGTrAAGTAA ATCGTCTGAA GTCACATAGT AGGTAAGGCA CTAAGGCTAT ACCTATGTGC AAAGCTGGGG CCTGTGTCAT ACT.AATC.AGA TTTCCAGTIT ATAACTGACC AACGATTTT AAACTTTAAA ATAAGTGTTA TAACTTTI'TA CTTTGTC.ATT TTAGGTACGG CAAAGAGCAA AAGAATGTTC CCAAGCTCTC TCATGGAAAG CCAGTGAAGA ATGCATTAGA TTT.AAAGrT AAGCTATAAA AAGTAATTIT TCTCTTGTCC TACAGTTCTT AAACAGAGCC AGGATI"rGGA TATGGTAGCA AGTAATAGTC CCCAAATACA GCTTCTACCT TCCTTC'ITCT AATAATTATA AGTCTGATTC TTGACATI'GA GATATGGAAC AACTGAAGGT TATTGTrTT TGTCATTTAA WO 99/09147PCUS7142 PCT/US97/14212 -101- TITrCTGCCT ATA ITGCAAG GTACAATATG ATAAAGGGCT GCAACCAGCC CCTCCCCAAT GCGCACACAC AGACACACAA AGCAGTACAG GTAAAGTATT GCAGCAATGA AGAATGCATr ATCTrGGACT AGATATGAAT GCAAAGTTAG TCAGTTT 480 540 577 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 396 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: 108. .199 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATCAATGTAT TTACCATCCC CATGAAATGA ACAATTATAT GATTGACAAA TAACACCACG AAATAGCTAT AAATITATAT CATGCT TTT T CAAATAGGAC GGGAAAAAAT AGC.AGTAATT GGACATrCTr ITGGTGGAGC AACGGTTA'rr GTGAAGATCA GAGATTCAGG TAAGAAAATA AGATAGTAAA GCAAGAGAAT TGGAAGAAAT TATATIGTGA GATATAATrT TrATrCAAAT TCTTAGTGAA CTCTTGGAGT TTATAAGGCT ATTCTTTTL-GC CCCCATAAAA TACTCTATAT AGGCTAAAAC ATCTCCTCTC CTGCTATTAA AATCTC INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 519 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: 181. .351 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CTTACAAAGT TAATCATATC CCTTTCCCAC ATTGAAGTAT GATACCTCTT AGATAACCCA TAATAAACTG GT.ATGGTGCG TGTCCA.CC.AA TCCTAGCATT CCTCAATGTT GGCTAGTATG TAACCAGTTT AATTCATCA TTGTCAACAA ATGTGGTATT GCCCTGG.ATG CATGGATGTT TCCACTGGGT GATGAAGTAT TCCTCAGCCC CTCTITITTA TCAACTCTGA ATATTTCCAA TATCCTGCTA AATGAAAAAA TGCTACTCAC CTGATAAAGA AAGAAAGATG ATTACAATCA AGTGACTTAT ITCATTATGT GAAACAAACT TGAAGCTTGG GTAAATATCA

TCATTTCTTC

TCTATTGATA

CAGACTCTTA

AGTAAATTAT

GGAAGGGGAT

ACATITCCT

TATTCCAATC

ATI'AGGATGT

ATATCTACAG

ATTCCAGAAT

ATATCATAAA

GGTAAGTATT

ATCGATATCA

120 180 240 300 360 396 120 180 240 300 360 420 WO 99/09147PC[S/122 PCTIUS97/14212 -102- TTTGGTAACT ATI'AAAGAAT TGCTGAATTG GTTGTTAGA CTTTCA.ATAA. GGAGAGAATr AGATAATCTC AG'TTTCTAAG TACATTTAGT CTACTC TT 480 519 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 569 base pairs TYPE: nucleic acid STRA1NDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoinic) (ix) FEATURE: NAME/KEY: exon LOCATION: 156. .304 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TGAAACACAT CTAAGTAGAT CAAATTACAA G7 TTATTTC 'TCTrrGGTr AGACCAACAA GACCAGTACC T ITCCT'rACA CTCTAACTAA AAAAATAATA ACAATGTGAC TTTTAAATGT CTTGTTCTCT TTTAGGGGTT CAGTCCACCA G.AC'TCACTT ITGCAACTGG CAAAATAATT GGACACATGC TCAAATTAAA GATTCAAATG TAGCTATTGA TCITAGCAAC AAAGCTTCAT TAGCATTC TT TIAGGTAAGA AACTATTTTT TTCATGACCT AAACCGAGAT GAATCTCGAG TCTATCTI'AA TACAGCTTTA GTACTATITA AACTAT'ITCC AGTrGGTTTA AAGCAGTATA TCAA'rrTGAA AACAGAAATT TGAGAAAGTC AA'I =TGCTG CTATATCATA GAAAGCAAAT CAACTGTTAA AGGTAATATT CTTTGTATGA CTCATGTGAG GATATCGAAC GACGGTGCT r1'CAGTAAAC

ATTATCAA

GAATT'ITGCT

GGGAGACATA

ACAAAAGCAT

GACAAAGCTG

CAATGGAACA

CTITACATCT

GCTAGAGTGA

120 180 240 300 360 420 480 540 569 INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 469 base pairs TYPE: nucleic acid STRANqDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: exon LOCATION: 137. .253 (xi) SEQUENCE DESCRIPTION: SEQ ID GATACAGAGG C-ACATCGTCT CTACCATCCT AACGGAACTT GTGTAATrG TAAATCTrI'A TTGCCACCTA GGGGCATCCA AACTGTTrAA TGCTCTCAAA AGITTAATAT GTITGATTAAC ACTTTATATT TTATAGGACT TCATAAAGAT TTTGATCAGT GGGACTGCTT GATrGAAGGA GATGATGAGA ATCTTATTCC AGGGACCAAC ATTAACACAA CCAATCAACA CATCATGTTrA CAGAACTCTT CAGGAATAGA GAAATACAAT TAGGATTAAA ATAGGTTTFI' TAAAAGTCTT 120 180 240 300 WO 99/09147PCIS7142 PCT/US97/14212 -103- GTITCAAAAC TGTCTAAAAT TATGTGTGTG TGTGTGTGTG TGTGTGTGTG AGAGAGAGAG AGAGAGAGAG AGAGAGAATT TTAATGTATT ITCCCAAAGG ACTCATATrT TAAAATGTAG GCTATACTGT AATCGTGATT GAAGCTTGGA CTAAGAATTT TTTCCCTT INFORMATION FOR SEQ ID NO:2i: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1494 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATUJRE: N)AME/KEY: CDS LOCATION: 117. .1436 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GGCACGAGCT AGGATCTGAC TCGCTCTGGT GGC.ATTGCTG CGCTCAGGGT TCTGGGTATC CGGGAGTCAG TGCAGTGACC AGAACATCAA. ACTGAAGCCA CTGCTCAGCT CCTAAG16 116 ATG GTA CCA CTC Met Vai Pro Leu 1 CTG CAG GCG CTT TTC TGC CTC CTC TGC Leu Gin Ala Leu Phe Cys Leu Leu Cys 10 TGC CTC Cys Leu CCA TGG GTC Pro Trp Val AGG CCG TCA Arg Pro Ser

CAT

His CCT TTT CAC TGG Pro Phe His Trp

CAA

Gin GAC ACA TCT TCT TTT GAC 'ITC Asp Thr Ser Ser Phe Asp Phe GTA ATG TI'T CAC AAG CTC CAA TCG GTG Val Met Phe His Lys Leu Gin Ser Val

ATG

Met TCT GCT GCC Ser Ala Ala GGC TCT Gly Ser GGC CAT AGT AAA Gly His Ser Lys CCC AAA GGA AAT Pro Lys Gly Asn TCG TAC CCC GTC Ser Tyr Pro Val

GGT

Gly TGT ACA GAT CTG Cys Thr Asp Leu

ATG

Met 70 TTC GGT TAT GGG Phe Gly Tyr Gly

AAT

Asn GAG AGC GTC TTC Giu Ser Val Phe CGT 'ITG TAC TAC Arg Leu Tyr Tyr

CCA

Pro GCT CAA GAT CAA Ala Gin Asp Gin

GGT

Gly 90 CGC CTC GAC ACT Arg Leu Asp Thr GTT TGG Val Trp ATC CCA AAC AAA GAA TAT TIT TTG Ile Pro Asn Lys Giu Tyr Phe Leu 100

GGT

Gly 105 CTT AGT ATA TTT Leu Ser Ile Phe CTT GGA ACA Leu Gly Thr 110 CCC AGT ATT Pro Ser Ile 115 ACT CCT GCA Thr Pro Ala 130 GTA GGC AAT ATT Val Gly Asn Ile

ETA

Leu 120 CAC CTC TTA TAT His Leu Leu Tyr GGT TCT CTG ACA Gly Ser Leu Thr 125 GAA AAA TAC CCG Giu Lys Tyr Pro AGC TGG AAT Ser Trp Asn

TCT

Ser 135 CCT TTA AGG ACT Pro Leu Arg Thr

GGA

Gly 140 ATT GTC TTT TCT Ile Val Phe Ser

CAT

His 150 GGT CTC GGA GCC Giy Leu Gly Ala AGG ACG ATT TAT Arg Thr Ile Tyr WO 99/09147 PCT/US97/1 4212 -104- GCT ATT GGC ATT Ala Ile Gly Ile

GGC

Gly 165 TTG GGA TCT Leu Ala Ser AAT GGG Asn Gly 170 GCA ACT Ala Thr 185 TTT ATA GTG GCC Phe Ile Val Ala ACT GTC Thr Val 175 GAA CAC AGA Glu His Arg GTG GCT GCA Val Ala Ala 195

GAG

Asp 180 AGA TCT GGA TCG Arg Ser Ala Ser TAC TTT TT Tyr Phe Phe GAA GAC CAG Glu Asp Gin 190 AGA AAA GTA Arg Lys Val AAA GTG GAA AAC Lys Val Glu Asn

AGG

Arg 200 TCT TGG CTT TAG Ser Trp Leu Tyr

CTG

Leu 205 AAA GAA Lys Gin 210 GAG GAG TCG GAA Glu Giu Ser Glu

AGT

Ser 215 GTC GGG AAA GAA Val Arg Lys Glu

GAG

Gin 220 GTr CAG CAA AGA Val Gin Gin Arg

GCA

Ala 225

GGA

Gly ATA GAA TGT TCC Ile Giu Gys Ser GAG CGA AAA GAG Asp Pro Lys Glu 245

CGG

Arg 230 GCT GTC AGT GCG Ala Leu Ser Ala CTT GAG ATT GAA Leu Asp Ile Glu

CAT

His 240 AAT GTA GTA GGT Asn Vai Leu Gly

TCA

Ser 250 GCT TT GAG ATG Ala Phe Asp Met AAA GAG Lys Gin 255 CTG AAG GAT Leu Lys Asp TTT GGA GGA Phe Gly Gly 275 GCT ATT GAT GAG ACT AAA ATA GCT TTG ATG GGA CAT TCT Ala Ile Asp Giu Thr Lys Ile Ala Leu Met Gly His Ser 260 265 270 GCA AGA GTT CTT Ala Thr Val Leu

CAA

Gin 280 GCG CT AGT GAG Ala Leu Ser Glu

GAC

Asp 285 GAG AGA TTC Gin Arg Phe AGA TGT Arg Gys 290 GGA GTT GGT CTT Gly Val Ala Leu

GAT

Asp 295 CCA TGG ATG TAT Pro Trp Met Tyr

CCG

Pro 300 GTG AAG GAA GAG Val Asn Giu Glu

CTG

Leu 305 TAG TCC AGA ACC Tyr Ser Arg Thr

CTC

Leu 310 GAG CCT CTC CTG Gin Pro Leu Leu ATC AAG TGT GCC Ile Asn Ser Ala

AAA

Lys 320 TTC CAG ACT CCA Phe Gin Thr Pro

AAG

Lys 325 GAG ATG GGA AAA Asp Ile Ala Lys

ATG

Met 330 AAA AAG TTC TAG Lys Lys Phe Tyr GAG CCT Gin Pro 335 GAG AAG GAA Asp Lys Glu TTT GAG GAG Phe Asp Asp 355 AAA AAT GAT TAG Lys Asn Asp Tyr

AAT

Asn 345 GAA GGG CTC AGG Gin Gly Leu Arg CAC CAG AAG His Gin Asn 350 AAG AAG CTG Asn Lys Leu TI' ACT TTT GTA Phe Thr Phe Val GGC AAA ATA ATT Gly Lys Ile Ile 1028 1076 1124 1172 1220 1268 1316 1364 1412 ACA CTG Thr Leu 370 AAA GGA GAA ATG Lys Gly Giu Ile

GAT

Asp 375 TCG AGA GTA GCG Ser Arg Val Ala

ATG

Ile 380 GAG CTC AGC AAG Asp Leu Thr Asn

AAA

Lys 385 GCT TGG ATG GCT Ala Ser Met Ala

TTC

Phe 390 ITA CAA AAG CAT Leu Gin Lys His TTA GGG CIT GAG AAA GAG Leu Gly Leu Gin Lys Asp 395 400 TTT GAT GAG TGG Phe Asp Gin Trp

GAC

Asp 405 CCT GTG GTG GAA Pro Leu Val Glu

GGA

Gly 410 GAT GAT GAG AAC Asp Asp Glu Asn CTG ATT Leu Ile 415 GCT GGG TCA Pro Gly Ser TT GAG GGA GTG Phe Asp Ala Val

ACC

Thr 425 GAG GCC CCG GCT Gin Ala Pro Ala GAG CAA CAC Gin Gin His 430 WO 99/09147 PCT/US97/14212 -105- TCT CCA GGA TCA CAG ACC CAG AAT TAGAAGAACT TGCTTGTTAC ACAGTTGCCT Ser Pro Gly Ser Gin Thr Gin Asn 435 440 TTTAAAAGTA GAGTGACATG AGAGAGAG INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 2191 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 92..1423 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CCGCGCGCTC CGGCCGGGGG ACCCTGGTTC CGGCGAGCGG CTCAGCGCGG CGCCCGGAAG TTTAAGCTGA AACCACTGCT CAGCTTCCAA G ATG TTG CCA CCC AAA CTG CAT Met Leu Pro Pro Lys Leu His 1466 1494 112 160 208 GCG CTT TTC TGC CTC TGC AGC Ala Leu Phe Cys Leu Cys Ser

TGC

Cys CTC ACA CTG GTT Leu Thr Leu Val CCT ATT GAC Pro Ile Asp TGG CAA Trp Gin GAC CTA AAT CCT Asp Leu Asn Pro

GTT

Val 30 GCC CAT ATT AGA Ala His Ile Arg

TCA

Ser TCA GCA TGG GCC Ser Ala Trp Ala

AAT

Asn AAA ATA CAA GCT Lys Ile Gin Ala ATG GCT GCT GCA Met Ala Ala Ala

AGT

Ser 50 ATT AGG CAA AGT Ile Arg Gin Ser

AGA

Arg ATT CCC AAA GGA AAT GGA TCT TAT TCT Ile Pro Lys Gly Asn Gly Ser Tyr Ser GTC GGT TGT ACA GAT TTG ATG Val Gly Cys Thr Asp Leu Met 65 TTT GAT TAT Phe Asp Tyr CAA GAG GAT Gin Glu Asp AAT AAG GGC ACC Asn Lys Gly Thr TTG CGT TTG TAT Leu Arg Leu Tyr TAT CCA TCG Tyr Pro Ser AAA GAA TAT Lys Glu Tyr GAC CAC TCT GAC Asp His Ser Asp

ACG

Thr CTT TGG ATC CCA Leu Trp Ile Pro TTT TTT Phe Phe 105 GGT CTT AGT AAA Gly Leu Ser Lys

TAT

Tyr 110 CTT GGA ACA CCC Leu Gly Thr Pro CTT ATG GGC AAA Leu Met Gly Lys 448 496

ATA

Ile 120 TTG AGC TTC TTT Leu Ser Phe Phe

TTT

Phe 125 GGT TCA GTG ACA Gly Ser Val Thr

ACT

Thr 130 CCT GCG AAC TGG Pro Ala Asn Trp

AAT

Asn 135 TCC CCT CTG AGG Ser Pro Leu Arg

ACT

Thr 140 GGT GAA AAA TAT Gly Glu Lys Tyr

CCA

Pro 145 CTG ATT GTT TTT Leu Ile Val Phe TCT CAT Ser His 150 GGT CTT GGA GCA TTC CGG ACA ATT TAT TCT GCT ATT GGC ATT GAT CTA Gly Leu Gly Ala Phe Arg Thr Ile Tyr Ser Ala Ile Gly Ile Asp Leu WO 99/09147 PCTIUS97/14212 -106- 165 GCA TCA CAT Ala Ser His 170 GGG TTC ATC GTT Gly Phe Ile Val

GCT

Ala 175 GCT ATA GAA CAC Ala Ile Glu His AGA GAT GGA TCC Arg Asp Gly Ser 180 GCA GAA ATA GGG Ala Giu Ile Gly GCC TCT Ala Ser 185 GCG ACT TAC TAT Ala Thr Tyr Tyr AAG GAC CAG TCT Lys Asp Gin Ser

GCT

Ala 195

AAC

Asn 200 AAA TCT TGG TCT Lys Ser Trp Ser

TAT

Tyr 205 CTT CAA GAA CTA Leu Gin Giu Leu

AAA

Lys 210 CCA GGG GAT GAG Pro Gly Asp Glu

GAG

Glu 215 ATA CAT OTT CGA le His Val Arg

AAT

Asn 220 GAG CAG GTA CAG Glu Gin Val Gin AGG GCA AAG GAG Arg Ala Lys Glu TGC TCC Cys Ser 230 CAA GCT CTC Gin Ala Leu AAT OTA CTA Asn Val Leu 250 TTG ATT CTG GAC Leu Ile Leu Asp

ATT

Ile 240 GAT CAT OGA AGG Asp His Gly Arg CCA ATT AAG Pro Ile Lys 245 GAC TCT ATT Asp Ser Ile GAC TTA GAG TTT Asp Leu Glu Phe

GAT

Asp 255 GTG GAA CAA CTG Val Glu Gin Leu

AAG

Lys 260 GAC AGG Asp Arg 265 GAT AAA ATA GCA Asp Lys Ile Ala

OTA

Val 270 ATT OGA CAT TCT Ile Gly His Ser

TTT

Phe 275 GGT GGA GCC ACA Oly Gly Ala Thr

GTT

Val 280 CTr CAG GCT CTT Leu Gin Ala Leu

AGT

Ser 285 GAA GAC CAG AGA Olu Asp Gin Arg

TTT

Phe 290 AGG TGC GGG ATT Arg Cys Gly Ile 0CC Ala 295 TTG GAT OCA TGG Leu Asp Ala Trp

ATG

Met 300 CTT CCA CTG OAT Leu Pro Leu Asp OCA ATA TAT TCC Ala Ile Tyr Ser AGA ATC Arg Ile 310 CCT CAG CCC Pro Gin Pro AAT ATC AAA Asn Ile Lys 330

CTC

Leu 315 TTT ITT ATT AAC Phe Phe Ile Asn

TCG

Ser 320 GAA CGG TTC CAA Glu Arg Phe Gin TT CCT GAG Phe Pro Glu 325 GAA AGA AAA Glu Arg Lys AAA ATG AAA AAA Lys Met Lys Lys

TGC

Cys 335 TAC TCA CCT OAC Tyr Ser Pro Asp

AAA

Lys 340 ATG ATT Met Ile 345 ACA ATC AGG OGT Thr Ile Arg Oly

TCA

Ser 350 GTC CAT CAG AAC Val His Gin Asn

TT

Phe 355 OCT OAT TIC ACT Ala Asp Phe Thr 1024 1072 1120 1168 1216 1264 1312 1360

TTT

Phe 360 ACA ACT GGC Thr Thr Gly AAA ATA Lys Ile 365 OTA OCA Val Ala 380 GTT GGA TAC ATA Val Gly Tyr Ile ACA TTA AAA GGA Thr Leu Lys Gly

OAT

Asp 375 ATA GAT TCA AAT Ile Asp Ser Asn ATT OAT CTT Ile Asp Leo

TGC

Cys 385 AAC AAA OCT Asn Lys Ala TCA TTG GCA Ser Leu Ala 390 Trr TTA CAA Phe Leu Gin TCT TTG AT Ser Leu Ile 410

AAG

Lys 395 CAT TTA GGA CTG His Leu Gly Leu CGG AAA GAT TTT OAT CAG TGG GAT Arg Lys Asp Phe Asp Oin Trp Asp 400 405 GAA OGA AAA GAC Glu Oly Lys Asp

GAA

Glu 415 AAT CTT ATG CCA Asn Leo Met Pro

GGG

Oly 420 ACC AAC AT Thr Asn Ile WO 99/09147 WO 9909147PCTIUS97/1 4212 -107- AAC ATC ACC AAC GAA CAT GAC ACT CTA CAG AAC TCT CCA GAA GCA GAG Asn Ile Thr Asn Giu His Asp Thr Leu Gin Asn Ser Pro Glu Ala Glu 425 430 435 AAA TCG AAT TTA GAT TAAAAGCACT TT-ITT-1AAAGA. TCTrGTTTA.A AAACTGTCAA Lys Ser Asn Leu Asp

AAAATGTGTG

TATCCCATAA

TGTCTATCGA

TTAATAACTA

CGTGTTGCAA

ATAA'ITGAGT

TI'GGTCATG

T1'AGGGATAC

A'ITAAAGCCA

GCATTTACTT

CAGGGATTCC

TGGGAGTGGA

AAAAAAAA

TATGACTT

AAGTGATI'GA

AATCATGCCA

CTTTTACATT

TGACATAACA

TTI'AGCAACA

ATAAGAAAAA

TGAACAATT

AGGCAAAGGC

GATGGTrTAT

AGTIATAACA

ITAGCACAAT

AATATA'TT

AGC'ITGGACT

GCCTAAATTT

CTTTAATGGA

ATCCCTAAAA

TGTTATGCTA

TTAGATCAAG

CACTATGGTA

AGCAGATrAG

CTCATGGATT

CA ETATTCAC

AGAGGCATAT

CTCAAATAAC

AGGAGGTTTT

TAATTTTACT

CAAGTATAAC

ATACAGATGT

GGTAGAATT

CAAATGATAA

ACTGAATGGG

AATGGATTAA

CATGAGTCAA

CCAAAGGGTT

GTTGCTTTAA

TCATATTGGA

TTTCTITAAA.

AAAATGATGC

AGGCACAAGG

TCTrGCCTCT

GGAAGCACTT

AAGCAGTGTT

GAGTGACCAA

AGAGAGTTTA

GAAAGGTGCG

CTTTAAITrCT

AAATGTAGGC

GAAAGATTGG

TGTGTCAAAA.

CTAATGAAAA

rTI'TCTA'TT CCC'rITGACT

ITACCAAGGA

GGGTAAAAAT

TAATITGTrrr

TAGGACAGGC

GTATGAGTAT

14 08 1463 1523 1583 1643 1703 1763 1823 1883 1943 2003 2063 2123 2183 2191 INFORMATION FOR SEQ ID NO:23: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1533 base pairs TYPE: nucleic acid STRAINDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: CDS LOCATION: 62. .1394 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CCGCGAGCAG TTCACCGCGG CGTCCGGAAG GITAAGCTGA AACGGCAGCT CAGCTTCGGA G ATG 'ITA CCG TCC AAA 'rrG CAT GCG CTT 1"rC TGC CTC TGC ACC TGC Met Leu Pro Ser Lys Leu His Ala Leu Phe Cys Leu Cys Thr Cys 1 5 10 CIT GCA CTG GTT TAT CCT TTT GAC TGG CAA GAC CTG AAT CCA GTT GCC Leu Ala Leu Val Tyr Pro Phe Asp Trp Gin Asp Leu Asn Pro Val Ala 25 TAT ATT GAA TCA CCA GCA TGG GTC AGT AAG ATA CAA GCT CTG ATG GCT Tyr Ile Giu Ser Pro Ala Trp Val Ser Lys Ile Gin Ala Leu Met Ala 40 WO 99/09147 WO 9909147PCTIUS97/1 4212 -108- GCT GCA AAC Ala Ala Asn ATr GGT CAA TCT Ile Gly Gin Ser

AAA

Lys ATC CCC AGA GGA Ile Pro Arg Gly GGA TCT TAT Gly Ser Tyr TCC GTC Ser Val GGT TGT ACA GAC Gly Cys Thr Asp ATG rrr GAT TAC Met Phe Asp Tyr

ACT

Thr AAT AAG GGC ACC Asn Lys Gly Thr TrC Phe TTG CGT TTG TAT Leu Arg Leu Tyr

TAT

Tyr 85 CCA TCT CAA GAT Pro Ser Gin Asp GAT GAT C-AC! TCC GAC ACC Asp Asp His Ser Asp Thr 90 CTT TGG ATC CCA Leu Trp Ile Pro AAA GAA TAT TTT Lys Glu Tyr Phe

TTG

Leu 105 GGT CTT AGT AAA Gly Leu Ser Lys iTr cTT- Phe Leu 110 GGA ACA CAC Gly Thr His ATG ACA ACT Met Thr Thr 130

TGG

Trp 115 CTT GTG GGC AAA Leu Val Gly Lys

ATT

Ile 120 ATG GGC TTA TTC Met Gly Leu Phe TTC GGT TCA Phe Giy Ser 125 GGG GAA AAA Gly Giu Lys CCT GCA GCC TGG Pro Ala Ala Trp

AAT

Asn 135 GCA CAT CTG AGG Ala His Leu Arg

ACT

Thr 140 TAC CCA Tyr Pro 145 CTA ATT ATT TT Leu Ile Ile Phe CAT GGT CTT GGA His Gly Leu Gly

GCA

Ala 155 TTC AGG ACG ATT Phe Arg Thr Ile

TAT

Tyr 160 TCT GCT ATT GGC Ser Ala Ile Gly

ATT

Ile 165 GAT CTG GCA TCC Asp Leu Ala Ser GGG ATA GTT Gly Phe Ile Val

GCT

Ala 175 250 298 346 394 442 490 538 586 634 682 730 778 826 874 922 970 GCT GTA GAA CAC Ala Val Giu His

AGG

Arg 180 GAT GGC TCT GCA Asp Gly Ser Ala

TCC

Ser 185 TCG ACA TAC TAT Ser Thr Tyr Tyr Trc AAG Phe Lys 190 GAC CAG TCT Asp Gin Ser ACC CTG AAG Thr Leu Lys 210

GCT

Ala 195 GTA GAA ATA GGC Val Giu Ile Gly AAG TCT TGG CTC Lys Ser Trp Leu TAT CTC AGA Tyr Leu Arg 205 GAG CAG ITA Glu Gin Leu CGA GGA GAG GAG Arg Giy Giu Giu

GAG

Giu 215 TIT CCT TTA CGA Phe Pro Leu Arg

AAT

Asn 220 CGG CAA Arg Gin 225 CGA GCA AAG GAA Arg Ala Lys Glu

TGT

Cys 230 TCT CAA GCT CTC Ser Gin Ala Leu

AGT

Se r 235 TTG AFI' CTG GAC Leu Ile Leu Asp

ATT

Ile 240 GAT CAC GGG AGG Asp His Gly Arg

CCA

Pro 245 GTG ACG AAT GTA Val Thr Asn Val CTA GAT TTA GAG FIT GAT Leu Asp Leu Giu Phe Asp 250 255 GTG GAA CAG CTG Val Giu Gin Leu

AAG

Lys 260 GAC TCT ATT GAT Asp Ser Ile Asp

AGG

Arg 265 GAT AAA ATA GCC Asp Lys Ile Ala ATT ATT Ile Ile 270 GGA CAT TCT TTT GGT GGA GCC ACA GTT ATT CAG ACT CTT AGT GAA GAC Gly His Ser Phe Gly Gly Ala Thr Val Ile Gin Thr Leu Ser Giu Asp 275 280 285 CAG AGA TTC Gin Arg Phe 290 AGG TGT GGC ATT Arg Cys Gly Ile CTG GAT GCA TGG Leu Asp Ala Trp

ATG

Met 300 rTTr CCC GTG Phe Pro Val GGT GAT Gly Asp 305 GAA GTA TAT TCC Giu Val Tyr Ser

AGA

Arg 310 ATT CCT CAA CCC CTC 'ITT ATC AAC Ile Pro Gin Pro Leu Phe Phe Ile Asn 1018 WO 99/09147 WO 9909147PCTIUS97/1 4212 -109-

TCG

Ser 320 TrC Phe GAA CGA TTC CAA Giu Arg Phe Gin

TAC

Tyr 325

GAA

Giu CCT TCT AAT ATC Pro Ser Asn Ile

ATA

Ile 330 AGA ATG AAA AAA Arg Met Lys Lys

TGC

Cys 335 TTA CCT GAT Leu Pro Asp

AGA

Arg 340

GTT

Val CGA AAA ATG Arg Lys Met

ATT

Ile 345 ACA ATC AGG GGT Thr Ile Arg Giy TCG GTC Ser Vai 350 CAT CAG AAT His Gin Asn TAC CTA TTC Tyr Leu Phe 370 C'rr AGC AAC Leu Ser Asn TTr Phe 355

ACA

Thr GAC TTC ACT Asp Phe Thr GCC ACT AGC AAA Aia Thr Ser Lys CTG AAA GGA Leu Lys Giy

GAC

Asp 375 GAT TCC AAT Asp Ser Asn

GTA

Val 380

CAT

His ATA ATT GGC Ile Ile Giy 365 GCC ATC AGC Aia Ile Ser TTA GGA CT Leu Giy Leu AAA GCT TCC Lys Aia Ser 385 CAG AAA Gin Lys

TI'A

Leu 390

TGG

Trp GCG ITC TrA CAA Aia Phe Leu Gin 1066 1114 1162 1210 1258 1306 i3 54 i4 04 1464 1524 1533 GAT TIT GAT Asp Phe Asp

CAG

Gin 405 GAT TCT TTA Asp Ser Leu 400

AAT

Asn GTr Vai 410

ACC

Thr GGC GAA GAT Giy Giu Asp

CAC

His 415

ATT

Ile CTT ATT CCA Leu Ile Pro GGG ACC Giy Thr 420 ACA GGA Thr Giy AAC ATT AAC ACA Asn Ile Asn Thr 425 ATA GAG AGA CCA Ile Giu Arg Pro 440 AAC CAC CAA Asn His Gin

GCC

Aia 430 CTG CAG AAC Leu Gin Asn AAT TTA GAT Asn Leu Asp T AAAAGAGCTr TTAAAAAGT T'ITG TTT ACG AACTTGTCTA AAAGTGTGTG TTTCTCAAAT AGCTCATATr AAAAAATGTA GGCTATAGCA

AAAAAAAAA

TGTGTATGAT TTAAATGTAT INFORMATION FOR SEQ ID NO:24: Wi SEQUENCE CHARACTERISTICS: LENGTH: i876 base pairs TYPE: nucieic acid STRANDEDNESS: singie TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (ix) FEATURE: NME/KEY: CDS LOCATION: 468. .1734 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CGGCGGGCTG CTGGCCCTTC CCGGCTGTTC GTAGAGCCGG ATCCTGCAGC GCCCCTGAGA CGAACCGCCC CGATGCGGTG CTCCTCAGCG CCACGGGACG CAGCCGGGGC CGGCCGTGT' GGCGCAGCTC CCACGACGTA CGCITCCTTT CCAGGCTCGA GGAAAGCCTC TCCCACAAAC ACCGTCCCAG CTGGGAAGTG AGGCGGAGTT TTGGTCCCTC CCCTCCGGCA GCGCCCGGCA TTCCGTCCGT CCGTCCGTCC GTCCGTGCGG CGCACGGCGC CCTGCAGAGC CGGGACACCG 120 180 240 300 WO 99/09147 WO 9909147PCT/US97/1 4212 -110- CAGCAGGGTA GGAGGACCCG GAGGTGGTGT GCAGCCACAG GTI'TCCATCC TGCCCCCACC TCCCGGGGAG CAGCCCTGTG CTATACCCAA CCCCCCGCAC AGAGCACTGA GCCGGCTGCT GCCTGCCTGC ACCCCGCCGT GGGACCTI'CT GCTO'ITCCCA ACAAGTG ATG GCA TCG Met Ala Ser 1 CTG TGG Leu Trp GTG AGA GCC AGG Val Arg Ala Arg

AGG

Arg GTG TTC ATG AAA Val Phe Met Lys CGT GCT TCA GGT Arg Ala Ser Gly 'rrC Phe TOG GCG AAG GCG Ser Ala Lys Ala ACG GAG ATG GGG Thr Glu Met Gly

AGC

Ser 30 GGC GGC GCG GAG Gly Gly Ala Glu

AAG

Lys GGO TAT CGG ATC Gly Tyr Arg Ile

CCC

Pro GCC GGG AAG GGC COG CAO GOC GTG GGC Ala Gly Lys Gly Pro His Ala Val Gly 45 TGO AOG Cys Thr GAT CTG ATG Asp Leu Met TAC CTA TCG Tyr Leu Ser

ACC

Thr GGC GAC GOG GCC Gly Asp Ala Ala

GAG

Glu 60 GGA AGC TT TTG Gly Ser Phe Leu OGC CTG TAT Arg Leu Tyr TGG ATT COA Trp Ile Pro TGT GAC GAO ACA Cys Asp Asp Thr ACT GAA GAG ACA Thr Glu Giu Thr 000 Pro GAT AAA Asp Lys GAG TAC TAC CAG Glu Tyr Tyr Gin

GGG

Gly CTG TCT GAC TTC Leu Ser Asp Phe AAC GTG TAO CGG Asn Val Tyr Arg

GC

Ala 100 CTG GGA GAA AGG Leu Gly Glu Arg TTC O.AG TAO TAO Phe Gin Tyr Tyr GTT GGO TCA GTG ACC TGT Val Gly Ser Val Thr Cys 110 115 CCT GOA AAA TCA Pro Ala Lys Ser

AAC

Asn 120 GOT GOT TTT AAG Ala Ala Phe Lys GGA GAG AAA TAO Gly Giu Lys Tyr OCA OTG Pro Leu 130 OTO GTT TrT Leu Val Phe ATO TGO ATA Ile Cys Ile 150

TOO

Ser 135 CAT GGA OTT GGA His Gly Leu Gly

GOT

Al a 140 TTT OGG ACC ATO Phe Arg Thr Ile TAT TOT GOT Tyr Ser Ala 145 GAG ATG GOT TOT Glu Met Ala Ser CAA GGO TTT OTA GTG GCA GOT GTG GAG Gin Gly Phe Leu Val Ala Ala Val Giu i55 160 CAC AGA His Arg 165 GAT GAA TOG GOT Asp Giu Ser Ala

TCA

Ser 170 GOA AOG TAT TTO Ala Thr Tyr Phe TGT AAA AAG AAG Cys Lys Lys Lys 175 GTG GAG AAG GAG Val Giu Lys Giu

GOT

Ala

TGG

Trp 195

GAT

Asp 180 TOT GAG 0014 GAG Ser Giu Pro Glu

GAG

Glu 185 GAT CAA A01A TCA Asp Gin Thr Ser

GGC

Gly 190 ATO TAO TAC AGG Ile Tyr Tyr Arg

AAG

Lys 200 OTO AGA GCA GGA Leu Arg Ala Gly GAG GAG CGO TGT Glu Giu Arg Cys CTG OGT Leu Arg 210 1004 1052 1100 1148 1196 CAO AAG GAG His Lys Gin OTO ATT OTT Leu Ile Leu 230

GTA

Val 215 GAG GAG AGA GGA Gin Gin Arg Ala

GAG

Gin 220 GAG TGC ATO AAA Glu Cys Ile Lys GOG CTC AAC Aia Leu Asn 225 GTG CTG AAO Val Leu Asn AAG ATO AGT TGA Lys Ile Ser Ser

GGA

Gly 235 GAG GAA GTG ATG Giu Giu Val Met

AAT

Asn 240 WO 99/09147 PCTIUS97/1 4212 -111- TCA GAC Ser Asp 245 TT GAC TGG AAC Phe Asp Trp Asn

CAC

His 250 CTG AAG GAT TCT Leu Lys Asp Ser

GTT

Val 255 GAT ACT AGC AGA Asp Thr Ser Arg

ATA

Ile 260 GCT GTG ATG GGA Ala Val Met Gly

CAC

His 265 TCT TTT GGT GGT Ser Phe Gly Gly

GCT

Ala 270 ACA GTr ATT GAG Thr Val Ile Glu

AGC

Ser 275 CTC AGC AAA GAA Leu Ser Lys Glu

ATT

Ile 280 AGA TTT AGG TGT Arg Phe Arg Cys

GGC

Gly 285 ATT GCC CIT GAT Ile Ala Leu Asp GCG TGG Ala Trp 290 ATG CTC CCG Met Leu Pro CTG CTC TTT Leu Leu Phe 310

GTA

Val 295 GGC GAT GAC ACT Gly Asp Asp Thr

TAC

Tyr 300 CAA AGC AGT GTG Gin Ser Ser Val CAG CAA CCA Gin Gin Pro 305 AAT ATC TTA Asn Ile Leu ATT AAT TCC Ile Asn Ser GAA AAA Glu Lys 315 TTC CAG TGG GCT Phe Gin Trp Ala

GCC

Ala 320 AAG ATG Lys Met 325 AAG AAG CIT AGC Lys Lys Leu Ser AAT GAT ACC AAC Asn Asp Thr Asn

AAG

Lys 335 AAA ATG ATC ACC Lys Met Ile Thr

ATC

Ile 340 AAA GGA TCG GTA Lys Gly Ser Val

CAT

His 345 CAG AGC TIT CCT Gin Ser Phe Pro

GAT

Asp 350 TTT ACT TTT GTG Phe Thr Phe Val

AGT

Ser 355 1244 1292 1340 1388 1436 1484 1532 1580 1628 1676 1724 1774 1834 1876 GGA GAA ATC ATT Gly Glu Ile Ile

GGA

Gly 360 AAG TIT TTC AAG Lys Phe Phe Lys

TTA

Leu 365 AAA GGA GAA Lys Giy Glu AAT GAA GCT Asn Glu Ala AAA CAT CTG Lys His Leu 390

ATT

Ile 375 GAT ATA TGC AAC Asp Ile Cys Asn GCT TCA ITG GCC Ala Ser Leu Ala ATA GAC CCA Ile Asp Pro 370 TTC CTG CAG Phe Leu Gin 385 TCA CTC GTG Ser Leu Val AGT CTT AAG AGA Ser Leu Lys Arg

GAT

Asp 395 TTT GAT AAG TGG Phe Asp Lys Trp

GAT

Asp 400 GAT GGC Asp Gly 405 ATA GGA CCC AAT Ile Gly Pro Asn

GTT

Val 410 ATT TCT GGT ACC Ile Ser Gly Thr ATC GAC TTA TCT Ile Asp Leu Ser CCA ACT GAG Pro Thr Glu 420 T AAGGAGTACA AGAAGTACTG CAAAGGCCAC CAGCAGCAGG ACACCAACGT TGGCCACACA TTGCTTGGAG CTGAGATAGC ACTGGCCTCC CACACAGCTr TTGGAGTGTG AAACAACAAA AAAAAAAATC ACAGGGGAGC CG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 517 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 2..514 WO 99/09147 WO 9909147PCT/US97/I 4212 -112- (xi) SEQUENCE DESCRIPTION: SEQ ID G GOG CAT TCT TTr GGA GGA GCA ACA GTT TTT CAA GCC CTA AGT GAA Gly His Ser Phe Gly Gly Ala Thr Val Phe Gin Ala Leu Ser Glu 1 5 10 GAC CAG AGA rrC Asp Gin Arg Phe TGT GGG ATT GCC Cys Gly Ile Ala GAT CCG TGG ATG Asp Pro Trp Met Trr CCC Phe Pro GTG AGT GAG Val Ser Giu AAC TCT GCC Asn Ser Ala

GAG

Giu CTG TAC TCC AGA Leu Tyr Ser Arg

GT

Vai 40 CCT CAG CCT CTC Pro Gin Pro Leu TTC TI' ATC Phe Phe Ile ATG AAA AAC Met Lys Asn GAA TTC CAG ACT Glu Phe Gin Thr AAG GAC ATT GCA Lys Asp Ile Ala

AAA

Lys TTC TAC Phe Tyr CAG CCT GAC AAG Gin Pro Asp Lys GAA AGG AAA ATG ATT ACG ATC AAG GGC TCA Giu Arg Lys Met Ile Thr Ile Lys Gly Ser 70

GTG

Val

GGA

Gly CAC GAG AAT TIT His Gin Asn Phe AAC AAG CTG TCA Asn Lys Leu Ser 100

GCT

Ala 85 GAC GGG ACT 'TT Asp Giy Thr Phe

GTA

Val ACT GGC AAA ATA Thr Gly Lys Ile CTG AAA GGA GAC Leu Lys Gly Asp

ATA

Ile 105 GAC TCC AGA GTT Asp Ser Arg Val GCC ATA Ala Ile 110 GAC CTC ACC Asp Leu Thr CT1' CAT AAA Leu His Lys 130

AAC

Asn 115 AAG GCT TCC 'ITG Lys Ala Ser Leu

GCT

Al a 120 'ITC 'ITA CAA AAA Phe Leu Gin Lys CAT TTA GGA His Leu Gly 125 GAC TIT GAT GAG Asp Phe Asp Gin TGG GAC TGT CTG GTG GAG GGA GAG AAC Trp Asp Cys Leu Val Glu Gly Glu Asn 135 140 GAG AAC Glu Asn 145 CTC ATC CCG GGG Leu Ile Pro Gly

TCA

Ser 150 CCC 'iTT GAT GTA Pro Phe Asp Val ACC GAG TCC CCG Thr Gin Ser Pro

GCT

Ala 160 CTG GAG, AGT TCT Leu Gin Ser Ser

CCC

Pro 165 GGA TCA CAC AAC Gly Ser His Asn GAG AAT TAG Gin Asn 170 INFORMATION FOR SEQ ID NO:26: Wi SEQUENCE CHARACTERISTICS: LENGTH: 580 base pairs TYPE: nucleic acid STRPANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NTAME/KEY: CDS LOCATION: 1. .580 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CAA GTA CTG ATG GCT GCT GCA AGC TIT GGC GAA CGT AAA ATC CCT AAG Gin Val Leu Met Ala Ala Ala Ser Phe Gly Giu .Arg Lys Ile Pro Lys WO 99/09147 WO 9909147PCT/US97/1 4212 -113- GGA AAT GGG Gly Asn Gly ACT AAA AAG Thr Lys Lys

CCT

Pro TAT TCC GT GGT Tyr Ser Val Gly ACA GAC TTA ATG Thr Asp Leu Met TTT GAT TAC Phe Asp Tyr CAA GAT GAT Gin Asp Asp GGC ACC rrC TTG Gly Thr Phe Leu TTA TAT TAT CCA Leu Tyr Tyr Pro

TCC

Ser GAT CGC Asp Arg CTT GAC ACC CTr Leu Asp Thr Leu ATC CCA AAT AAG Ile Pro Asn Lys TAT TTr TGG GGT Tyr Phe Trp Gly

CTT

Leu AGC AAG TAT CT Ser Lys Tyr Leu

GGA

Gly 70 AAA CAC TGG CT Lys His Trp Leu ATG GGC AAC ATT 'ITG AGT Met Gly Asn Ile Leu Ser 75 ITA CTC 'iTT GGT TCA GTG ACA ACT CCT GCA AAC TGG AAT TCC Leu Leu Phe Gly Ser Val Thr Thr Pro Ala Asn Trp Asn Ser CCT CTG Pro Leu AGG CCT GGT Arg Pro Gly GCA 'rrC AGG Ala Phe Arg 115

GAA

Glu 100 AAA TAC CCA CT Lys Tyr Pro Leu GTT TTT TCT CAT Val Phe Ser His GGT CIT GGA Gly Leu Gly 110 GCA TCT CAT Ala Ser His ACA ATT TAT TCT Thr Ile Tyr Ser A'IT GGC ATT GAC Ile Gly Ile Asp

CTG

Leu 125 GGG 'rir Gly Phe 130 ATA GTT GCT GCT Ile Val Ala Ala GAA CAC AGA GAT Glu His Arg Asp

AGA

Arg 140 TCT GCA TCT GCA Ser Ala Ser Ala

ACT

Thr 145 TAC TAT TTC AAG Tyr Tyr Phe Lys

AAC

Asn 150 CAA TCT GCT GCA Gin Ser Ala Ala

GAA

Giu 155 ATA GGG AAA AAG Ile Gly Lys Lys

TCT

Ser 160 TGG CTC TAC CTT Trp Leu Tyr Leu AGA ACC Arg Thr 165 CTG AAA GAA Leu Lys Giu GAG GAG ATA CAT Giu Glu Ile His ATA CGA Ile Arg 175 AAT AAG CAG Asn Lys Gin

GTA

Val 180 CGA CAA AGA GCA Arg Gin Arg Ala

AAA

Lys 185 GAA TGT TCC Glu Cys Ser CAA GCT CTC AGT Gin Ala Leu Ser 190 528 576 580 CTG A Leu INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Gly Xaa Ser Xaa Gly 1 WO 99/09147 WO 9909147PCTIUS97/1 4212 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: TATTCTAGAA TrATGATACA AGTATTAATG GCTGCTGCA.A G INFORMATION FOR SEQ ID NO:29: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: ATI'GATATCC TAATTGTATT TCTCTATrCC TG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1335 base pairs TYPE: nucleic acid STP.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID ATGGTACCCC CAAAGCTGCA CGTCCTGTTT TGTCTGTGTG GATGTCTCGC

CCCTTCGATT

AAGATCCAGG

GGCCCCTACA

CTGAGACTGT

AAAGAATATT

TTGAGGTTAC

GGTGAAAAAT

TCTGCTATTG

GATAGATCTG

AAGTCTTGGC

CAGGTACGGC

GGCAGTATAT

TGCTCATGGC

GCGTGGGCTG

ACTACCCCAG

TI'TGGGGTCT

TC'FTTGGTTC

ATCCACTTGT

GCATTGACCT

CATCTGCAAC

TCTACCTTAG

AAAGAGCAAA

CAACCCCGTG

CGC.ACCAAGC

CACCGATCTG

CCAGGACAAC

TAGCAAATT

AATGACAACT

TGTTI'CT

GGCATCTCAT

TTACTATTTC

AACCCTGAAA

AG.AATGTTCC

GCTCACATGA

TTCGGTCAGA

ATGTTCGACC

GACAGACTGG

CTTGGAAC.AC

CCTGCAAACT

CATGGTCTTG

GGGTTTATAG

AAGGACCAAT

CAAGAGGAGG

CAAGCTCTCA

AGAGCAGCGC

CCAAGATTCC

ATACCAACAA

ATACTCTGTG

ACTGGCTTAT

GGAATTCCCC

GGGCATTCAG

TTGCTGCTGT

CTGCTGCAGA

AGACACATAT

GTCTGATTCT

CGTCGTGTAC

CTGGGTGAAT

TAGAGGCAAC

AGGAACTTr

GATCCCAAAT

GGGCAACATr

TCTGAGGCCT

GACAC'ITTAT

AGAACACAGA

AATAGGGGAC

ACGAAATGAG

TGACATTGAT

120 180 240 300 360 420 480 540 600 660 720 780 CATGGAAAGC CAGTGAAGAA TGCATTAGAT TTAAAGTTTG ATATGGAACA ACTGAAGGAC WO 99/09147PCUS/I41 PCTIUS97/14212 -115-

TCTATTGATA

CAGACTCTTA

CCACTGGGTG

TATTTCCAAT

AGAAAGATGA

ACTGGCAAAA

A7TGATC'ITA

GATITI'GATC

AACATTAACA

AATTAGGATT

GGGAAAAAAT

GTGAAGATCA

ATGAAGTATA

ATCCTGCTAA

TI'ACAATCAG

TAATTGGACA

GCAACAAAGC

AGTGGGACTG

CAACCAATCA

CTAGA

AGCAGTAATr

GAGATTCAGA

TrCCAGAATT

TATCATAAAA

GGGTI'CAGTC

CATGCTCAAA

TI'CATTAGCA

CTTGAT'rGAA

ACACATCATG

GGACATTCTT

TGTGGTATTG

CCTCAGCCCC

ATGAAAAAAT

CACCAGAATT

TTAAGGGAG

TTCTTACAAA

GGAGATGATG

TTACAGAACT

TTGGTGGAGC

CCCTGGATGC

TC IT'TTAT

GCTACTCACC

TI'GCTGACTr

ACATAGATTC

AGCATTTAGG

AGAATCTTAT

CTTCAGGAAT

AACGGTTATT

ATGGATGTT

CAACTCTGYAA

TGATAAAGAA

CACTTGCA

AAATGTAGCT

ACTTCATAAA

TCCAGGGACC

AGAGAAATAC

840 900 960 1020 1080 1140 1200 1260 1320 1335

Claims (15)

1. A purified and isolated human plasma platelet-activating factor acetylhydrolase (PAF-AH) polypeptide fragment which is lacking up to the first twelve N-terminal amino acids of the mature human PAF-AH amino acid sequence set out in SEQ ID NO: 8.

2. The PAF-AH polypeptide fragment of claim 1 selected from the group consisting of: polypeptides having Met 4 6 of SEQ ID NO: 8 as the initial N-terminal amino acid; polypeptides having Ala 4 7 of SEQ ID NO: 8 as the initial N-terminal amino acid; polypeptides having Ala 4 8 of SEQ ID NO: 8 as the initial N-terminal amino acid;

3. The PAF-AH polypeptide fragment of any of claims 1 or 2 which is lacking up to 30 C-terminal amino acids of the amino acid sequence of SEQ ID NO:

8. 4. The PAF-AH polypeptide fragment of any of claims 1 or 2 having as its C-terminal residue a residue of SEQ ID NO: 8 selected from the group consisting of: 11e 4 2 9 Leu 4 3 1 and Asn44 1 -117- A variant of the PAF-AH polypeptide fragment of claim 1 which has an amino acid replacement in the sequence of SEQ ID NO: 8 selected from the group consisting of: S 108 A, S 273 A, D 286 A, D 286 N, D 296 A, D 304 A, D 338 A, H 351 A, H 395 A, H 399 A, C 67 S, C 229 S, C 291 S, C 334 S,and C 407 S. 6. A purified and isolated human PAT-AH polypeptide variant which has an amino acid replacement in the sequence of SEQ ID NO: 8 selected from the group consisting of: D 286 A D 286 N D 304 A 7. An isolated polynucleotide encoding a PAF-AH polypeptide fragment, variant or variant fragment according to any of claims 1 or 6. -118- 8. An isolated polynucleotide encoding a human PAF-AH fragment or variant fragment having Met 4 6 of SEQ ID NO: 8 as the N-terminal residue and Ile 429 or Asn441 as the C-terminal residue.

9. The polynucleotide of any of claims 7 or 8 which is a DNA.

10.A DNA vector comprising a DNA of claim 9. 11 .A host cell stably transformed or transfected with a DNA according to claim 9 in a manner allowing expression in said host cell of a PAF-AH polypeptide fragment, variant or variant fragment.

12.A method of producing a PAF-AH polypeptide fragment, variant or variant fragment of plasma PAF-AH comprising growing a host cell according to claim 11 in a suitable nutrient and isolating said PAF-AH fragment, variant or variant fragment from said cell or the medium of its growth.

13.A PAF-AH polypeptide fragment, variant or variant fragment produced by the method of claim 12.

14.A pharmaceutical composition comprising the PAF-AH fragment, variant or variant fragment of any of claims 1, 6 or 13 and a pharmaceutically acceptable diluent, adjuvant or carrier. of a pharmaceutical composition according to claim 14 in an amount sufficient to supplement PAF-AH activity and to inactivate pathological effects of PAF-Ah for preparing a medicament to treat a mammal susceptible to or suffering from a PAF-medicated pathological condition.

16.A method according to claim 15 wherein said pathological condition is pleurisy, asthma, rhinitis, necrotizing enterocolitis, acute respiratory distress syndrome, acute pancreatitis or neurological disease associated with HIV infection. -119-

17.A purified and isolated PAF-AH polypeptide fragment or variant according to claims 1 or 6 substantially as herein before described in reference to the examples.

18.An isolated polynucleotide according to claim 7 or 8 substantially as herein before described in reference to the examples.

19.A DNA vector according to claim 10 substantially as herein before described in reference to the examples. host cell according to claim 11 substantially as herein before described in reference to the examples.

21.A method of producing a PAF-AH polypeptide fragment, variant or variant fragment according to claim 12 substantially as herein before described in reference to the examples.

22.A pharmaceutical composition according to claim 14 substantially as herein before described in reference to the examples. Dated this SECOND day of JULY 2002. ICOS Corporation Applicant Wray Associates Perth, Western Australia Patent Attorneys for the Applicant