WO1987002037A1 - Proteines tensio-actives pulmonaires - Google Patents

Proteines tensio-actives pulmonaires Download PDF

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Publication number
WO1987002037A1
WO1987002037A1 PCT/US1986/002034 US8602034W WO8702037A1 WO 1987002037 A1 WO1987002037 A1 WO 1987002037A1 US 8602034 W US8602034 W US 8602034W WO 8702037 A1 WO8702037 A1 WO 8702037A1
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Prior art keywords
proteins
protein
sequence
dna
pulmonary surfactant
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PCT/US1986/002034
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English (en)
Inventor
H. William Taeusch, Jr.
Kenneth A. Jacobs
D. Randall Steinbrink
Joanna Floros
Davis S. Phelps
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Genetics Institute, Inc.
Brigham And Women's Hospital
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Publication of WO1987002037A1 publication Critical patent/WO1987002037A1/fr

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    • 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
    • C07K14/785—Alveolar surfactant peptides; Pulmonary surfactant peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to proteins isolated from human lung lavage, methods for obtaining said proteins and uses thereof.
  • Hyaline Membrane Disease and Respiratory Distress Syndrome (RDS) are synonymous terms denoting the clinical condition of pulmonary dysfunction in premature infants.
  • the disease is attributable to the absence of surface active material (surfactant) which lines the air-alveolar interface in the lung and prevents collapse of the alveoli during respiration.
  • surfactant surface active material
  • Current therapy is predominantly supportive.
  • bovine-derived surfactant into the lungs of the neonate.
  • pulmonary surfactant This complex consists of phospholipid and 5-10% protein (King, 1982).
  • the protein fraction of the surfactant is composed of nonserum and serum proteins.
  • the major surfactant associated protein is reportedly a 35,000 dalton nonserum, sialoglycoprotein (Shelly et al., 1982; Bhattacharyya et al, 1975; Sueishin and Benson 1981; King et al, 1973, Katyal & Singh, 1981). This protein reportedly seems to be important for the normal function of the pulmonary surfactant (King et. al., 1983; Hawgood et.al., 1985).
  • the present invention relates to a new group of proteins recovered and purified from lung lavage of patients with alveolar proteinosis, methods for obtaining the proteins, corresponding recombinant proteins, antibodies to the proteins for use in diagnostic products, compositions containing the novel proteins, and methods for using the compositions, e.g. in the treatment of infants afflicted with conditions such as Respiratory Distress Syndrome (RDS), as a drug delivery vehicle in the administration of other therapeutic materials to the lungs or other organs and in the treatment of adult RDS, which can occur during cardiopulmonary operations or in other situations when the lungs are filled with fluid and natural pulmonary surfactant production and/or function ceases.
  • RDS Respiratory Distress Syndrome
  • This invention relates to novel proteins useful for enhancing pulmonary surfactant activity, methods for obtaining said proteins and compositions containing one or more of the proteins.
  • the proteins of this invention include the following:
  • a protein characterized by a molecular weight of about 35 kd and by being encoded for by the DNA sequence depicted in Table 1.
  • a protein characterized by a molecular weight of about 35 kd and by being encoded for by the DNA sequence depicted in Table 2.
  • the proteins of this invention were obtained by subjecting pulmonary lavage material from an alveolar proteinosis patient to a combination of separation techniques followed by chromatographic purification. More specifically, the lavage material was centrifuged, and the protein-containing pellet so obtained was washed with buffer and extracted with a solvent such as 1-butanol to remove lipids and lipidassociated proteins. The butanol extract was set aside and treated as described below. The 1-butanol-insoluble material was then washed, redissolved in buffer and purified chromatographically. Two proteins were thus obtained which are characterized by a molecular weight of about 35 kd.
  • One of the 35 kd proteins is encoded for by the DNA sequence depicted in Table 1; the second 35 kd protein is encoded for by the DNA sequence depicted in Table 2.
  • Butanol-soluble proteins were obtained by cryoprecipitation. More specifically, storage of the 1-butanol extract at -20°C yielded a precipitate which was purified chromatographically to yield a protein characterized by an apparent molecular weight of about 6 kd (as determined by SDS-PAGE) and the observed amino acid composition set forth in Table 3.
  • a second 6 kd (as determined by SDS-PAGE) protein was obtained by concentrating the supernatant to dryness and purifying the residue chromatographically. The observed amino acid
  • composition of the latter 6kd protein is set forth in Table 4.
  • the two approximately 6kd proteins differ significantly from each other with respect to amino acid composition, as well as from the protein described by Tanaka, Chem. Pharm. Bull. 311:4100 (1983). Additionally, the N-terminal peptide sequence of the cold butanol-insoluble 6 kd protein was determined (Table 5). For the sake of simplicity, both low molecular weight PSP proteins are referred to hereinafter as "6k" proteins based on their approximate apparent molecular weights as determined by conventional SDS-PAGE. It should be understood, however, that the actual molecular weights of these proteins are in the range of 5.5-9 kilodaltons.
  • DNAs from two of these positive clones were subcloned into M13 for DNA sequencing, thus generating the clones MPSAP-1A and MPSAP-6A.
  • the nucleotide sequence for the cDNA clones encoding each of the two 35kd surfactant proteins was thereby elucidated and is presented above in Tables 1 and 2, respectively.
  • the sequences of subclones encoding the two 35 kd proteins are similar to each other but not identical.
  • the sequence differences result in restriction fragment polymoirphism between the two clones with respect to the coding region recognized by the restriction enzyme Pstl. Considerably more nucleotide variation between the two clones was found in their 3' untranslated regions.
  • Plasmids PSP35K-1A-10 and PSP35K-6A-8 were constructed by inserting the approximately 940-950 nucleotide EcoRI fragments depicted in Tables 1 and 2, respectively, into the EcoRI site of plasmid SP65 (see infra).
  • PSP35K-1A-10 contains the polylinker site adjacent to the EcoRI site at cDNA position 1
  • PSP35K-6A-8 contains the polylinker site adjacent to the EcoRI site at cDNA position 947.
  • PSP35K-1A-10 and PSP35K-6A-8 have been deposited with the American Type Culture Collection (ATCC), Rockville, MD under accession Nos. ATCC 40243 and 40244, respectively.
  • oligonucleotide probes based on the N-terminal sequence of the cold butanol-insoluble 6K protein (See Table 5) were synthesized and were used to screen a cDNA library prepared from human lung mRNA (Toole et al., 1984) as described in greater detail in Example 4, below. Several clones which hybridized to the probes were identified. Based on hybridization intensity one clone was selected, subcloned into M13 and sequenced. Plasmid PSP6K-17-3 was constructed by inserting the cloned cDNA so identified as an EcoRI fragment into the EcoRI site of plasmid SP65 (D.A.
  • PSP6K-17-3 has been deposited with the ATCC under accession No. ATCC 40245.
  • the nucleotide sequence of the cloned cDNA insert is shown in Table 6.
  • the cDNA insert in PSP6K-17-3 contains an open reading frame encoding a protein having a molecular weight of over 40kd. It is presently believed that the primary translation product is further processed, e.g., by Type II pneumocytes (Alveolar Type II cells), to yield the approximately 6K protein. It is contemplated that the cloned cDNA, portions thereof or sequences capable of hybridizing thereto may be expressed in host cells or cell lines by conventional expression methods to produce "recombinant" proteins having surfactant or surfactant enhancing activity.
  • this invention encompasses vectors containing a heterologous DNA sequence encoding the characteristic peptide sequence lie through Cys corresponding to nucleotides A-656 through C757 of the sequence shown in Table 6, i.e., IKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQC.
  • One such vector contains the nucleotide sequence
  • vectors of this invention contain a heterologous DNA sequence encoding the characteristic peptide sequence substantially as depicted in the underlined peptide region of Table 6, i.e., FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRWPLVAGGICQCLAERYSVILLDTLLGRML.
  • One such vector contains the DNA sequence substantially as depicted in the underlined nucleotide sequence of Table 6, i.e., TTC CCC ATT CCT CTC CCC TAT TGC TGG CTC TGC AGG GCT CTG ATC AAG CGG ATC CAA GCC ATG ATT CCC AAG GGT GCG CTA GCT GTG GCA GTG GCC CAG GTG TGC CGC GTG GTA CCT CTG GTG GCG GGC GGC ATC TGC CAG TGC CTG GCT GAG CGC TAC TCC GTC ATC CTG CTC GAC ACG CTG CTG GGC ATG CTG GGC ATG CTG
  • Another exemplary vector contains a heterologous DNA sequence, such as the nucleotide sequence depicted in Table 6, which encodes the full-length peptide sequence of Table 6.
  • DNA inserts for such vectors which comprise a DNA sequence shorter than the full-length cDNA of PSP6K-17-3, depicted in Table 6, may be synthesized by known methods, e.g. using an automated DNA synthesizer, or may be prepared from the full-length cDNA sequence by conventional methods such as loop-out mutagenesis or cleavage with restriction enzymes and ligation. Vectors so prepared may be used to express the subject proteins by conventional means or may be used in the assembly of vectors with larger cDNA inserts. In the former case the vector will also contain a promoter to which the DNA insert is operatively linked and may additionally contain an amplifiable and/or selectable marker, all as is well known in the art.
  • the proteins of this invention may thus be produced by recovering and purifying the naturally-occuring proteins from human pulmonary lavage material as described herein.
  • the corresponding "recombinant" proteins may be produced by expression of the DNA sequence encoding the desired protein by conventional expression methodology using microbial or insect or preferably, mammalian host cells.
  • Suitable vectors as well as methods for inserting therein the desired DNA are well known in the art.
  • Suitable host cells for transfection or transformation by such vectors and expression of the cDNA are also known in the art.
  • Mammalian cell expression vectors for example, may be synthesized by techniques well known to those skilled in this art.
  • the components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like may be obtained from natural sources or synthesized by known procedures. See Kaufman, Proc. Natl. Acad. Sci. 82: 689-693 (1985).
  • Established cell lines including transformed cell lines, are suitable as hosts.
  • Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants are also suitable.
  • Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is dominantly acting.
  • the host cells preferably will be established mammalian cell lines.
  • CHO (Chinese hamster Ovary) cells are generally preferred.
  • the vector DNA may include all or part of the bovine papilloma virus genome (Lusky et al., Cell, 36:391-401 (1984) and be carried in cell lines such as C127 mouse cells as a stable episomal element.
  • Other usable mammalian cell lines include HeLa, COS-1 monkey cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines and the like.
  • Cell lines derived from Alveolar Type II cells may be preferred in certain cases such as expression of the 6K protein (alone or with one or more other proteins of this invention) using the cDNA insert from PSP6K-13-7 or a fragment thereof.
  • Stable transformants then are screened for expression of the product by standard immunological or enzymatic assays.
  • the presence of the DNA encoding the proteins may be detected by standard procedures such as Southern blotting.
  • Transient expression of the DNA encoding the proteins during the several days after introduction of the expression vector DNA into suitable host cells such as COS-1 monkey cells is measured without selection by activity or immunological assay of the proteins in the culture medium.
  • the DNA encoding the protein may be further modified to contain preferred codons for bacterial expression as is known in the art and preferably is operatively linked in-frame to a nucleotide sequence encoding a secretory leader polypeptide permittng bacterial secretion of the mature variant protein, also as is known in the art.
  • the compounds expressed in mammalian, insect or microbial host cells may then be recovered, purified, and/or characterized with respect to physicochemical, biochemical and/or clinical parameters, all by known methods.
  • One or more of the proteins of this invention may be combined with a pharmaceutically acceptable fatty acid or lipid such as dipalmitoylphosphatidyl choline or with mixtures of such fatty acids or lipids which may be obtained from commercial sources or by conventional methods, or with natural surfactant lipids to provide a formulated pulmonary surfactant composition.
  • Natural surfactant lipids may be extracted by known methods from lung lavage, e.g. bovine or human lung lavage. Typically the weight ratios of total lipids to total proteins in the composition will be about 20:1 to about 100:1. At the levels currently being tested in clinical trials, one dose of the surfactant composition corresponds to 1-2 mg of total protein and 98-99 mg. of total lipid.
  • Pulmonary lavage (50 ml) from an alveolar proteinosis patient was centrifuged at 10,000 x g for 5 min. The pellet was collected and washed 5 times in 20 mm Tris HCl, 0.5 M NaCl, pH 7.4. The lipids and lipid-associated proteins were extracted from the washed pellet by shaking with 50 ml 1-butanol for 1 hr at room temperature. The butanol-insoluble material was collected by centrifugation, washed with distilled water and dissolved in 50 mM sodium phosphate, pH 6.0 and 6M guanidine HCl.
  • the protein was applied to a Vydac C4 reverse phase column and eluted with a gradient of acetonitrile: 2-propanol (2:1,v:v) containing 0.1% trifluoroacetic acid.
  • the major protein peak eluting at 50% B was collected and evaporated to dryness.
  • the proteins present were analyzed by SDS-PAGE (Laemmli, 1970).
  • the protein so obtained (approx. 50ug) was taken up and reduced in 200mM Tris, ImM EDTA, 6M guanidine HCl, 20mM DTT, pH8.5 at 37°C for 2 hrs. Solid iodacetamide was added to a final concentration of 60mM and the reaction incubated at 0°C for 2 hrs under argon in the dark. The reaction was stopped and the reagents removed by dialysis into 0.1M NH 4 HCO 3 , 50mM 2-mercaptoethanol, pH7.5 followed by further dialysis into 100mM NH 4 HCO 3 , pH7.5.
  • the alkylated protein was digested with trypsin (3% trypsin by weight) at 37°C for 16 hrs and the digest chromatographed over a C18 Vydac Reverse phase HPLC column (4.6x250mm).
  • the tryptic peptides were eluted with a linear gradient of 95% acetonitrile and 0.1% TFA, collected and subjected to N-terminal Edman degradation using an Applied-Biosystems Model 470A protein sequencer.
  • the PTH-amino acids were analyzed by the method of Hunkapillar and Hood (1983). Sequence data so obtained for tryptic fragments T19, T26 and T28 is presented below in Table 7.
  • the butanol extract obtained in Example 1 was stored at -20°C causing precipitation of one of the low MW proteins.
  • the precipitate was collected by centrifugation and dried under vacuum.
  • the butanol layer containing butanol-soluble protein was evaporated to dryness.
  • the precipitated cold butanol insoluble protein and the cold butanol-soluble protein were then purified in parallel by the same method as follows.
  • Each crude protein was separately dissolved in CHCI 3 : MeOH (2:1, v/v), applied to Sephadex LH20 columns and eluted with CECl 3 :MeOH (2:1).
  • the proteins were then analyzed by SDS-PAGE. Fractions containing the protein were pooled and evaporated to dryness.
  • Amino acid composition was determined by hydrolysis in 6 N HCl at 110°C for 22 hrs followed by chromatography on a Beckman model 63000 amino acid analyzer. N-terminal sequence was determined on an Applied Biosystems 470A sequencer. Molecular weights were determined on 10-20% gradient SDS polyacrylamide gels.
  • oligonucleotide probe Based on the amino acid sequence of tryptic fragment T28, (Table 7) an oligonucleotide probe was synthesized. The probe consisted of four pools of 20 mers and each pool contained 32 different sequences. The sequences of the 20 mers are depicted in Table 8.
  • a cDNA library from human lung mRNA was prepared as described in Toole et al., (1984) and screened with thetotal mixture of the four pools using tetramethyl ammoniumchloride as a hybridization solvent (Jacobs et al., 1985) Between 0.5-1% of the phage clones were positive with this probe.
  • the two clones differed in nucleotide sequence at three positions out of 250 nucleotides. Both clones were completely sequenced by generating an ordered set of deletions with Bal 31 nuclease, recloning into other M13 vectors and sequencing via the dideoxynucletide chain terminationprocedure (Viera and Messing, 1982; Sanger et al., 1977).
  • One clone corresponded to a full-lenth copy of the type referred to as 1A (Table 1), the second to an incomplete copy of the type referred to as 6A (Table 2).
  • Table 1A the second to an incomplete copy of the type referred to as 6A
  • 6A Table 2
  • MPSAP-1A represents the M13 subclone of a 0.9kb ECORI fragment in one orientation in M13.
  • MPSAP-1B represents the same fragment cloned in the opposite orientation in M13.
  • Each filter (1cm 2 ) was cut into nine equal size pieces and each piece was used in a 20-30ul hybridization reaction.
  • Each reaction contained human lung RNA (5mg/ml), 50% deionized formamide (Fluka AG Chemical Corp.), 10mM PIPES [(Piperazine-N, N'-bis) (2-ethanesulfonic acid)] pH 6.4 and 0.4M NaCl (Miller et al., 1983). The source and preparation of the RNA have been reported previously (Floros et al., 1985).
  • Each hybridization reaction was routinely incubated at 50°C for 3 hrs. At the end of the incubation period each filter was washed for five minutes with 1ml 1XSSC (.15M NaCl, 0.015M sodium citrate, 0.5% SDS at 60°C five times.
  • RNA was eluted by boiling for one minute in 300ul of ImM EDTA pH 7.9 and 10ug of yeast tRNA (Boehringer, Mannheim). The precipitated RNA was translated, immunoprecipitated and subjected to one and two dimensional gel electrophoresis as described in Floros et al., 1985.
  • Table 5 an oligonucleotide probe was synthesized.
  • the probe consisted of six pools of 17 mers. Three of the pools each contained 128 different sequences, and three of the pools each contained 64 different sequences. Based on the first seven amino acids two pools of 20 mers were synthesized. These pools contained either 384 or 192 different sequences.
  • a cDNA library from human lung mRNA was prepared as described in Toole et al., (1984) and screened with the total mixture of the six pools using tetramethylammoniumchloride as a hybridization solvent (Jacobs et al., 1985). Approximately 100,000 phage were screened, and 100 phage which hybridized to the probe were plaque purified. The phage were then pooled into groups of 25 and screened with the individual 17 mer and 20 mer pools. Six phage which hybridized most intensely to one of the 20 mer oligonucleotide probes and one of the corresponding 17 mer pools (pool 1447 containing 128 different sequences) were plaque purified.
  • the 17 mer pool 1447 was divided into four pools of 32 different sequences and hybridized to a dot blot of DNA prepared from these phage. Based on the hybridization intensity, DNA from one of these six phage were subcloned into M13 for DNA sequence analysis. A sequence corresponding in identity and position to the amino acids shown in Table 5 was obtained, confirming that the isolated clone coded for the approximately 6kd cold butanol-insoluble protein found in the lavage material of alveolar proteinosis patients (see above).
  • the first clone obtained was presumed to be an incomplete copy of the mRNA because it lacked an initiating methionine, and was used to isolate longer clones.
  • Two clones were completely sequenced by generating an ordered set of deletions with Bal 31 nuclease, recloning into other M13 vectors and sequencing via the dideoxynucleotide chain termination procedure (Viera and Messing, 1982; Sanger et al., 1977).
  • One clone corresponded to a full-length copy of the type referred to as 17 (Table 6), the second began at nucleotide 148 of clone 17.
  • Sequence of the 5' end of a third clone confirmed the sequence of the 5' end of clone 17.
  • the clones are identical throughout the coding region and differ only at two positions in the 3' untranslated region.
  • the proteins of this invention include two separate proteins characterize by molecular weights of about 35 kd and two separate proteins characterized by molecular weights of about 5.5-9 kd.
  • This invention relates to proteins isolated from human lung lavage, methods for obtaining said proteins and uses thereof.
  • Hyaline Membrane Disease and Respiratory Distress Syndrome (RDS) are synonymous terms denoting the clinical condition of pulmonary dysfunction in premature infants.
  • the disease is attributable to the absence of surface active material (surfactant) which lines the air-alveolar interface in the lung and prevents collapse of the alveoli during respiration.
  • surfactant surface active material
  • Current therapy is predominantly supportive.
  • bovine-derived surfactant into the lungs of the neonate.
  • pulmonary surfactant This complex consists of phospholipid and 5-10% protein (King, 1982).
  • the protein fraction of the surfactant is composed of nonserum and serum proteins.
  • the major surfactant associated protein is reportedly a 35,000 dalton nonserum, sialoglycoprotein (Shelly et al., 1982; Bhattacharyya et al, 1975; Sueishin and Benson 1981; King et al, 1973, Katyal & Singh, 1981). This protein reportedly seems to be important for the normal function of the pulmonary surfactant (King et. al., 1983; Hawgood et.al., 1985).
  • the present invention relates to a new group of proteins recovered and purified from lung lavage of patients with alveolar proteinosis, methods for obtaining the proteins, corresponding recombinant proteins, antibodies to the proteins for use in diagnostic products, compositions containing the novel proteins, and methods for using the compositions, e.g. in the treatment of infants afflicted with conditions such as Respiratory Distress Syndrome (RDS), as a drug delivery vehicle in the administration of other therapeutic materials to the lungs or other organs and in the treatment of adult RDS, which can occur during cardiopulmonary operations or in other situations when the lungs are filled with fluid and natural pulmonary surfactant production and/or function ceases.
  • RDS Respiratory Distress Syndrome
  • This invention relates to novel proteins useful for enhancing pulmonary surfactant activity, methods for obtaining said proteins and compositions containing one or more of the proteins.
  • the proteins of this invention include the following:
  • a protein characterized by a molecular weight of about 35 kd and by being encoded for by the DNA sequence depicted in Table 1.
  • a protein characterized by a molecular weight of about 35 kd and by being encoded for by the DNA sequence depicted in Table 2.
  • the proteins of this invention were obtained by subjecting pulmonary lavage material from an alveolar proteinosis patient to a combination of separation techniques followed by chromatographic purification. More specifically, the lavage material was centrifuged, and the protein-containing pellet so obtained was washed with buffer and extracted with a solvent such as 1-butanol to remove lipids and lipidassociated proteins. The butanol extract was set aside and treated as described below. The 1-butanol-insoluble material was then washed, redissolved in buffer and purified chromatographically. Two proteins were thus obtained which are characterized by a molecular weight of about 35 kd.
  • One of the 35 kd proteins is encoded for by the DNA sequence depicted in Table 1; the second 35 kd protein is encoded for by the DNA sequence depicted in Table 2.
  • Butanol-soluble proteins were obtained by cryoprecipitation. More specifically, storage of the 1-butanol extract at -20°C yielded a precipitate which was purified chromatographically to yield a protein characterized by an apparent molecular weight of about 6 kd (as determined by SDS-PAGE) and the observed amino acid composition set forth in Table 3.
  • a second 6 kd (as determined by SDS-PAGE) protein was obtained by concentrating the supernatant to dryness and purifying the residue chromatographically. The observed amino acid
  • composition of the latter 6kd protein is set forth in Table 4.
  • the two approximately 6kd proteins differ significantly from each other with respect to amino acid composition, as well as from the protein described by Tanaka, Chem. Pharm. Bull. 311:4100 (1983). Additionally, the N-terminal peptide sequence of the cold butanol-insoluble 6 kd protein was determined (Table 5). For the sake of simplicity, both low molecular weight PSP proteins are referred to hereinafter as "6k" proteins based on their approximate apparent molecular weights as determined by conventional SDS-PAGE. It should be understood, however, that the actual molecular weights of these proteins are in the range of 5.5-9 kilodaltons.
  • DNAs from two of these positive clones were subcloned into M13 for DNA sequencing, thus generating the clones MPSAP-1A and MPSAP-6A.
  • the nucleotide sequence for the cDNA clones encoding each of the two 35kd surfactant proteins was thereby elucidated and is presented above in Tables 1 and 2, respectively.
  • the sequences of subclones encoding the two 35 kd proteins are similar to each other but not identical.
  • the sequence differences result in restriction fragment polymorphism between the two clones with respect to the coding region recognized by the restriction enzyme Pstl. Considerably more nucleotide variation between the two clones was found in their 3' untranslated regions.
  • Plasmids PSP35K-1A-10 and PSP35K-6A-8 were constructed by inserting the approximately 940-950 nucleotide EcoRI fragments depicted in Tables 1 and 2, respectively, into the EcoRI site of plasmid SP65 (see infra).
  • PSP35K-1A-10 contains the polylinker site adjacent to the EcoRI site at cDNA position 1
  • PSP35K-6A-8 contains the polylinker site adjacent to the EcoRI site at cDNA position 947.
  • PSP35K-1A-10 and PSP35K-6A-8 have been deposited with the American Type Culture Collection (ATCC), Rockville, MD under accession Nos. ATCC 40243 and 40244, respectively.
  • oligonucleotide probes based on the N-terminal sequence of the cold butanol-insoluble 6K protein (See Table 5) were synthesized and were used to screen a cDNA library prepared from human lung mRNA (Tooie et al., 1984) as described in greater detail in Example 4, below. Several clones which hybridized to the probes were identified. Based on hybridization intensity one clone was selected, subcloned into M13 and sequenced. Plasmid PSP6K-17-3 was constructed by inserting the cloned cDNA so identified as an EcoRI fragment into the EcoRI site of plasmid SP65 (D.A.
  • PSP6K-17-3 has been deposited with the ATCC under accession No. ATCC 40245.
  • the nucleotide sequence of the cloned cDNA insert is shown in Table 6.
  • the cDNA insert in PSP6K-17-3 contains an open reading frame encoding a protein having a molecular weight of over 40kd. It is presently believed that the primary translation product is further processed, e.g., by Type II pneumocytes (Alveolar Type II cells), to yield the approximately 6K protein. It is contemplated that the cloned cDNA, portions thereof or sequences capable of hybridizing thereto may be expressed in host cells or cell lines by conventional expression methods to produce "recombinant" proteins having surfactant or surfactant enhancing activity.
  • this invention encompasses vectors containing a heterologous DNA sequence encoding the characteristic peptide sequence lie through Cys corresponding to nucleotides A-656 through C757 of the sequence shown in Table 6, i.e., IKRIQAMIPKGALAVAVAQVCRVVPLVAGGICQC.
  • One such vector contains the nucleotide sequence
  • vectors of this invention contain a heterologous DNA sequence encoding the characteristic peptide sequence substantially as depicted in the underlined peptide region of Table 6, i.e., FPIPLPYCWLCRALIKRIQAMIPKGALAVAVAQVCRWPLVAGGICQCLAERYSVILLDTLLGRML.
  • One such vector contains the DNA sequence substantially as depicted in the underlined nucleotide sequence of Table 6, i.e.,
  • Another exemplary vector contains a heterologous DNA sequence, such as the nucleotide sequence depicted in Table 6, which encodes the full-length peptide sequence of Table 6.
  • DNA inserts for such vectors which comprise a DNA sequence shorter than the full-length cDNA of PSP6K-17-3, depicted in Table 6, may be synthesized by known methods, e.g. using an automated DNA synthesizer, or may be prepared from the full-length cDNA sequence by conventional methods such as loop-out mutagenesis or cleavage with restriction enzymes and ligation. Vectors so prepared may be used to express the subject proteins by conventional means or may be used in the assembly of vectors with larger cDNA inserts. In the former case the vector will also contain a promoter to which the DNA insert is operatively linked and may additionally contain an amplifiable and/or selectable marker, all as is well known in the art.
  • the proteins of this invention may thus be produced by recovering and purifying the naturally-occuring proteins from human pulmonary lavage material as described herein.
  • the corresponding "recombinant" proteins may be produced by expression of the DNA sequence encoding the desired protein by conventional expression methodology using mi ⁇ robial or insect or preferably, mammalian host cells.
  • Suitable vectors as well as methods for inserting therein the desired DNA are well known in the art.
  • Suitable host cells for transfection or transformation by such vectors and expression of the cDNA are also known in the art.
  • Mammalian cell expression vectors for example, may be synthesized by techniques well known to those skilled in this art.
  • the components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like may be obtained from natural sources or synthesized by known procedures. See Kaufman, Proc. Natl. A ⁇ ad. Sci. 82: 689-693 (1985).
  • Established cell lines including transformed cell lines, are suitable as hosts.
  • Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants are also suitable.
  • Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is dominantly acting.
  • the host cells preferably will be established mammalian cell lines.
  • CHO (Chinese hamster Ovary) cells are generally preferred.
  • the vector DNA may include all or part of the bovine papilloma virus genome (Lusky et al., Cell, 36:391-401 (1984) and be carried in cell lines such as C127 mouse cells as a stable episomal element.
  • Other usable mammalian cell lines include HeLa, COS-1 monkey cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines and the like.
  • Cell lines derived from Alveolar Type II cells may be preferred in certain cases such as expression of the 6K protein (alone or with one or more other proteins of this invention) using the cDNA insert from PSP6K-13-7 or a fragment thereof.
  • Stable transformants then are screened for expression of the product by standard immunological or enzymatic assays.
  • the presence of the DNA encoding the proteins may be detected by standard procedures such as Southern blotting.
  • Transient expression of the DNA encoding the proteins during the several days after introduction of the expression vector DNA into suitable host cells such as COS-1 monkey cells is measured without selection by activity or immunological assay of the proteins in the culture medium.
  • the DNA encoding the protein may be further modified to contain preferred codons for bacterial expression as is known in the art and preferably is operatively linked in-frame to a nucleotide sequence encoding a secretory leader polypeptide permittng bacterial secretion of the mature variant protein, also as is known in the art.
  • the compounds expressed in mammalian, insect or microbial host cells may then be recovered, purified, and/or characterized with respect to physicochemical, biochemical and/or clinical parameters, all by known methods.
  • One or more of the proteins of this invention may be combined with a pharmaceutically acceptable fatty acid or lipid such as dipalmitoylphosphatidyl choline or with mixtures of such fatty acids or lipids which may be obtained from commercial sources or by conventional methods, or with natural surfactant lipids to provide a formulated pulmonary surfactant composition.
  • Natural surfactant lipids may be extracted by known methods from lung lavage, e.g. bovine or human lung lavage. Typically the weight ratios of total lipids to total proteins in the composition will be about 20:1 to about 100:1. At the levels currently being tested in clinical trials, one dose of the surfactant composition corresponds to 1-2 mg of total protein and 98-99 mg. of total lipid.
  • Pulmonary lavage (50 ml) from an alveolar proteinosis patient was centrifuged at 10,000 x g for 5 min. The pellet was collected and washed 5 times in 20 mm Tris HCl, 0.5 M NaCl, pH 7.4. The lipids and lipid-associated proteins were extracted from the washed pellet by shaking with 50 ml 1-butanol for 1 hr at room temperature. The butanol-insoluble material was collected by centrifugation, washed with distilled water and dissolved in 50 mM sodium phosphate, pH 6.0 and 6M guanidine HCl.
  • the protein was applied to a Vydac C4 reverse phase column and eluted with a gradient of acetonitrile: 2-propanol (2:1,v:v) containing 0.1% trifluoroacetic acid.
  • the major protein peak eluting at 50% B was collected and evaporated to dryness.
  • the proteins present were analyzed by SDS-PAGE (Laemmli, 1970).
  • the protein so obtained (approx. 50ug) was taken up and reduced in 200mM Tris, 1mM EDTA, 6M guanidine HCl, 20mM DTT, pH8.5 at 37°C for 2 hrs. Solid iodacetamide was added to a final concentration of 60mM and the reaction incubated at 0°C for 2 hrs under argon in the dark. The reaction was stopped and the reagents removed by dialysis into 0.1M NH 4 HCO 3 , 50mM 2-mercaptoethanol, pH7.5 followed by further dialysis into 100mM NH 4 HCO 3 , pH7.5.
  • the alkylated protein was digested with trypsin (3% trypsin by weight) at 37°C for 16 hrs and the digest chromatographed over a C18 Vydac Reverse phase HPLC column (4.6x250mm).
  • the tryptic peptides were eluted with a linear gradient of 95% acetonitrile and 0.1% TFA, collected and subjected to N-terminal Edman degradation using an Applled-Biosystems Model 470A protein sequencer.
  • the PTH-amino acids were analyzed by the method of Hunkapillar and Hood (1983). Sequence data so obtained for tryptic fragments T19, T26 and T28 is presented below in Table 7.
  • the butanol extract obtained in Example 1 was stored at -20°C causing precipitation of one of the low MW proteins.
  • the precipitate was collected by centrifugation and dried under vacuum.
  • the butanol layer containing butanol-soluble protein was evaporated to dryness.
  • the precipitated cold butanol insoluble protein and the cold butanol-soluble protein were then purified in parallel by the same method as follows.
  • Each crude protein was separately dissolved in CHCl 3 : MeOH (2:1, v/v), applied to Sephadex LH20 columns and eluted with CHCl 3 :MeOH (2:1).
  • the proteins were then analyzed by SDS-PAGE. Fractions containing the protein were pooled and evaporated to dryness.
  • Amino acid composition was determined by hydrolysis in 6 N HCl at 110°C for 22 hrs followed by chromatography on a Beckman model 63000 amino acid analyzer. N-terminal sequence was determined on an Applied Biosystems 470A sequencer. Molecular weights were determined on 10-20% gradient SDS polyacrylamide gels.
  • oligonucleotide probe Based on the amino acid sequence of tryptic fragment T28, (Table 7) an oligonucleotide probe was synthesized. The probe consisted of four pools of 20 mers and each pool contained 32 different sequences. The sequences of the 20 mers are depicted in Table 8.
  • a cDNA library from human lung mRNA was prepared as described in Toole et al., (1984) and screened with the total mixture of the four pools using tetramethyl ammoniumchloride as a hybridization solvent (Jacobs et al., 1985) Between 0.5-1% of the phage clones were positive with this probe.
  • the two clones differed in nucleotide sequence at three positions out of 250 nucleotides. Both clones were completely sequenced by generating an ordered set of deletions with Bal 31 nuclease, recloning into other M13 vectors and sequencing via the dideoxynucletide chain termination procedure (Viera and Messing, 1982; Sanger et al., 1977).
  • One clone corresponded to a full-lenth copy of the type referred to as 1A (Table 1), the second to an incomplete copy of the type referred to as 6A (Table 2).
  • Table 1A the second to an incomplete copy of the type referred to as 6A
  • 6A Table 2
  • MPSAP-1A Very dilute (10-15 ug/ml) single stranded DNA from either subclone MPSAP-1A or MPSAP-1B was applied to nitrocellulose paper (10 ug/cm ) under vacuum (Kafatos et al., 1979).
  • MPSAP-IA represents the M13 subclone of a 0.9kb ECORI fragment in one orientation in M13.
  • MPSAP-1B represents the same fragment cloned in the opposite orientation in M13.
  • Each filter (1cm 2 ) was cut into nine equal size pieces and each piece was used in a 20-30ul hybridization reaction.
  • Each reaction contained human lung RNA (5mg/ml), 50% deionized formamide (Fluka AG Chemical Corp.), 10mM PIPES [(Piperazine-N, N'-bis) (2-ethanesulfonic acid)] pH 6.4 and 0.4M NaCl (Miller et al., 1983). The source and preparation of the RNA have been reported previously (Floros et al., 1985).
  • Each hybridization reaction was routinely incubated at 50°C for 3 hrs. At the end of the incubation period each filter was washed for five minutes with 1ml 1XSSC (.15M NaCl, 0.015M sodium citrate, 0.5% SDS at 60°C five times.
  • RNA was eluted by boiling for one minute in 300ul of ImM EDTA pH 7.9 and 10ug of yeast tRNA (Boehringer, Mannheim). The precipitated RNA was translated, immunoprecipitated and subjected to one and two dimensional gel electrophoresis as described in Floros et al., 1985.
  • an oligonucleotide probe was synthesized.
  • the probe consisted of six pools of 17 mers. Three of the pools each contained 128 different sequences, and three of the pools each contained 64 different sequences.Based on the first seven amino acids two pools of 20 mers were synthesized. These pools contained either 384 or 192 different sequences.
  • a cDNA library from human lung mRNA was prepared as described in Toole et al., (1984) and screened with the total mixture of the six pools using tetramethylammoniumchloride as a hybridization solvent (Jacobs et al., 1985). Approximately 100,000 phage were screened, and 100 phage which hybridized to the probe were plaque purified. The phage were then pooled into groups of 25 and screened with the individual 17 mer and 20 mer pools, six phage which hybridized most intensely to one of the 20 mer oligonucleotide probes and one of the corresponding 17 mer pools (pool 1447 containing 128 different sequences) were plaque purified.
  • the 17 mer pool 1447 was divided into four pools of 32 different sequences and hybridized to a dot blot of DNA prepared from these phage. Based on the hybridization intensity, DNA from one of these six phage were subcloned into M13 for DNA sequence analysis. A sequence corresponding in identity and position to the amino acids shown in Table 5 was obtained, confirming that the isolated clone coded for the approximately 6kd cold butanol-insoluble protein found in the lavage material of alveolar proteinosis patients (see above).
  • the first clone obtained was presumed to be an incomplete copy of the mRNA because it lacked an initiating methionine, and was used to isolate longer clones.
  • Two clones were completely sequenced by generating an ordered set of deletions with Bal 31 nuclease, recloning into other M13 vectors and sequencing via the dideoxynucleotide chain termination procedure (Viera and Messing, 1982; Sanger et al., 1977).
  • One clone corresponded to a full-length copy of the type referred to as 17 (Table 6), the second began at nucleotide 148 of clone 17.
  • Sequence of the 5' end of a third clone confirmed the sequence of the 5' end of clone 17.
  • the clones are identical throughout the coding region and differ only at two positions in the 3' untranslated region.

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Abstract

Nouvelles protéines destinées à améliorer l'activité tensio-active pulmonaire, procédés pour obtenir lesdites protéines et compositions contenant une ou plusieurs desdites protéines. Celles-ci se composent de deux protéines séparées caractérisées par un poids moléculaire d'environ 35 kd et de deux protéines séparées caractérisées par un poids moléculaire d'environ 5,5-9 kd.
PCT/US1986/002034 1985-09-26 1986-09-26 Proteines tensio-actives pulmonaires WO1987002037A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
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WO1988004324A1 (fr) * 1986-12-08 1988-06-16 Abbott Laboratories Proteines associees a des tensioactifs hydrophobes pulmonaires
EP0273916A1 (fr) * 1986-05-06 1988-07-13 Children's Hospital Medical Center Proteine associee a un agent tensio-actif hydrophobe pulmonaire d'un poids moleculaire de 6000 daltons, et ses multimeres
EP0290516A1 (fr) * 1986-10-24 1988-11-17 WHITSETT, Jeffrey A. Proteines associees a des agents tensioactifs hydrophobes pulmonaires
WO1989002915A1 (fr) * 1987-09-28 1989-04-06 Children's Hospital Medical Center Lignees cellulaires humaines provenant de l'adenocarcinone epithelial pulmonaire, proteines humaines et procedes
EP0413957A2 (fr) * 1989-08-22 1991-02-27 Abbott Laboratories Fragments de la protéine de tensio-actif pulmonaire
US5013720A (en) * 1986-05-06 1991-05-07 Abbott Laboratories SAP-6-Val proteins and methods
EP0458167A1 (fr) * 1990-05-21 1991-11-27 Abbott Laboratories Conjugés acide gras- tensioactif pulmonaire
EP0491033A1 (fr) * 1990-07-10 1992-06-24 California Biotechnology Inc Isolation et purification de proteine de surfactant pulmonaire.
WO2004084930A1 (fr) * 2003-03-26 2004-10-07 Develogen Aktiengesellschaft Utilisation de proteines associees a la saposine pour la prevention et le traitement de l'obesite, du diabete et/ou du syndrome metabolique
WO2009035395A1 (fr) 2007-09-14 2009-03-19 Marcus Larsson Produit surfactant pulmonaire
US8551700B2 (en) 2001-09-05 2013-10-08 The Brigham And Women's Hospital, Inc. Diagnostic and prognostic tests

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US4615974A (en) * 1981-08-25 1986-10-07 Celltech Limited Yeast expression vectors
US4603124A (en) * 1983-03-10 1986-07-29 Tokyo Tanabe Company, Limited Surfactant and pharmaceutical compositions containing same
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GB2143535A (en) * 1983-07-19 1985-02-13 Suntory Ltd Improved plasmid vector and use thereof
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See also references of EP0240550A4 *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5013720A (en) * 1986-05-06 1991-05-07 Abbott Laboratories SAP-6-Val proteins and methods
EP0273916A1 (fr) * 1986-05-06 1988-07-13 Children's Hospital Medical Center Proteine associee a un agent tensio-actif hydrophobe pulmonaire d'un poids moleculaire de 6000 daltons, et ses multimeres
EP0273916A4 (fr) * 1986-05-06 1989-11-30 Childrens Hosp Medical Center Proteine associee a un agent tensio-actif hydrophobe pulmonaire d'un poids moleculaire de 6000 daltons, et ses multimeres.
EP0290516A1 (fr) * 1986-10-24 1988-11-17 WHITSETT, Jeffrey A. Proteines associees a des agents tensioactifs hydrophobes pulmonaires
EP0290516A4 (fr) * 1986-10-24 1989-11-14 Jeffrey A Whitsett Proteines associees a des agents tensioactifs hydrophobes pulmonaires.
EP0307513A2 (fr) * 1986-12-08 1989-03-22 Abbott Laboratories Protéines hydrophobes pulmonaires à tensioactivité associée
EP0307513A3 (fr) * 1986-12-08 1990-01-10 Abbott Laboratories Protéines hydrophobes pulmonaires à tensioactivité associée
WO1988004324A1 (fr) * 1986-12-08 1988-06-16 Abbott Laboratories Proteines associees a des tensioactifs hydrophobes pulmonaires
WO1989002915A1 (fr) * 1987-09-28 1989-04-06 Children's Hospital Medical Center Lignees cellulaires humaines provenant de l'adenocarcinone epithelial pulmonaire, proteines humaines et procedes
EP0313224A1 (fr) * 1987-09-28 1989-04-26 Children's Hospital Medical Center Lignées cellulaires humaines d'adénocarcinome épithélial pulmonaire, protéines humaines et méthodes
US5302581A (en) * 1989-08-22 1994-04-12 Abbott Laboratories Pulmonary surfactant protein fragments
EP0413957A3 (en) * 1989-08-22 1991-10-16 Abbott Laboratories Pulmonary surfactant protein fragments
EP0413957A2 (fr) * 1989-08-22 1991-02-27 Abbott Laboratories Fragments de la protéine de tensio-actif pulmonaire
AU639937B2 (en) * 1989-08-22 1993-08-12 Abbott Laboratories Pulmonary surfactant protein fragments
US5238920A (en) * 1989-08-22 1993-08-24 Abbott Laboratories Pulmonary surfactant protein fragments
EP0458167A1 (fr) * 1990-05-21 1991-11-27 Abbott Laboratories Conjugés acide gras- tensioactif pulmonaire
EP0491033A4 (fr) * 1990-07-10 1994-03-30 California Biotechnology, Inc.
US5258496A (en) * 1990-07-10 1993-11-02 Scios Nova Inc. Isolation and purification of lung surfactant protein
EP0491033A1 (fr) * 1990-07-10 1992-06-24 California Biotechnology Inc Isolation et purification de proteine de surfactant pulmonaire.
US5403915A (en) * 1990-07-10 1995-04-04 Scios Nova Inc. Isolation and purification of lung surfactant protein
US8551700B2 (en) 2001-09-05 2013-10-08 The Brigham And Women's Hospital, Inc. Diagnostic and prognostic tests
WO2004084930A1 (fr) * 2003-03-26 2004-10-07 Develogen Aktiengesellschaft Utilisation de proteines associees a la saposine pour la prevention et le traitement de l'obesite, du diabete et/ou du syndrome metabolique
WO2009035395A1 (fr) 2007-09-14 2009-03-19 Marcus Larsson Produit surfactant pulmonaire
EP2197459A1 (fr) * 2007-09-14 2010-06-23 Marcus Larsson Produit surfactant pulmonaire
EP2197459A4 (fr) * 2007-09-14 2011-03-09 Marcus Larsson Produit surfactant pulmonaire

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