WO1986006100A1 - Proteine anti-inflammatoire d'inhibition de la phospholipase humaine - Google Patents

Proteine anti-inflammatoire d'inhibition de la phospholipase humaine Download PDF

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Publication number
WO1986006100A1
WO1986006100A1 PCT/US1986/000772 US8600772W WO8606100A1 WO 1986006100 A1 WO1986006100 A1 WO 1986006100A1 US 8600772 W US8600772 W US 8600772W WO 8606100 A1 WO8606100 A1 WO 8606100A1
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hpip
pip
protein
fraction
active
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PCT/US1986/000772
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English (en)
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Lorin F. Johnson
John P. Longenecker
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Biotechnology Research Partners, Limited
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Publication of WO1986006100A1 publication Critical patent/WO1986006100A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4721—Lipocortins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/71—Expression systems using regulatory sequences derived from the trp-operon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the field' of treating inflammation in humans and animals. More particularly, the invention concerns a pure preparation of a phospholipase inhibitory protein (PIP) which is effective in controlling inflammation.
  • PIP phospholipase inhibitory protein
  • inflammation may occur as a chronic inflammatory disorder, such as, for example, rheumatoid arthritis or systemic lupus erythomatosis. Such conditions are debilitating in themselves and can result in often life threatening, acute episodes.
  • the inflammation associated with another such disorder, asthma may also result in death due to constriction of the bronchia.
  • the picture is a complex one. Part of the initiation process is mediated by peptide kinins, such as bradykinin, which are liberated by kallikrein proteases upon tissue destruction.
  • the kinins or other peptide messengers act on specific cell receptors at the inflammation site to activate the phospholipase enzymes A2 and/or C, to initiate the arachidonate cascade.
  • the eicosanoids include the leukotrienes and prostaglandins, which are released into the extracellular environment to exert their effects directly on the inflammatory site. They have relatively short half lives. However, their physiological effects are varied and dramatic, and include vasodilation (e.g., prostacylin and leukotrienes LTC, and LTD ⁇ ),
  • vasoconstriction e.g., thromboxane and LTB
  • hista ine release e.g., hista ine release
  • lipocortin a human form of the inhibitor termed lipocortin has been identified in human fibroblasts (Errasfa, M. , et al. Biochim Biophys Acta (1985)
  • the invention provides purified human phospholipase inhibitory protein (hPIP) and materials useful in its production by recombinant technology.
  • hPIP human phospholipase inhibitory protein
  • a material purified to apparent homogeneity from human peritoneal dialysis fluid having a single 40 kd band on SDS-PAGE contains substantial amounts of apolipoprotein IV (apoAIV) and significant PIP activity.
  • apoAIV apolipoprotein IV
  • Immunization of rabbits with protein eluted from this 40 kd band raises antisera capable of reacting both with apoAIV and with PIP. These antibodies are thus appropriate for screening recombinant and other cells for hPIP production.
  • Human PIP free from other proteins normally in association with it is obtained in two ways, by direct purification from peritonyl dialysis fluid, and by production using recombinant hosts. Accordingly, in one aspect, the invention relates to human PIP in substantially pure form. In another aspect, the invention relates to a protein having human PIP activity which comprises the amino acid sequence set forth in Figure 13. In still another aspect, the invention relates to a process for the preparation of purified hPIP from peritoneal dialysis fluid and to the product of that process. The product of the purification process is characterized by a 36 kd or 40 kd molecular weight in the nonglycosylated or glycosylated form, respectively, and by PIP activity.
  • Pure hPIP may also be produced using recombinant techniques. Accordingly, other aspects of the invention relate to this recombinantly produced hPIP in both nonglycosylated and glycosylated form, to expression systems which permit production of this protein in recombinant hosts, to vectors containing the expression system, to hosts transformed with the system and to a method of producing hPIP by culturing recombinant host cells.
  • the invention relates to the antibodies produced in response to administration of the 40 kd mixture of apoAIV and PIP and of the PIP protein of the invention itself, to pharmaceutical compositions containing PIP, and to methods for ameliorating inflammation in human and veterinary subjects using such compositions or purified PIP*. It appears that PIP may be stabilized by the presence of apoAIV, and compositions containing PIP in admixture with apoAIV are specifically useful as pharmaceuticals.
  • Figure 1 shows the arachidonate cascade and the eicosanoid products of the reaction scheme.
  • Figure 2 shows an outline of the purification procedure used herein.
  • Figure 3 shows elution patterns obtained when the 40%-60% saturation ammonium sulfate fraction of peritoneal dialyzate is subjected to Affi-gel blue, concanavalin A-sepharose. and DEAE cellulose chromatography.
  • Figure 4 shows the results of SDS-PAGE on unpurified and purified fractions of peritoneal dialysis fluid.
  • FIG. 5 shows relative PIP activity at various stages of the purification scheme.
  • Figure 6 shows the results of analytical reverse-phase high performance liquid chromatography (RP-HPLC) performed on the activity-containing fractions from a concanavalin A-sepharose column.
  • Figure 7 displays the ability of polyclonal antisera directed against purified hPIP to detect hPIP in various fractions during the purification procedure.
  • Figure 8 shows detection of hPIP bound to PA2 by anti-40kd antibodies.
  • Figure 9 shows the results of Western Blot for cells producing hPIP.
  • Figure 10 shows the nucleotide and deduced amino acid sequence for the insert in pU200.
  • Figure 11 shows the nucleotide and deduced amino acid sequence for the insert in pU500.
  • Figure 12 shows the nucleotide and deduced amino acid sequence for the insert in p600.
  • Figure 13 shows the nucleotide and deduced amino acid sequence for the insert in pLE-1 having the complete coding sequence for hPIP.
  • Figure 14 shows the relationship of various cloned hPIP cDNA and genomic segments.
  • Figure 15 shows suppression of PA2 activity in hPIP-transformed CHO cells.
  • Figure 16 shows PA2 inhibition by recombinant hPIP,
  • Figure 17 shows comparative amino acid- sequences for hPIP and phospholipase.
  • Figure 18 shows the activity of the 40 kd protein in an jLn vitro assay measuring- PGE release.
  • Figure 19 shows the results of an in vivo rat pleurisy inhibition assay for hPIP activity.
  • Figure 20 shows the activity of the hPIP injected simultaneously with carrageenin in an in vivo paw-edema assay.
  • Figure 21 shows the relative abilities of 100 ⁇ g of purified hPIP, 1 mg of indomethacin and 200 ⁇ g of dexamethasone to inhibit hind paw edema when administered into the peritoneal cavity.
  • Figure 22 shows the relative abilities of various doses of the purified hPIP to inhibit hind paw edema when administered to rats via the femoral vein.
  • Figure 23 displays the relative abilities of purified hPIP and dexamethasone (200 ⁇ g) , when administered to rats intramuscularly, to inhibit joint swelling associated with adjuvant induced arthritis.
  • the protein of the invention has been prepared using as a starting material, human peritoneal dialysis fluid obtained from dialysis patients.
  • a purification procedure is presented whereby the protein has been obtained in homogeneous form, free of impurities normally associated with the protein as found in its native state. While the purification procedure is successful in obtaining this protein in sufficient purity to be used therapeutically per se. the availability of the purified material is also a significant step in the development of alternative modes of preparation. Recombinant techniques for the preparation of hPIP are described herein. Accordingly, the PIP of the invention includes not only the hPIP prepared as herein described, but proteins of substantially similar structure obtained using alternative approaches.
  • substantially similar is meant that the activity of the protein in inhibiting phospholipase A2 (PA2 inhibition assay), as described in the in vitro enzymatic assay procedure set forth hereinbelow, is retained.
  • PA2 inhibition assay phospholipase A2
  • the amino acid sequence set forth in Figure 13 is known to exhibit this activity.
  • amino acid sequences may be modified in various ways and still retain their fundamental activity.
  • proteins since proteins contain ionizable hydrogen, the ionization state of the protein as neutral or salt form, is dependent on the pH of the surrounding medium, if the protein is in solution, or on the pH of the solution from which the protein is prepared in solid form.
  • the purified or recombinant PIP protein of the invention is defined functionally, but all embodiments are expected to retain extensive primary sequence homology with the hPIP exempli-fied in Figure 13. The level of homology is expected to be above 40% considering both conservative changes and exact homologies in the region of interest encoded by nucleotides 490-852 of Figure 13. Additional variation is acceptable in other regions of the protein. All of the foregoing modifications are within the definition so long as.the activity as exhibited in the in vitro phospholipase A2 (PA2) inhibition assay, set forth hereinbelow, is not destroyed.
  • PA2 in vitro phospholipase A2
  • Active PIP fragment refers to a peptide comprising, with respect to PIP. only the sequence encoded by the above-mentioned nucleotides 490-852 of Figure 13. If only this fragment is used, somewhat less than 40% homology in the primary structure is required when the secondary structure is sufficiently similar, as determined by application of the standard algorithms of Chou-Fasman or Kyte-Doolittle.
  • the peptide encoded by nucleotides designated 31-381 in the lipocortin disclosed by Wallner et al (supra) provides a secondary structure filling this requrirement, though the primary amino acid sequence clearly has less than 40% homology in this region. Therefore, this particular fragment is also claimed, as well as those derived from the hPIP sequence disclosed herein.
  • operably linked refers to a juxtaposition wherein the components are configured so as to perform their usual function.
  • control sequences operably linked to coding sequences are capable of effecting the expression of the coding sequence.
  • Control sequence refers to a DNA sequence or sequences which are capable, when properly ligated to a desired coding sequence, of effecting its expression in hosts compatible with such sequences.
  • control sequences include promoters in both procaryotic and eucaryotic hosts, and in procaryotic organisms also include ribosome binding site sequences, and, in eucaryotes. termination signals. Additional factors necessary or helpful in effecting expression may subsequently be identified.
  • control sequences simply refers to whatever DNA sequence may be required to effect expression in the particular host used.
  • Cells or “recombinant host cells” or “host cells” are often used interchangably as will be clear from the context. These terms include the immediate subject cell, and, of course, the progeny thereof. It is understood that not all progeny are exactly identical to the parental cell, due to chance mutations or differences in environment. However, such altered progeny are included when the above terms are used.
  • Dialysis fluid sufficient to yield approximately 2 1 of fluid is conveniently obtained in one batch from patients undergoing continuous ambulatory peritoneal dialysis. Successive 2 1 batches are processed as described below to obtain the desired quantity of pure product.
  • the fluid is first subjected to increasing concentrations of ammonium sulfate: the fraction precipitated at approximately 40%-60% ammonium sulfate saturation contains the activity and is subjected to further purification. It is helpful to pre-precipitate proteins which are insoluble at less than 40% ammonium sulfate.
  • the precipitates are recovered by centrifugation and then subjected to further purification.
  • the ammonium sulfate-containing fraction is dissolved in a suitable buffer of approximately pH 8 and then dialyzed against the same or comparable buffer at low temperature.
  • the dialyzate is then subjected to a treatment which removes the albumin component, the largest quantity contaminant. Chromatography on the affinity support Affi-gel blue previously equilibrated in the same buffer is suitable for this purpose.
  • the desired PIP-containing fraction is not bound to the column under these conditions and appears in the flow-through volume, although contaminating proteins in the solution are retained by the column and are eluted at higher salt concentration.
  • the PIP fractions are treated with a lectin support, such as, for example, concanavalin-A sepharose, lentil lectin-sepharose. or peanut agglutinin-sepharose. preferably concanavalin-A sepharose equilibrated with similar pH 8 buffer. Again the desired activity does not adhere to the column under these conditions.
  • a lectin support such as, for example, concanavalin-A sepharose, lentil lectin-sepharose. or peanut agglutinin-sepharose. preferably concanavalin-A sepharose equilibrated with similar pH 8 buffer. Again the desired activity does not adhere to the column under these conditions.
  • the PIP-containing flow through fractions are further purified by anion-exchange chromatography. using, for example, DEAE cellulose, QAE-cellulose, or SP-cellulose, preferably DEAE cellulose, equilibrated in a similar buffer at approximately pH 8.
  • the hPIP adsorbs to the column and fractions are eluted using a convenient salt gradient.
  • the PA2 inhibitory activity of the eluate fractions is measured to ascertain the desired PIP fractions.
  • SDS-PAGE When the active fractions are subjected to preparative SDS-PAGE, SDS-PAGE yields an 18 kD band of inactive protein, and an active 40 kD band.
  • the 40 kD band was eluted and reconstituted in the absence of detergent, to obtain protein exhibiting the desired activity.
  • the preparative procedure is conducted substantially as described by Laemli, U.K., Nature (1970) 23_:680-685; and an approximately 3 mm 12.5% gel is appropriate to purify a mixture containing 10 mg total protein.
  • the 40 kd band obtained as described has high PIP activity as demonstrated in a number of in vitro and in vivo assays as described in section D below, but contains, in addition to PIP, apoAIV and additional protein(s) of similar molecular weight. It is believed that apoAIV following PIP through this purification procedure confers stability on PIP, and that PIP* may occur in situ as an apoAIV complex. Once isolated from apoAIV as described below, PIP becomes relatively labile. Therefore, it may be advisable to prepare PIP in pharmaceutical compositions in the form of this complex. The association of PIP activity with a 40 kd protein was further confirmed by performing analytical RP-HPLC on the active fractions resulting from concanavalin-A sepharose treatment.
  • the 40 kd band when injected into rabbits, yielded polyclonal antisera capable of immunoreaction so as to bind PA2 inhibitory activity and so as to bind protein capable of forming a complex with phospholipase A. Therefore, although the immunogen also contains apoAIV, the elicited antibodies are reactive with PIP.
  • the PIP proteins of the invention show in vitro activity as phospholipase inhibitors, are capable of suppressing the production of PGE in cell culture, and are effective in several in vivo models in combatting inflammation. Accordingly, the proteins of the invention are useful in ameliorating, treating, or reducing undesirable inflammatory symptomologies in human and veterinary subjects.
  • the proteins of the invention are useful both to control inflammatory responses which are excessive, but formed in response to external stimuli such as infection or wounding, and to treat inflammatory disorders such as rheumatoid arthritis, asthma, unwanted edema, dermatitis, arthritis, conjunctivitis, allergies, and lupus erythomatosis.
  • Administration of the proteins of the invention is in a general dosage range of approximately 0.1-100 ⁇ g/kg, more preferably 0.1-10 ⁇ g/kg per host body weight.
  • the quantity administered depends, of course, on the nature of the subject, the severity of the condition to be treated, and on the mode of administration. Intravenous injection, for example, generally requires smaller amounts than alternative routes.
  • the PIP protein may be administered in a -single dose, over several partial doses, or by constant infusion over a extended time period until the desired benefits are obtained.
  • the protein may be administered in aqueous solution or in the presence of additional pharmaceutical excipients, depending on the mode of administration desired.
  • Protein pharmaceuticals are desirably administered by injection, such as subcutaneous, intravenous, or intramuscular injection, or parenterally through a membrane transfer. Aerosol or oral administration may also be possible in the presence of stabilizing compositions. Complexation with apoAIV is stabilizing to PIP in these compositions, as well as in forms intended for injection.
  • Injectables are prepared in conventional forms as liquid solutions or suspensions, in solid form suitable for reconstitution prior to injection, or as emulsions, either of PIP or of its apoAIV complex.
  • Suitable excipients are, for example, water, saline, dextrose, and the like. Minor amounts of auxilliary substances such as buffering agents, emulsifying agents, and so forth may also be included.
  • Suppository administration may additionally employ binders and carriers such as polyalkylene glycols and triglycerides; aerosol administration, which would be especially suitable for relief of bronchial problems generally utilizes the PIP protein or its complex in finely divided form along with a surfactant and propellant.
  • Typical surfactants include fatty acid esters; typical propellants are the lower alkanes or fluorinated alkanes, such as freon. Topical administration in the form of lotions or salves is also practical, and is preferred in the case of localized treatment.
  • Both the 40 kd SDS-PAGE eluate containing both PIP and apoAIV and the purified PIP protein of the invention are also useful in order to prepare antisera or monoclonal antibodies for immunoassays useful in diagnosis and in monitoring of therapy.
  • Design of immunoassays is well understood in the art, and can take a.variety of permutations.
  • Either competitive antigen or antibodies can be labeled using radioactive materials, fluorescent materials, or enzymes.
  • the assay may be conducted as a direct detection of the antigen-antibody complex, as a competition assay for imraunocomplexing, or as a sandwich assay wherein * the complex is further immunoreactive with an additional antibody.
  • the assays may employ standard procedures; the contribution of the invention is the provision of suitable antigen, both for direct use as standards or competitive antigen for conduct of such assays and as antibody raising material for the preparation of suitable antisera. D. Examples
  • the precipitate which contained the PA2 inhibition activity, was dissolved in 20 mM ammonium bicarbonate, pH 7.8 buffer (buffer A) containing 1 mM phenyl methyl sulfonyl fluoride (PMSF) and aprotinin at 50 ⁇ g/ml.
  • buffer A pH 7.8 buffer
  • PMSF phenyl methyl sulfonyl fluoride
  • aprotinin at 50 ⁇ g/ml.
  • the reconstituted solution was dialyzed against buffer A at 4°C to lower the total salt concentration, and shown to be active in the in vitro PA2 inhibition assay set forth below.
  • the active fractions were pooled, lyophilized. and then reconstituted and applied to a 2.5 cm x 12 cm concanavalin-A sepharose column pre-equilibrated in buffer A ' . Elution at a flow rate of 0.2 ml/min yielded the elution profile shown in Figure 3b. Again, all activity, as assayed by the n vitro PA2 inhibition assay, was found in the flow through volume.
  • the active fractions were pooled and lyophilized. • A small portion of the active fraction pool was set aside for RP-HPLC analysis, as described below.
  • the active DEAE eluate fractions were pooled, concentrated, desalted, and approximately 10 mg of the protein was loaded onto a 3 mm 12.5% polyacrylamide gel for electrophoresis separation according to the procedure of Laemli (supra) except that the disulfide bonds were not reduced with ⁇ -mercaptoethanol prior to fractionation. Protein bands were detected by a 3 min staining procedure using 0.1% Coomassie blue in 50% trichloroacetic acid, followed by 5 min of destain in 5% acetic acid solution. Only two major protein bands resulted: one at 18 kD and the other at 40 kD.
  • Figure 4 shows the comparative results of SDS-PAGE performed on the original extract, on the preparation at various stages of purification, and on the purified protein.
  • the bands were developed using. silver stain (BioRad Labs. Richmond, CA) by two successive oxidation and staining procedures according to the manufacturer's instructions.
  • Lane 1 contains molecular weight markers; lane 2 is the dialysis- fluid before ammonium sulfate treatment; lane 3 is the 40%-60% ammonium sulfate precipitate; lane 4 contains the pooled active fractions from Affi-gel blue chromatography; lane 5 contains pooled active fractions from concanavalin-A sepharose chromatography; lane 6 contains pooled active fractions from DEAE chromatography; lanes 7 and 8 contain 1 ⁇ g and 50 ng respectively of the 40 kD band obtained from preparative gel electrophoresis. The entire procedure results in a purification of approximately 500 fold as shown by the results illustrated in Figure 5.
  • the specific activity of the crude extract is approximately 0.2% of that for the purified 40 kD band.
  • a portion of the active fractions resulting from concanavalin-A sepharose treatment described above was subjected to RP-HPLC using a C 8 column and elution with a gradient of acetonitrile in 0.1% trifluoroacetic acid.
  • Figure 6 shows the elution profile for the column. A multiplicity of protein fractions are obtained, only one contains the activity.
  • This active fraction was then subjected to analytical SDS-PAGE using a 12.5% gel, and the developed gels were stained with Coomassie blue G-250 or with silver reagent. Only a 40 kd band was observed in each case.
  • the pooled active eluate from the anion exchange resin above was dialyzed against pH 5.5 sodium citrate buffer. Protein which precipitated was. removed by centrifugation and the proteins in the supernatant were subjected to preparative SDS-PAGE. The 40 kd band is then recovered as described above. Alternatively the supernatant can be brought to pH 7.5 and passed through a column of PLA2-Sepharose as described by. Parente, L.. et al. Life Sci (1985) 3_6_:1225-1231. After washing the column with 150 mM NaCl the bound hPIP is eluted with either 1.0 M NaCl or 0.1 M acetic acid in pure form.
  • the 40 kd band above was subsequently shown to be a mixture of proteins whcih includes hPIP and apolipoprotein AIV (apoAIV). as will be further described below. However, this mixture was capable of raising antibodies specifically reactive with hPIP.
  • apolipoprotein AIV apolipoprotein AIV
  • To obtain anti-hPIP New England white rabbits were injected subcutaneously or intramuscularly with 200 ⁇ g of the eluted 40 kd band fraction contained in complete Freunds adjuvant. The rabbits were boosted at 3-week intervals with the same vaccine.
  • the rabbits were then bled from the ear vein 7-10 days after the boost, and the resulting serum was tested for its ability to bind the 40 kd eluate described above, using non-immune serum as control.
  • 500 ng of purified hPIP-containing 40 kd protein was immobilized on individual wells of a polystyrene plate, and varying dilutions of antisera added to the wells. The quantity of specifically bound antibodies was quantified using
  • the immunoreactivity of the antisera was further used to show the progress of purification by Western blot.
  • the samples to be tested against the antisera were diluted 1:1 with 2 x SDS:sample buffer (Laemli, supra) and 10-25 ⁇ g of protein is fractionated on 1.5 mm thick SDS-polyacrylamide gels (12.5% acrylamide).
  • the fractionated proteins were electro-transferred to nitrocellulose sheets and immuno eactive 40 kd protein is detected with anti-hPIP
  • Figure 7 shows the results of Western blot performed on the same fractions shown in Figure 4 obtained at various purification stages.
  • Lane 1- is 43
  • lane 2 is the dialysis fluid
  • the 40 kd band is specifically detected throughout purification.
  • PA2 phospholipase A2
  • Figure 8 PA2 (150 pmol) was immobilized on polystyrene wells and excess sites were blocked with human serum albumin. Increasing quantities of 40 kd protein were then added in the absence and presence of 1500 pmol free PA2 and binding continued for 30 min at
  • preincubation of the antisera at 1:100 dilution with 10 ⁇ g of the 40 kd mixture neutralized its ability to detect protein bound to immobilized PA2.
  • Candidates for screening include cultured human inflammatory cells such as the monocyte-macrophage cell lines U937 and HL60, and isolated human blood cells, such as polymorphonuclear cells (PMN) , as well as lung tissue which is known to contain ⁇ 20% macrophages.
  • the cells were incubated .in appropriate culture media overnight, either containing serum or serum-free, in the presence and absence of dexamethasone (0.1 ⁇ M) .
  • Cell supernatants or the cells themselves, as appropriate were assayed for protein immunoreactive with anti-hPIP. For cell lysates.
  • the lysate was collected from the culture dish and nuclei were removed by centrifugation (1000 x g, 5 min) .
  • the samples (supernatants or lysates) to be tested against the antisera were diluted 1:1 with 2 X SDS:sample buffer (Laemmli, U.K., supra) and 50-100 ⁇ g of protein was fractionated on 1.5 mm thick SDS-polyacrylamide gels (12.5% acrylamide) .
  • the gels were developed as above with the addition of using anti-hPIP antisera preincubated with 50 ⁇ g of purified 40 kd protein as a control for nonspecific binding of the antibody.
  • the hPIP immunoreactivity in several cell-types can be quantitated by densitometric scanning of the autoradiogram.
  • hPIP is " secreted into the culture media.
  • Partially purified hPIP from such culture media are assayed as above.
  • the conditioned media may be subjected to ammonium sulfate fractionation and chromatography on Affi-gel blue and Con-A sepharose. Fractions which do not bind to the Con-A sepharose column are lyophilized and analyzed for hPIP imraunoreactivity on Western gels as described.
  • Figure 9 shows the results obtained from both serum-containing and serum-free media conditioned for 48 hours by U937 cells.
  • the PIP protein appears as a 37 kd band in the medium from serum-free cultured cells.
  • the immunoreactive band at ⁇ 40,000 daltons is most probably apoAIV, as it is present in serum and in serum-containing U937-conditioned media, but not in serum-free U937-conditioned media.
  • the immunoreactive band seen at ⁇ 68,000 daltons represents albumin -contained in the serum.
  • the 50,000 dalton band is the IgG heavy chain, as it is only seen in serum-containing media and is also detected when nonimmune antiserum is used; the IgG heavy chain can
  • 125 bind I-protein A independent of the antibody used to probe the Western.
  • two separate cDNA libraries can be constructed, one using random primers (P. L. Biochemicals) and the other using an oligo-dT primer.
  • the first strand synthesis uses 15 ⁇ g of poly A RNA with the appropriate primer and is synthesized by reverse transcriptase (Avian Myeloblastosis Virus) using standard reaction conditions.
  • the second strand of each cDNA preparation is synthesized using RNAse H (Gubber, U., et al. Gene
  • Polymeric linkers are reduced to monomers by subsequent digestion with EcoRI, and the fragments can be screened to a specific size class by electrophoresis through low melting point agarose (1.5% agarose) and recovered, if desired.
  • the resulting double stranded cDNA is ligated into the unique EcoRI site of the .bacteriophage. expression vector ⁇ gtll as described by Young. R.A. and Davis, R.W. , Science (1983) 222:778-782.
  • any cDNA whose reading frame and orientation are also correct with respect to the EcoRI site may be expressed as a fusion protein with ⁇ -galactosidase upon induction with isopropyl thiogalactoside (IPTG).
  • IPTG isopropyl thiogalactoside
  • the random frequency of this occurring is one in six. (Two orientations times three reading frames.)
  • the proteins expressed by such induced phage plaques were also screened with polyclonal antisera directed against hPIP prepared using the 40 kd protein above.
  • the recombinant DNA was packaged in vitro using a commercial packaging extract (Amersham, Inc.) and the recombinant phage were titered on the host strain E. coli C600Hf1 as described by Young, R.A.. et al (supra). About 200.000 recombinants were obtained.
  • the cDNA library was screened by antibodies
  • the filters were processed by first blocking nonspecific binding with 5% nonfat dry milk dissolved in PBS, 0.1% NP-40.
  • the filters were incubated with a 1:100 dilution of the antiserum prepared above in PBS, 0.1% NP-40, 5% BSA, 5% ovalbumin. for 2 hr and then washed in PBS, 0.1% NP-40.
  • 125 5 antibody was detected using I-protein A (10 cpm/filter) and autoradiography of the washed filters.
  • Each cDNA library was prepared for screening by growing approximately 10 phage on E. coli C ⁇ OOHfl, and transferring plaque lysates to nitrocellulose filters. The filters were processed for hybridization . as described by Benton, W.E., et al. Science (1977) 196:180-182. The probes were labeled by nick translation according to Maniatis, T.. et al. Cloning
  • the U937 ⁇ gtlO library yielded several hybridizing cDNAs, of which the longest was about 500 bp and designated ⁇ U500.
  • the nucleotide sequence of " ⁇ .U500 was determined by dideoxy sequencing and is shown in Figure 11. Comparison of Figure 11 with Figure 10 shows that U200 and ⁇ U500 share approximately 50 bp at the 5' ends of each and then diverge. This divergence was later shown to be due to intron splicing, as described below.
  • the ⁇ gtlO PMN library yielded four hybridizing positives having sizes of 400-600 bp; the longest cDNA was designated PMN600 and dideoxy sequenced.
  • the sequence determined for PMN600, along with the deduced amino acid sequence, is shown in Figure 12.
  • PMN600 not only shares the 50 bp segment common to ⁇ U200 and ⁇ U500, but its homology with ⁇ U500 extends for another approximately 200 bp. These sequences then also diverge. This divergence was shown to be due to the presence of optional 3 ' terminal exons in the gene., as set forth below.
  • the fetal lung library was probed as described above using a synthetic oligonucleotide of the sequence 5 ' -GAAGGTAGCCACAGCCACGG-3 ' , which represents the bases 23-42 of the sequence of PMN600 ( Figure 12).
  • a positively hybridizing cDNA contained additional upstream sequences which could, in fact, be mapped onto an upstream exon of the genomic clone described below.
  • This cDNA was subcloned into pBR329. designated pSR-1 and used as follows to determine the size of the PIP encoding mRNA:
  • Primer extension analysis using a 586 bp BamHI fragment from pSR-1 as the primer, showed that an additional 500 bases were required to obtain full-length cDNA.
  • the adult human lung cDNA library was therefore screened using two probes: PMN600 cloned into pBR329 (p600). and a synthetic oligonucleotide 5'-ATGAGCTGTGAGAGGGGCCG-3• . which maps to the 5• end of pSR-1.
  • Four positively hybridizing plaques were purified and sized, and found to contain either 1360 or 1340 bp.
  • These cDNAs were cloned into M13mp8 and M13mp9 for dideoxy sequencing, and one of them shown to contain the full-length coding sequence for hPIP.
  • Figure 13 shows the complete DNA sequence and the deduced amino acid sequence encoded by this clone.
  • pLE-1 which ' corresponds to a primary translation product of 331 amino acids representing a protein of approximately 36.5 kd. The mature protein is believed to begin at nucleotide 112-114, encoding leucine.
  • RNA -polymerase SP6 System, Amersham Corporation. Arlington Heights, IL
  • the translation product showed the predicted size of 36 kd. It is believed that the discrepancy between the 36 kd protein encoded and the 40 kd protein associated with peritoneal fluid is due to glycosylation.
  • the sequence of pLE-1 has four canonical glycosylation sites (Asn-X-Ser/Thr) beginning at bases 625, 595, 709. and
  • RNA transcribed as above from SP6-pLE-l into Xenopus laevis oocytes (which can glycosylate the primary translation product) and localizing the mature 40 kd product in the membrane fraction of the oocytes.
  • D.5. Isolation of Genomic Clones Encoding hPIP The complete DNA sequence encoding hPIP was also isolated from a human genomic library. The pU200 and the Ncol fragment containing bases 198-590 from p600 were used as probes; they have no common sequences. The library, in ⁇ Charon phage, was constructed from
  • exon V appears to be provided in an alternate form VA, as shown in Figure 14.
  • Exon VA is not present in ⁇ l2-3, but is found in ⁇ l2-l, a genomic clone which hybridizes to the Ncol fragment and pU500.
  • the cDNA clone encoding hPIP is conveniently used to produce the recombinant protein in a variety of hosts, as set forth in 1FE.1 below. However, expression in mammalian systems is favored as the host is capable of post translational processing analogous to that experienced by the natively produced protein. Either cDNA or genomic sequences may be used, as the host is also capable of processing introns.
  • the full length cDNA hPIP encoding clone, pLE-1 is inserted as an EcoRI fragment into the mammalian expression vector pHSl as described below.
  • pHSl the Host Expression Vector
  • the plasmid pHSl contains 840 bp of the hMT-II sequence from p84H (Karin, M.. et al. Nature (1982) 299: 297-802) which spans from the Hind.III site at position -765 of the hMT-II gene to the BaraHI cleavage site at base + 70.
  • Plasmid p84H was digested to completion with BamHI. treated with exonuclease Bal-31 to remove terminal nucleotides. and then digested with Hindlll. The desired 840 bp fragment was ligated into pUC8 (Vieira, J.. et al.
  • pHSl contains the hMT-II control sequences upstream of a polylinker containing convenient restriction sites.
  • pLE-2 lacks the sequence at the C-terminal region of the hPIP protein, which is believed to be responsible for its binding to the cellular membrane.
  • pLE-2 was constructed by subjecting M13 phage harboring the pLE-1 sequence shown in Figure 13 to site-directed rautagenesis to place a TAA stop codon at base 951 to replace TAC (encoding a tyrosine residue).
  • the oligonucleotide 5'-CACTGCGTTACTGGA-3 ' was used as a primer and the mutated sequences were retrieved by screening the recombinant phage plaques using kinased oligonucleotide 5 '-GCAGCCCCACTGCGTTACTGGACATCCAG-3 ' as a probe, under stringent washing of 55°C, 3 M tetramethyl ammonium chloride.
  • the mutagenesis was confirmed by dideoxy sequencing and the single-stranded form converted to double-stranded DNA. Correct functioning of the stop codon was also confirmed by subcloning the EcoRI insert into the SP6 vector system, transcription with RNA polymerase, followed by translation in the reticulocyte lysate system.
  • SDS-PAGE of the isolation product shows a 34 kd protein, as compared to the 36.4 kd protein obtained from full-length hPIP-encoding sequences.
  • the SP6 mRNA was also injected into Xenopus oocytes. Media conditioned by the injected cells for 18 hr showed PIP activity as assayed by the release of labeled arachidonate from zymosan-stimulated mouse peritoneal macrophages. Media conditioned by oocytes injected with mRNA from the LE-1 sequences cloned into SP6 failed to show activity in this assay.
  • the EcoRI insert was transferred into pHS-1, as described above for the construction of pLE-1 to give pMT-PIP(-).
  • pLE-3 contains an alternate 3'-terminal sequence encoded by the VA exon which had been retrieved in pUSOO above.
  • pLE-3 was constructed by replacing the downstream sequences in pLE-1 with the appropriate fragment from pUSOO: ⁇ LE-1 was digested with Ncol and EcoRI and the unique 718 bp fragment purified.
  • pUSOO was digested with EcoRI and Ncol and the unique 419 bp downstream portion of the cDNA insert purified. Ligation of these fragments resulted in a 1137 bp fragment which was inserted into the EcoRI site of pUC8 and transfected into E.
  • Plasmids containing the correct orientation and placement of the insert were designated pLE-3.
  • pLE-3 coding sequences were modified by addition of a polyadenylation signal to the 3* untranslated sequence by digesting pLE-3 with Avrll. blunting, and further digesting with EcoRI to obtain the hPIP encoding fragment.
  • the unique 1037 bp cDNA insert was then isolated.
  • a poly-A addition site derived from apoAI cDNA was isolated by digesting plasmid pBL13Al (Seilhamer. J.. et al, DNA (1984) 3.:309-317) with Narl.
  • an alternative signal sequence such as that derived from human growth hormone, human alolipoprotein Al, human lung surfactant, or human renin. This can be accomplished using standard procedures i.e., by digesting pLE-1, pLE-2 or pLE-3 with SphI to remove the 5' end of the cDNA, replacing lost codons of the mature protein, and ligating the appropriate signal sequences upstream therefrom.
  • hPIP is predicted to be the leucine beginning at base 112 of Figure 13 and SphI cuts at base 124 it is necessary to reconstruct the first four amino acids using oligonucleotides encoding Leu-Arg-Cys-Met.
  • Production of hPIP by Mammalian Recombinants Chinese hamster ovary (CHO)-Kl cells were grown on medium composed of a 1:1 mixture of F12 medium and DME medium with 12% fetal calf serum.
  • the competent cells are co-transformed with pMT-PIP, pMT-PIP(-), pMT-PIP/A or pAc-PIP/A and pSV2:NEO (Southern, P., et al.
  • pSV2:NEO contains a functional gene conferring resistance to the neomycin analog G418.
  • 500 ng of pSV2-NEO and 5 ⁇ g of pMT-hPIP are applied to a 16 mm dish of cells in a calcium phosphate-DNA co-precipitate according to the protocol of Wigler, M.. et al. Cell (1979) .16.: 777-785, with the inclusion of a two minute "shock" with 15% glycerol after four hours of exposure to the DNA.
  • the cells are subjected to 1 mg/ml G418 to provide a pool of G418-resistant colonies.
  • Successful transformants, also having a stable inheritance of PIP-encoding plasmid were plated at low density for purification of clonal isolates. For transformants containing PIP sequences under control of the MT promoter, small amounts of these isolates were
  • hPIP production was assayed by demonstration of inhibition of induced PA'2 activity in the cultured cells.
  • the media were harvested and concentrated by Amicon UM-100 ultrafiltration. To obtain the membrane, the cells were treated with 1 mM EDTA and collected b'y centrifugation at 100 x g for 5 min. The cell pellet was res.uspended in 2 ml of 10 mM Tris, pH 8, 250 mM sucrose. 150 mM NaCl. 1 mM EDTA. 1 mM PMSF, and broken in a glass homogenizer. monitoring the cell rupture by phase contrast microscopy.
  • the nuclei were removed by centrifugation for 5 min at 1000 x g, and the supernatant lysate was fractionated into soluble and membrane fractions by centrifugation at 100,000 x g for 1 hr.
  • the nuclear fraction was extracted with 0.1% Tween-20, and insoluble material removed by centrifugation.
  • the membrane pellet from the lysate supernate was resuspended in homogenizing buffer, which further included 10% glycerol.
  • the media, cell lysate soluble fraction, membrane, and nuclear extract fractions were then assayed for PIP activity using two separate assays: Inhibition of the release of labeled arachidonic acid from the cellular membranes of zymosan-stimulated mouse resident peritoneal macrophages as described by Bonney, R.J.. et al. Biochem J (1979) 176:433-440: and inhibition of release of C-14-labeled oleic acid incorporated into E. coli membranes in an in vitro assay using porcine pancreatic PA-2, as described by Vadas, P., et al. Life Sci (1985) 3_6:579-583. Only the membrane fraction shows activity in both of these assays.
  • the hPIP secreted into the medium can be purified according to the procedures set forth above for the native protein, or by other standard methods known in the art.
  • the sets of pairs were ligated together with T4 ligase and the ligated DNA recovered and digested with EcoRI and PstI.
  • the resulting DNA fragments were visualized by wet gel autoradiography and a 100 bp fragment was eluted and dideoxy sequenced to confirm the designed double stranded sequence above which contains promoter and operator regions of the trp operon and the ribosome binding site of the trp leader peptide.
  • Plasmid pKK233-2 (Amann. E.. et al. Gene (1985) 40.:183-190) was digested to completion with Ndel blunted, and religated to obtain the corresponding pla ' smid lacking the Ndel site, pKK-233-2-Nde.
  • Ten ng pKK-233-2-Nde was digested to completion with EcoRI and PstI, treated with CIP, and mixed with 50 ng of the synthetic EcoRI/PstI trp promoter/operator sequence described above. The mixture was ligated with T4 DNA-ligase, followed by transformation into E. coli
  • Transformants were screened for the- presence of plasmid DNA containing the desired insert; designated pTRP-233.
  • the hPIP encoding segments lacking the native signal sequence were removed from pLE-1,. pLE-2, and pLE-3 by digestion with SphI. which cuts at nucleotide 114, as shown in Figure 13, blunting with Klenow, and then digestion with Hindlll which cuts at a vector site just 3' of the insert.
  • the SphI(blunt)/HindIII fragments were ligated into the KpnI(blunt)/HindIII digested pTRP233. placing the coding sequences under control of the trp promoter, to give pTRP-PIP, pTRP-PIP(-), and pTRP-PIP/A, respectively.
  • the ligation mixtures were transformed into E coli HBlOl to verify correct orientation, and these transformants were grown under standard M9 salts plus 0.5% casamino acids (Difco) to OD-550 of 0.1 before induction with 25 ⁇ g/ml IAA with further growth to OD 1.0.
  • the bacteria were then harvested, lysed with a French press or by sonication and the lysate assayed for PA2 inhibition using the in vitro assay of Vadas et al (supra).
  • the hPIP protein is obtained in nonglycosylated form and can be purified using standard procedures, following the purification by the PA2 inhibition assay.
  • a fraction precipitating at 40-60% saturated ammonium sulfate is resolublilized in 25 mM Tris-HCl, pH 8.0, 2 mM EDTA. and subjected to DEAE Sephadex equilibrated with the same buffer.
  • the protein is eluted in a NaCl gradient at 0.125 M. and purified to homgeneity on a C8 HPLC column or other hydrophopbic column, from which active fractions elute at about 50% acetonitrile* on a 20-100% acetonitrile gradient in 1% TFA.
  • the dried protein in the active fractions is resolubilized in 20 mM Tris, pH 8.0, and reoxidized, if necssary. to the disulfide according to the method of Van Scherrenberg, G.M.. et al. Hoppe-Seyler's Z Physiol Chem (1980) 361:571-576.
  • FIG. 17 shows a comparison of the amino acid sequences of the hPIP of Figure 13 between nucleotide positions 490-852 with the known sequences of pancreatic and C. atrox venom phospholipases. This region of homology is suggested as sufficient for activity, by analogy with the Vipera system, which also shows homology between enzyme and inhibitor (Mancheva, I., et al, Hoppe-Seyler's Z Physiol Chem (1984) 345:885-894) . This region of hPIP with various downstream sequences is obtained as a BstEII(blunt)/HindIII fragment from pLE-1, pLE-2, and pLE-3.
  • any of the above vectors can be modified to encode only the desired fragment representing the desired homologous region. This is accomplished by coverting the CAA glutamine codon at nucleotide positions 853-855 to a TAA stop codon by site specific mutagenesis.
  • pTRP-PIP(F) is digested with Hindlll and EcoRI and cloned into Hindlll/EcoRI digested M13mpl8.
  • the single stranded DNA is treated with DNA polymerase using 5 '-GCATGTTAGCACATT-3 ' as primer, and the resulting double stranded DNA transfected into competent cells.
  • the mutagenized phage are probed with
  • the protein obtained in 1TD.1 as a 40 kd band on SDS-PAGE contained hPIP in N-terminal blocked form and apoAIV with an N-terminus available for sequencing. When the entire fraction was subjected to sequencing using Applied Biosystems 470A gas phase sequencer, the presence of the apoAIV N-terminus was confirmed. Approximately 50 ⁇ g of the protein were subjected to N-terminal sequencing, and PTH amino acids were identified with a Beckman 334T HPLC using an IBM CN column as described by Hunkapiller, M.W. and Hood, L.E., Meth Enzym (1983) ⁇ :486-492. The N-terminal sequence obtained was
  • a minor secondary sequence of the 40 kd mixture could also be read.
  • the sequence obtained was: Glu-Asn-Leu-Pro-Gln-Asn-Gly. Presumably the sequence arises from an internal proteolytic clip of the blocked hPIP molecule, which contains extensive disulfide bonding. This sequence begins at base 662 of the sequence shown in Figure 13 and ends at base 693.
  • Portions 25-50 ⁇ l of the reaction mixture were applied to silica G type TLC plates for analysis in an ascending solvent system containing chloroform, methanol, acetic acid (90:10:1).
  • Arachidonic acid was used as a standard to locate the arachidonic acid band; the phosphatidyl choline band remains at the origin.
  • the spots were visualized by staining with iodine vapor, and the labeled arachidonate and phosphatidyl choline spots were scraped from the plate and radioactivity determined using scintillation counting in toluene-omnifluor (New England Nuclear).
  • the percent hydrolysis was computed as a fraction 100 x cpm arachidonic/(cpm arachidonic + cpm phosphatidyl choline)
  • the hPIP was also shown to be capable of inhibiting the production of PGE by cultured fibrosarcoma cells.
  • confluent cultures of mouse fibrosarcoma cells obtained from ATCC (ATCC #CCL148) were preincubated with various concentrations of the protein to be assayed and 25 ⁇ l phosphate buffered saline in 150 ⁇ l HAMMs F10 medium (Gibco) for 15 min at 37°.
  • HAMMs F10 medium Gibco
  • One hundred ⁇ l of.FlO medium containing 2% fetal calf serum was added, and the cells were incubated for an additional hour at 37° in a humidified incubator with a 5% CO /95% air atmosphere.
  • the media were removed and assayed for PGE using a commercial radioimmunoassay kit (Seragen, Inc., Boston, MA).
  • Figure 18 shows the inhibition of PGE production by purified (by SDS-PAGE) PIP and by DEX.
  • the 18 kD band from the gel (open circles) was used as a control.
  • the closed circles are the results using active fractions from the Con-A Sepharose column.
  • the squares are the results with protein from the 40 kD band; the triangles are results using DEX.
  • the response is dose-dependent for the active proteins and for DEX.
  • the addition of 100 ⁇ g of arachidonic acid to the cells (added at the same time PIP was applied) was able to override inhibition by both PIP and DEX.
  • rat pleurisy model Three in vivo models were employed to demonstrate the activity of PIP: the rat pleurisy model, the rat paw edema model, and the adjuvant-induced arthritis model.
  • the pleural cavities of three groups of rats were inflamed by the injection of 0.1 ml of 0.5% carrageenin in saline, and the- ability of the test substances to control the inflammation was measured.
  • 10 ⁇ g of PIP were injected along with the carrageenin.
  • Carrageenin injections containing 200 ⁇ g of DEX were used as positive controls.
  • Inhibitory activity was shown by decrease in the exudate volume and exudate protein.
  • Ten ⁇ g of the purified PIP was as effective as 200 ⁇ g DEX in suppressing both of these parameters, as shown in Figure 19.
  • the data on the left record the quantity of protein exuded in mg/cavity.
  • the quantity is reduced essentially to normal levels in the presence either of 50 ⁇ g DEX or 10 ⁇ g PIP.
  • the measure of exudate volume is shown on the right in ml. Again either 50 ⁇ g DEX or 10 ⁇ g PIP reduce the exuded fluid volume to normal levels.
  • test substances The anti-inflammatory activity of test substances was examined by administering the test substance in one of three ways: either intravenous injection, intr ' aperitoneal injection, or co-injection with the carrageenin. Except when specified, dexamethasone administered intraperitoneally 3 hr previously, was used as a control. Results were evaluated by measuring the thickness of the hind paw with a constant-pressure caliper starting at time zero
  • Figure 20 shows the results as increase in foot 2 pad thickness (ram x 10 ) of determinations using 0-5 ⁇ g of the purified hPIP, isolated -from SDS-PAGE, in experimental injections or 2.5 ⁇ g dexamethasone in control injections, each co-administered with the carrageenin.
  • the hPIP significantly reduced the edema in a dose-dependent manner.
  • the control DEX also reduced the inflammation, although the time dependence, as shown in Figure 20, is slightly different from that of PIP.
  • Figure 21 shows the results when injection of the test materials was made intraperitoneally 30 min prior to inducing inflammation. Indomethacin as well as DEX was used as a control. The data show that PIP and indomethacin are comparably effective in suppressing paw swelling, although both were slightly less effective than DEX. Accordingly, hPIP is able to enter the circulation and act at the site of inflammation.
  • Figure 22 shows the results when administration was made by injection into the femoral vein 2 min prior to the injection of carrageenin.
  • hPIP was effective in inhibiting inflammation over 3, 4, or 5 hr following IV administration in a dose-dependent manner, and 25 ⁇ g of PIP were equivalent to the effect of 200 ⁇ g DEX after 3 or 4 hr.
  • the results permitted an estimate of the biological half-life of 2-3 hr for PIP.
  • the remaining jln vivo assay was the adjuvant-induced arthritis model, according to the method of Colpaert, F.C., et al. Life Sci (1982) ⁇ :67-75.
  • hPIP as shown in Figure 23, was successful in producing a rapid diminution of hind paw and joint swelling which reached a maximum by 10 hr but the swelling returned to control levels after 28 hr.
  • DEX produced swelling reduction after 3 hr, which was maintained for the full 28 hr.experimental period.
  • procaryotic and eucaryotic systems may be used to express the PIP encoding sequences; procaryotic hosts are. of course, the most convenient for cloning procedures. Procaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used; for example, E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species by Bolivar, et al. Gene (1977) 2.:95. pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides additional markers which can be either retained or destroyed in constructing the desired vector.
  • procaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al. Nature (1977) 198:1056 and the tryptophan (trp) promoter system (Goeddel. et al Nucleic Acids Res (1980) 8_:4057 and the lambda derived P promoter and N-gene ribosome binding
  • eucaryotic microbes such as yeast
  • yeast may also be used as hosts.
  • Laboratory strains of Saccharomyces cerevisiae. Baker's yeast are most used although a number of other strains are commonly available.
  • Vectors employing, for example, the 2 ⁇ origin of replication of Broach, J. R. , Meth Enz (1983) 101:307. or other yeast compatible origins of replications (see, for example, Stinchcomb, et al. Nature (1979) 282:39. Tschempe. et al. Gene (1980) 10:157 and Clarke, L. et al. Meth Enz (1983) 101:300) may be used.
  • Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess, et al, J Adv Enzyme Reg (1968) 7.:149; Holland, et al. Biochemistry (1978) 17:4900) .
  • Additional promoters known in the art include the promoter for 3-phosphoglycerate kinase (Hitzeraan, et al. J Biol Chem (1980) 255:2073) , and those for other glycolytic enzymes.
  • Other promoters, which have the additional advantage of transcription controlled by growth conditions. are the promoter regions for alcohol dehydrogenase 2, isocytochrome C.
  • terminator sequences are desirable at the 3' end of the coding sequences. Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes. It is also, of course, possible to express genes encoding polypeptides in eucaryotic host cell cultures derived from multicellular organisms. See, for example. Axel, et al. U.S. patent 4,399,216. These systems have the additional advantage of the ability to splice out introns and thus can be used directly to express genomic fragments.
  • Useful host cell lines include VERO and HeLa cells, and Chinese hamster ovary (CHO) cells.
  • Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and late promoters from Simian Virus 40 (SV 40) (Fiers, et al. Nature (1978) 273:113), or other viral promoters such as those derived from polyoma. Adenovirus 2, bovine papiloma virus, or avian sarcoma viruses. The controllable promoter. hMT-II (Karin. M. , et al. Nature (1982) 222:797-802) --Y also be used.
  • transformation is done using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride, as described by Cohen, S.N.. Proc Natl Acad Sci (USA) (1972) 12: 110. or the RbCl method described in Maniatis, T. , et al. Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Press, p. 254 may be used for procaryotes or other cells which contain substantial cell wall barriers.
  • the calcium phosphate precipitation method of Graham and van der Eb, Virology (1978) 52.:546, optionally as modified by Wigler, M. , et al. Cell (1979) JL6.:777-785 may be used.
  • Transformations into yeast may be carried out according to the method of Van Solingen. P.. et al, J Bact (1977) 130:946 or of Hsiao. C.L., et al. Proc Natl Acad Sci (USA) (1979) 76:3829.
  • Plasmid plasmids DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired. Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g.. New England Biolabs, Product Catalog. In general, about 1 ⁇ g of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate.
  • Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA 5 polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20 to 25°C in 50 mM Tris pH 7.6. 50 mM NaCl. 6 mM MgCl , 6 mM DTT and 5-10 ⁇ M dNTPs. The Klenow fragment fills in at 5' sticky ends 10 but chews back protruding 3' single strands, even though the four dNTPs are present.
  • Klenow deoxynucleotide triphosphates
  • selective repair can be performed by supplying only one of the, or selected, dNTPs within the limitations dictated by the nature of the sticky ends.
  • the mixture is extracted with phenol/chloroform and ethanol precipitated.
  • Treatment under appropriate conditions with SI nuclease or Bal-31 results in hydrolysis of any single-stranded portion.
  • Synthetic oligonucleotides are prepared by the 20 method of Efimov, V.A., et al (Nucleic Acids Res (1982) 6875-6894). and can be prepared using commercially available automated oligonucleotide synthesizers. Kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g., 25 approximately 10 units of polynucleotide kinase to 1 nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl , 5 mM dithiothreitol.
  • Ligations are performed in 15-50 ⁇ l volumes under the following standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl , 10 mM DTT.
  • vector fragment In vector construction employing "vector fragments", the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in order to remove the .5' phosphate and prevent religation of the vector.
  • BAP bacterial alkaline phosphatase
  • CIP calf intestinal alkaline phosphatase
  • site specific primer directed mutagenesis is used for portions of vectors derived from cDNA or genomic DNA which require sequence modifications*. This is conducted using a primer synthetic oligonucleotide complementary to a single stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage.
  • 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence.
  • the resulting plaques are hybridized with kinased synthetic primer at a temperature which permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization.- Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered.
  • cDNA libraries can be prepared as described above in ⁇ gtll phage-, or double-stranded cDNA synthesized from mRNA isolated using standard techniques can be prepared for insertion into a plasmid vector such as pBR322 using homopolymeric tailing mediated by calf thymus terminal transferase (Sutcliffe, J.G.. Nucleic Acid Res (1978) 5_:2721-2732) .
  • First strand cDNA is synthesized by the RNA-dependent DNA polymerase from Avian Myeloblastosis Virus, by priming with oligo (dT) 12-18 on 5 ⁇ g mRNA.
  • RNA template is then liberated from the nascent DNA strand by denaturation at 100°C for 5 min. followed by chilling on ice.
  • Second strand DNA is synthesized by using the large fragment of DNA polymerase I of E. coli. relying on self-priming at the 3'-end of the first strand molecule, thereby forming a double-stranded hairpin DNA. These molecules are blunt-ended at the open-ended termini, and the hairpin loop is cleaved open with SI nuclease from Asperqillus oryzae.
  • SI nuclease digestion of the double-stranded cDNA takes place in 300 mM NaCl, 30 mM NaOAc, pH 4.5, 3 mM ZnCl for 30 rain at 37*°C with 600 units enzyme.
  • the cDNA is extracted with phenol:chloroform, and small oligonucleotides are removed by three ethanol precipitations in the presence of ammonium acetate. This is done as follows: a half volume of 7.5 M ammonium acetate and two volumes ethanol are added to the cDNA solution, which is precipitated at -70°C.
  • the blunt-ended, double-stranded cDNA is then fractionated by size using gel filtration through a column (0.3 x 14 cm) Sepharose 4B (Pharmacia Fine Chemicals, Piscataway. NJ) or by ultracentrifugation in 5-20% glycerol gradient followed by fractionation of the gradient.
  • cDNA roughly greater than the desired length, e.g.. 300 base pairs is retained and recovered by precipitation with 70% ethanol.
  • Short (10-30 nucleotides) polymeric tails of deoxycytosine are added to the 3' termini of the cDNA using a reaction containing 0.2 M potassium cacodylate, 25 mM Tris, pH 6.9. 2 mM dithiothreitol. 0.5 mM CoCl .
  • the tailed cDNA is annealed with a host vector such as pBR322 which has been cleaved with, for example, PstI and tailed with oligo dG.
  • a host vector such as pBR322 which has been cleaved with, for example, PstI and tailed with oligo dG.
  • 2.5 ⁇ g pBR322-dG DNA is annealed with the cDNA at a vector concentration of 5 ⁇ g/ml, and the hybrids are transferred into E. coli MC1061 by the CaCl -treatment described by Casabadan, M. , et al, Mol Biol (1980) 138:179-207.
  • cDNA or genomic libraries may be screened if desired using the colony or plaque hybridization procedures. Colonies or plaques are replicated onto duplicate nitrocellulose filter papers (S & S type BA-85) and colonies are allowed to grow at 37°C for 14-16 hr on L agar containing 15 ⁇ g/ml tetracycline. The colonies are lysed with 10% SDS and the DNA is fixed to the filter by sequential treatment for 5 min with 500 mM NaOH/1.5 M NaCl, then 0.5 M Tris HCl(pH 8.0)/1.5 M NaCl followed by 2 x standard saline citrate (SSC). Filters are air dried and baked at 80°C for 2 hr.
  • SSC standard saline citrate
  • the duplicate filters are prehybridized at 42°C for 16-18 hr with 10 ml per filter of DNA hybridization buffer (50% formamide (40% formamide if reduced stringency). 5 x SSC. pH 7.0, 5x Denhardt's solution (polyvinylpyrrolidine, plus
  • Samples are hybridized with nick-translated DNA probes at 42°C for 12-36 hr for homologous species and 37°C for heterologous species contained in 5 ml of this same DNA hybridization buffer.
  • the filters are washed two times for 30 min. each time* at 50°C. in 0.2 x SSC, 0.1% SDS for homologous species hybridization, and at 50°C in 3 x SSC, 0.1% SDS for heterologous species hybridization. Filters are air dried and autoradiographed for 1-3 days at -70°C.
  • the duplicate filters are prehybridized at 42°C for 2-8 hr with 10 ml per filter of oligo-hybridization buffer (6 x SSC, 0.1% SDS, 1 mM EDTA, 5x Denhardt's, 0.05% sodium pyrophosphate and 50 ⁇ g/ml denatured and sheared salmon sperm DNA) .
  • oligo-hybridization buffer 6 x SSC, 0.1% SDS, 1 mM EDTA, 5x Denhardt's, 0.05% sodium pyrophosphate and 50 ⁇ g/ml denatured and sheared salmon sperm DNA.
  • Typical conditions employ a temperature of 30-42°C for 24-36 hr with 5 ml/filter of this same oligo-hybridization buffer containing probe.
  • the filters are washed two times for 15 min at 23°C, each time with 6 x SSC, 0.1% SDS and 50 mM sodium phosphate buffer at pH 7, then are washed once for 2 min at the calculated hybridization temperature with 6 x SSC and 0.1% SDS, air dried, and are autoradiographed at -70°C for 2 to 3 days.
  • the DNA for insertion into the host vectors of the invention may be obtained either by synthetic means, or, if vectors containing such sequences are on deposit or available, by cloning such vectors.
  • alternating sense and anti-sense overlapping single stranded oligonucleotides are prepared, and the alternating sense and anti-sense single stranded portions filled in enzymatically by treating with DNA polymerase and dNTPs.
  • the oligoraers are prepared by the method of Efimov, V.A., et al (Nucleic Acids Res (1982) 6875-6894), and can be prepared using commercially available automated oligonucleotide synthesizers. Kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 1 nmole substrate in the presence of 50 mM Tris. pH 7.6.
  • E. coli strain MC1061 was used for cloning and sequencing, and for expression of construction under control of most bacterial promoters.
  • the cells used for mammalian expression are Chinese hamster ovary (CHO) cells.

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Abstract

Le clonage et l'expression du gène codant une protéine d'inhibition de la phospholipase humaine (hPIP) permettent la production d'une protéine anti-inflammatoire dans des quantités pratiques à l'aide de techniques de recombinaison.
PCT/US1986/000772 1985-04-15 1986-04-14 Proteine anti-inflammatoire d'inhibition de la phospholipase humaine WO1986006100A1 (fr)

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0209568A1 (fr) * 1985-01-10 1987-01-28 Biogen Nv Sequences d'adn, molecules d'adn recombinant et procedes de production de polypeptides similaires a la lipocortine humaine.
EP0213916A2 (fr) * 1985-09-04 1987-03-11 Teijin Limited Protéine à activité inhibitrice de la phospholipase A2
WO1988007552A1 (fr) * 1987-04-02 1988-10-06 Teijin Limited Proteine inhibant la phospholipase a2 provoquant des inflammations
WO1988007576A2 (fr) * 1987-03-28 1988-10-06 Boehringer Ingelheim International Gmbh Proteines vasculaires anticoagulantes, leurs adn de codage, production et utilisation
US4874743A (en) * 1985-01-10 1989-10-17 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing human phospholipase inhibitor-like polypeptides
US4879224A (en) * 1985-01-10 1989-11-07 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing human phospholipase inhibitor polypeptides
US4950646A (en) * 1985-01-10 1990-08-21 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing human lipocortin-like polypeptides
WO1991003544A1 (fr) * 1989-08-29 1991-03-21 The Regents Of The University Of California Nouveaux inhibiteurs d'enzymes hydrolytiques, substrats et analyses, methodes et coffrets d'analyses s'y rapportant
US5081019A (en) * 1985-01-10 1992-01-14 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing lipocortin-like polypeptides
US5298489A (en) * 1988-02-26 1994-03-29 Biogen, Inc. DNA sequences recombinant DNA molecules and processes for producing lipocortins III, IV, V and VI
US5891664A (en) * 1989-04-07 1999-04-06 Cancerforskningsfondet Af 1989 Vectors and methods for recombinant production of uPA-binding fragments of the human urokinase-type plasminogen receptor (uPAR)
US6113897A (en) * 1989-04-07 2000-09-05 Cancerforskiningsfonden Af 1989 Antibodies and their use
EP0467932B1 (fr) * 1989-04-07 2000-10-18 Cancerforskningsfonden af 1989 (fonden til fremme af eksperimentel cancerforskning) Recepteur de l'activateur du plasminogene du type urokinase
US6248715B1 (en) 1993-06-01 2001-06-19 Chiron Corporation Method of treating a urokinase-type plasminogen activator-mediated disorder
US6268341B1 (en) 1993-06-01 2001-07-31 Chiron Corporation Expression of urokinase plasminogen activator inhibitors
US6462170B1 (en) * 1997-03-20 2002-10-08 Fondazione Centro San Raffaele Del Monte Tabor UPAR mimicking peptide

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001999A1 (fr) * 1989-08-03 1991-02-21 Teijin Limited Proteine d'inhibition de la phospholipase a2 naissant dans une region inflammee, sa production et gene prevu a cet effet
US5344764A (en) * 1989-08-03 1994-09-06 Teijin Limited Protein inhibitors of phospholipase A2 purified from inflammatory sites and production process

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Publication number Priority date Publication date Assignee Title
ATE108830T1 (de) * 1985-01-10 1994-08-15 Biogen Inc Dns-sequenzen, rekombinante dns-moleküle und verfahren zur herstellung menschlicher lipocortinähnlicher polypeptide.
JPS6256429A (ja) * 1985-09-04 1987-03-12 Teijin Ltd フオスフオリパ−ゼａ↓２阻害活性を有する蛋白

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Biochemical and Biophysical Communications Vol. 111, issued March 16, 1983, HATTORI et al, "Differentiation of a Histocytic Lymphoma Cell Line by Lipomodulin, A Phopholipase Inhibitory Protein," pages 551-559 see particularly pages 556 and 557. *
Biochemical and Biophysical Research Communications Vol. 122, issued August 16, 1984, ETIENNE et al. "Inhibitory Factor of Phospholipase A2 In Normal Human Serum," pages 1117-1124, see particularly page 1119. *
Biochimica et al Biophysica Acta, Vol. 847, issued November 20, 1985, ERRASFA et al, "The Presence of Lipocortin in Human Skin Fibroblasts and its Regulation by Anti-Inflamatory Seroids", pages 247-254, see particularly pages 249 and 252. *
Proc. Natl. Acad. Sci USA Vol. 78 issued May 1981, HIRATA et al, "Presence of Autoantibody for Phospholipase Inhibitory Protein, Lipomodium, in Patients with Rheumatic Diseases," pages 3190-3194, see particularly page 1392. *
Science, Vol. 222, issued November 18, 1983, YOUNG et al, "Yeast RNA Polymerase II Genes: Isolation with Antibody Probes", pages 778-782, see particularly page 779. *
See also references of EP0218696A4 *
The Journal of Biological Chemistry, Vol. 256, issued August 10, 1981, HIRATA, "The Regulation of Lipomodulin, a Phospholipase Inhibitory Protein, in Rabbit Neutrophils by Phosphorylation", pages 7730-7733, see particularly page 7730. *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4874743A (en) * 1985-01-10 1989-10-17 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing human phospholipase inhibitor-like polypeptides
US5081019A (en) * 1985-01-10 1992-01-14 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing lipocortin-like polypeptides
EP0209568A1 (fr) * 1985-01-10 1987-01-28 Biogen Nv Sequences d'adn, molecules d'adn recombinant et procedes de production de polypeptides similaires a la lipocortine humaine.
US4950646A (en) * 1985-01-10 1990-08-21 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing human lipocortin-like polypeptides
EP0209568A4 (fr) * 1985-01-10 1988-10-27 Biogen Nv Sequences d'adn, molecules d'adn recombinant et procedes de production de polypeptides similaires a la lipocortine humaine.
US4879224A (en) * 1985-01-10 1989-11-07 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing human phospholipase inhibitor polypeptides
EP0213916A3 (fr) * 1985-09-04 1989-02-08 Teijin Limited Protéine à activité inhibitrice de la phospholipase A2
EP0213916A2 (fr) * 1985-09-04 1987-03-11 Teijin Limited Protéine à activité inhibitrice de la phospholipase A2
WO1988007576A3 (fr) * 1987-03-28 1989-01-26 Boehringer Ingelheim Int Proteines vasculaires anticoagulantes, leurs adn de codage, production et utilisation
WO1988007576A2 (fr) * 1987-03-28 1988-10-06 Boehringer Ingelheim International Gmbh Proteines vasculaires anticoagulantes, leurs adn de codage, production et utilisation
WO1988007552A1 (fr) * 1987-04-02 1988-10-06 Teijin Limited Proteine inhibant la phospholipase a2 provoquant des inflammations
US5116942A (en) * 1987-04-02 1992-05-26 Teijin Limited Protein having an inflammatory phospholipase a2 inhibitory activity
US5484711A (en) * 1988-02-26 1996-01-16 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing lipocortins III, IV, V & VI
US5298489A (en) * 1988-02-26 1994-03-29 Biogen, Inc. DNA sequences recombinant DNA molecules and processes for producing lipocortins III, IV, V and VI
US5891664A (en) * 1989-04-07 1999-04-06 Cancerforskningsfondet Af 1989 Vectors and methods for recombinant production of uPA-binding fragments of the human urokinase-type plasminogen receptor (uPAR)
US6113897A (en) * 1989-04-07 2000-09-05 Cancerforskiningsfonden Af 1989 Antibodies and their use
EP0467932B1 (fr) * 1989-04-07 2000-10-18 Cancerforskningsfonden af 1989 (fonden til fremme af eksperimentel cancerforskning) Recepteur de l'activateur du plasminogene du type urokinase
US6248712B1 (en) * 1989-04-07 2001-06-19 Cancerforskningsfondet Af 1989 Urokinase-type plasminogen activator receptor
WO1991003544A1 (fr) * 1989-08-29 1991-03-21 The Regents Of The University Of California Nouveaux inhibiteurs d'enzymes hydrolytiques, substrats et analyses, methodes et coffrets d'analyses s'y rapportant
US6248715B1 (en) 1993-06-01 2001-06-19 Chiron Corporation Method of treating a urokinase-type plasminogen activator-mediated disorder
US6268341B1 (en) 1993-06-01 2001-07-31 Chiron Corporation Expression of urokinase plasminogen activator inhibitors
US6462170B1 (en) * 1997-03-20 2002-10-08 Fondazione Centro San Raffaele Del Monte Tabor UPAR mimicking peptide

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JPH08187087A (ja) 1996-07-23
AU5770786A (en) 1986-11-05
JP2637709B2 (ja) 1997-08-06
ES553961A0 (es) 1987-11-16
EP0218696A4 (fr) 1989-08-16
EP0218696A1 (fr) 1987-04-22
ES8800691A1 (es) 1987-11-16
JPS63500561A (ja) 1988-03-03
JP2757987B2 (ja) 1998-05-25
CA1290267C (fr) 1991-10-08

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