WO2002074786A2 - Procedes de modulation de la production d'agents de surface - Google Patents

Procedes de modulation de la production d'agents de surface Download PDF

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WO2002074786A2
WO2002074786A2 PCT/US2002/002661 US0202661W WO02074786A2 WO 2002074786 A2 WO2002074786 A2 WO 2002074786A2 US 0202661 W US0202661 W US 0202661W WO 02074786 A2 WO02074786 A2 WO 02074786A2
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scd
polypeptide
vector
seq
nucleic acid
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PCT/US2002/002661
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WO2002074786A9 (fr
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Robert James Mason
Steven Neben
Michael R. Eckart
Malinda Longphre
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Bayer Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • This invention relates to methods of regulating surfactant production by modulating stearoyl coenzyme A desaturase (SCD) activity. More particularly, this invention relates to the use of SCD, or agonist or antagonist compounds capable of modulating SCD activity, to regulate surfactant production as a treatment for lung disease.
  • SCD stearoyl coenzyme A desaturase
  • SCD stearoyl coenzyme A desaturase
  • Stearoyl coenzyme A desaturase is an enzyme which catalyzes the production of unsaturated fatty acids which are required for lung surfactant synthesis.
  • Stearoyl CoA Desaturase introduces a double bond at ⁇ 9 in fatty acids such as palmitate and stearate, resulting in oleic and palmitoleic acids.
  • SCD is considered to be critical for normal surfactant lipogenesis (Kim, Y.C. et al. 2000. J Lipid Res. 41:1310-6).
  • Pulmonary surfactant is synthesized in and secreted by the Type II epithelial cells in the alveolar regions of the lungs and consists of 80-90% phospholipids and 10-20% surfactant proteins. It functions to reduce surface tension in the alveoli thus preventing their collapse, h addition, pulmonary surfactant maintains a key role in reducing the likelihood of bacterial or viral infection in the lungs and is crucial in protecting the lungs from inflammation due to exposure to atmospheric pollutants and antigens.
  • Alveolar proteinosis is characterized by an alveolar accumulation of proteinaceous materials also rich in phospholipids.
  • IRDS Infantile respiratory distress syndrome
  • Synthetic surfactants consisting of various combinations of phospholipids and surfactant proteins have been used extensively to prevent and to treat RDS in neonates (Avery, M.E.
  • ARDS adult respiratory distress syndrome
  • the present invention provides methods for modulating SCD activity in order to regulate surfactant production.
  • One aspect of the present invention involves using SCD polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • Another aspect of the present invention involves using SCD polypeptides to screen test compounds for the ability to modulate SCD activity, and using these agonist and antagonist compounds, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • the present invention further encompasses the use of SCD polypeptides and agonist or antagonist polypeptides for the treatment of a disease or clinical condition where surfactant production is relevant to the causation or treatment of the disease or clinical condition.
  • such diseases or conditions include lung diseases, such as adult/infant respiratory distress syndrome, acute lung injury, chronic obstructive pulmonary disease (emphysema and chronic bronchitis), asthma, alveolar proteinosis, small airway disease, pulmonary conditions related to maintaining airway patency, and still other related pulmonary conditions.
  • lung diseases such as adult/infant respiratory distress syndrome, acute lung injury, chronic obstructive pulmonary disease (emphysema and chronic bronchitis), asthma, alveolar proteinosis, small airway disease, pulmonary conditions related to maintaining airway patency, and still other related pulmonary conditions.
  • nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA, as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
  • processes for producing such polypeptides by recombinant techniques through the use of recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production of the polypeptides of the present invention, as well as recombinant prokaryotic and/or eukaryotic host cells comprising a nucleic acid sequence encoding a polypeptide of the present invention.
  • recombinant vectors such as cloning and expression plasmids useful as reagents in the recombinant production of the polypeptides of the present invention, as well as recombinant prokaryotic and/or eukaryotic host cells comprising a nucleic acid sequence encoding a polypeptide of the present invention.
  • gene therapy is defined as the process of providing for the expression of nucleic acid sequences of exogenous origin in an individual for the treatment of a disease condition within that individual.
  • compositions containing the SCD polypeptides and agonist or antagonist polypeptides and the use of such pharmaceutical compositions for the treatment of a disease or clinical condition where surfactant production is relevant to the causation or treatment of the disease or clinical condition, such as various lung diseases.
  • gene therapy is defined as the process of providing for the expression of nucleic acid sequences of exogenous origin in an individual for the treatment of a disease condition within that individual.
  • FIG. 1 depicts the cDNA sequence (SEQ ID NO: 1) and corresponding deduced amino acid sequence (SEQ ID NO: 2) of SCD.
  • FIG. 2 illustrates the amino acid sequence homology between human SCD and SCD from other species (SEQ ID NO:3-5). conserveed amino acids are readily ascertainable.
  • FIG. 3A illustrates early changes in SCD mRNA expression in rat alveolar type II cells in response to treatment with Keratinocyte Growth Factor (KGF, 20 ng/ml) for 1,3, 6, and 24 hours, a stimulus known to dramatically increase surfactant production after 7days. RNA expression was determined by hybridizing 5 ⁇ g of total RNA from treated cells to Affymetrix GeneChips. Data shown is the average of 3 separate experiments.
  • FIG. 3B illustrates SCD mRNA expression in rat alveolar type II cells in response to treatment with KGF (10 ng/ml) for 7 days. RNA expression was determined by hybridizing 5 ⁇ g of total RNA from treated cells to Affymetrix GeneChips. Data shown is the average of 3 separate experiments.
  • the present invention provides methods for modulating surfactant production using SCD polypeptides, or polynucleotides encoding such polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • the present invention also provides methods for modulating surfactant production using agonist or antagonist polypeptides that are capable of modulating SCD activity, or polynucleotides encoding such polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • Polypeptides having SCD activity from various species have been identified in International Patent Publication WO 00/09754, the disclosure of which is incorporated in its entirety by reference herein.
  • One aspect of the present invention therefore relates to isolated nucleic acid molecules (polynucleotides) which encode for the mature SCD polypeptide having the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2).
  • the polynucleotide encoding human SCD is structurally related to rat SCD, as well as that of the mouse, sheep, pig, cow and chicken.
  • Human SCD is comprised of a 1077 base pair coding region that encodes a putative protein of 359 amino acids with calculated molecular weight of 43.1 kDa.
  • Rat SCD is comprised of a 1077 base pair coding region that encodes a putative protein of 359 amino acids with a calculated molecular weight of 43.1 kDa and has approximately 83% nucleic acid identity, 80 % amino acid identity and 90% similarity over the full length human SCD sequences.
  • Mouse SCD is comprised of 1065 base pair coding region that encodes a putative protein of 355 amino acids with a calculated molecular weight of 42.6 kDa and has approximately 83% nucleic acid identity, 80% amino acid identity and 89% similarity over the full length human SCD sequences.
  • Sheep SCD is comprised of a 1077 base pair coding region that encodes a putative protein of 359 amino acids with a calculated molecular weight of 43.1 kDa and has approximately 86% nucleic acid identity, 81% amino acid identity and 89% similarity over the full length human SCD sequences.
  • Pig SCD is comprised of a 1077 base pair coding region that encodes a putative protein of 359 amino acids with a calculated molecular weight of 43.1 kDa and has approximately 86% nucleic acid identity, 84% amino acid identity and 90% similarity over the full length human SCD sequences.
  • Cow SCD is comprised of a 1077 base pair coding region that encodes a putative protein of 359 amino acids with a calculated molecular weight of 43.1 kDa and has approximately 85% nucleic acid identity, 81% amino acid identity and 89% similarity over the full length human SCD sequences.
  • Chicken SCD is comprised of a 1077 base pair coding region that encodes a putative protein of 359 amino acids with a calculated molecular weight of 43.1 kDa and has approximately 78% nucleic acid identity, 64% amino acid identity and 76% similarity over the full length human SCD sequences.
  • the homology of human SCD to rat, mouse, sheep, pig, cow and chicken SCD is depicted by the shaded boxes in Fig. 2.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double-stranded or single-stranded.
  • the coding sequence that encodes the mature SCD polypeptide may be identical to the coding sequence shown in FIG. 1 (SEQ ID NO: 1) or may be a different coding sequence as a result of the redundancy or degeneracy of the genetic code, encoding the same, mature SCD polypeptide as the DNA of FIG. 1, (SEQ ID NO:l).
  • the polynucleotides which encode for the mature SCD polypeptide of FIG. 1 may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the herein above-described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequence of FIG. 1 (SEQ ID NO: 2).
  • the variants of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the same mature SCD polypeptide as shown in FIG. 1 (SEQ ID NO:2) as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ ID NO: 2).
  • Such nucleotide variants include deletion variants, substitution variants and addition or insertion- variants .
  • the polynucleotide may have a coding sequence that is a naturally occurring allelic variant of the coding sequence shown in FIG. 1 (SEQ ID NO: 1).
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptides.
  • the present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptides may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
  • a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
  • the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotides may also encode for a proprotein which is the mature protein plus additional 5'amino acid residues.
  • a mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
  • the polynucleotides of the present invention may encode a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence).
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 31:161 (1984)).
  • the term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • Fragments of the full length SCD gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity.
  • Probes of this type preferably have at least 20 bases and may contain, for example, 50 or more bases.
  • the probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete SCD gene including regulatory and promotor regions, exons, and introns.
  • An example of a screen comprises isolating the coding region of the SCD gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • the present invention further relates to polynucleotides that hybridize to the hereinabove- described sequences if there is at least 70%, preferably at least 80%, more preferably at least 90%, and still more preferably at least 95% identity between the sequences.
  • the present invention particularly relates to polynucleotides that hybridize under stringent conditions to the hereinabove-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 90% and preferably 95% and more preferably at least 91% identity between the sequences.
  • polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO : 1 ) .
  • the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity.
  • such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO: 1 , for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
  • the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 80% identity, more preferably at least 90% and still more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at least 20 bases and preferably 30 bases and more preferably at least 50 bases and to polypeptides encoded by such polynucleotides.
  • the present invention further relates to a SCD polypeptide having the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2), as well as fragments, analogs and derivatives of such polypeptides.
  • fragment when referring to the polypeptide of FIG. 1 (SEQ ID NO:2) means polypeptides which retains essentially the same biological function or activity as such polypeptides.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.
  • SEQ ID NO:2 may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably 70% identity) to the polypeptide of SEQ ID NO:2, preferably at least 80% similarity (preferably 80% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least a 90% similarity (more preferably at least a 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least a 95% similarity (still more preferably a 95% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.
  • Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
  • the present invention also relates to vectors that include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells may be genetically engineered (transduced or transformed or fransfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the SCD genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotide of the present invention may be employed for producing a polypeptide by recombinant techniques.
  • the polynucleotide sequence maybe included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing a polypeptide.
  • Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector or plasmid may be used as long as they are replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease sites by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoter for example, LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as herein above described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • appropriate hosts there may be mentioned: bacterial cells, such as E.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation, hi a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a vector such as a plasmid or viral vector
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • Bacterial pQ ⁇ 70, pQE60, pQE-9 (Qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, ⁇ NH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, PRIT5 (Pharmacia).
  • Eukaryotic pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, PSVL (Pharmacia).
  • any other plasmid or vector may be used as long as they are replicable and viable in the host. Promoter regions can be selected from any desired gene using
  • CAT(chloramphenicol transferase) vectors or other vectors with selectable markers are pKK232-8 and pCM7.
  • Particular named bacterial promoters include laci, lacZ, T3, T7, gpt, lambda P , P and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention also relates to host cells containing the above-described construct.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate or liposome- based fransfection, DEAE-Dextran mediated fransfection, or elecfroporation (Davis, L., Dibner, M., Bartey, L, Basic Methods in Molecular Biology, (1986)).
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis- acting elements of DNA, usually from about 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), a cytomegalo virus early promoter enhancer, a polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S.
  • heterologous structural sequence is assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation, initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E.coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • Useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA).
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. After transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • polypeptide of the present invention may be recovered and purified from recombinant cell cultures by methods used heretofore, including ammom ' um sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • the polypeptide of the present invention may be a naturally purified product, or a product of chemical synthetic-procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture).
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture.
  • the polypeptides of the present invention maybe glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • the polypeptide of the present invention as a result of its ability to modulate surfactant production, maybe employed in treatment of various disease or clinical conditions where surfactant production is relevant to the causation or treatment of the disease or clinical condition, such as various lung diseases, including infant respiratory distress syndrome, chronic obstructive pulmonary disease (emphysema and chronic bronchitis), asthma and related pulmonary conditions.
  • various lung diseases including infant respiratory distress syndrome, chronic obstructive pulmonary disease (emphysema and chronic bronchitis), asthma and related pulmonary conditions.
  • This invention provides a method of screening compounds to identify those that modulate the action of the polypeptide of the present invention.
  • This method comprises incubating the SCD polypeptides or a cell fransfected with cDNA encoding SCD under conditions sufficient to allow the components to interact, and then measuring the effect of the compound or composition on SCD activity. Both agonist and antagonist compounds may be identified by this procedure; however agonist compounds are preferred.
  • Potential agonist and antagonist compounds include small molecules which stimulate or inhibit SCD polypeptides which in turn stimulates or inhibits surfactant production.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • Potential antagonist compounds further include antisense constructs prepared using antisense technology.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix-see Lee et al., Nucl. Acids Res. 3:173 (1979); Cooney et al, Science, 241:456 (1988); andDervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of the polypeptides of the present invention.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptide (Antisense—Okano, J.
  • oligonucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the polypeptide.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to the complementary target RNA, followed by endonucleolytic cleavage, thereby preventing transcription and the production of the polypeptides of the present invention.
  • Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the polypeptides, agonists and antagonists of the present invention may be employed in combination with a suitable pharmaceutical carrier to comprise a pharmaceutical composition for parenteral administration.
  • Such compositions comprise a therapeutically effective amount of the polypeptide, agonist or antagonist and a pharmaceutically acceptable carrier or excipient.
  • Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the polypeptides, agonists and antagonists of the present invention maybe employed in conjunction with other therapeutic compounds.
  • the pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, sublingual, intratracheal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes.
  • the pharmaceutical compositions can be administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 ⁇ g/kg body weight and in most cases they will be administered in an amount not in excess of about 10 mg/Kg body weight per day. hi most cases, the dosage is from about 0.1 ⁇ g/kg to about 100 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc. hi the specific case of topical administration, dosages are preferably administered from about 0.1 ⁇ g to 9 mg per cm .
  • polypeptide of the invention and agonist and antagonist polypeptides may also be employed in accordance with the present invention by expression of such polypeptide in vivo, which is often referred to as "gene therapy.”
  • cells may be engineered with a polynucleotide (DNA or RNA) encoding for the polypeptide ex vivo, the engineered cells are then provided to a patient to be treated with the polypeptide.
  • DNA or RNA polynucleotide
  • RNA encoding for the polypeptide ex vivo
  • Such methods are well-known in the art.
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding for the polypeptide of the present invention.
  • cells may be engineered in vivo for expression of the polypeptide in vivo, for example, by procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a simple retroviral particle, for example, a lentivirus, an adenovirus or an adeno-associated virus, which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • a simple retroviral particle for example, a lentivirus, an adenovirus or an adeno-associated virus, which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • Retioviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, B-type viruses represented by the mouse mammary tumor virus as well as mammalian C-type retioviruses such as Moloney murine leukemia virus, spleen necrosis virus, gibbon ape leukemia virus, Harvey sarcoma virus and myeloproliferative sarcoma virus.
  • plasmid vectors may be based on avian sarcoma/leukemia viruses such as Rous sarcoma virus and avian leukosis virus.
  • Retroviral constructs may also be generated from lentiviruses exemplified by the human immunodeficiency virus and spumaretroviruses such as the human foamy virus .
  • the vector includes one or more promoters.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, 7(9): 980-990 (1989), the T7 promoter, or RSV promoter, or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol HI, and ⁇ -actin promoters).
  • CMV human cytomegalovirus
  • viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvo virus promoters.
  • TK thymidine kinase
  • B19 parvo virus promoters The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • the nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the ⁇ -actin promoter; and human growth hormone promoters.
  • adenoviral promoters such as the adenoviral major late promoter
  • heterologous promoters such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; the albumin promoter; the ApoAI promoter; human globin promoter
  • Potentially useful inducible promoters include the lac operator-repressor system, the human and mouse metallothionein promoter, the heat-shock- inducible promoter, the tetR-based promoter and the mifepristone-activated promoter.
  • the promoter also may be the native promoter which controls the gene encoding the polypeptide.
  • the retioviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which maybe fransfected include, but are not limited to, the PE501, PA317, ⁇ -2, ⁇ -AM, PA12, T19-14X, VT-19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86,GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, 1: 5- 14 (1990), which is incorporated herein by reference in its entirety.
  • the vector may tiansduce the packaging cells through any means known in the art. Such means include, but are not limited to, elecfroporation, the use of liposomes, and CaPO 4 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides.
  • Such retioviral vector particles then may be employed, to tiansduce eukaryotic cells, either in vitro or in vivo.
  • the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • This invention is also related to the use of the genes of the present invention as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the nucleic acid sequences encoding the polypeptide of the present invention.
  • Individuals carrying mutations in a gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from ' blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose.
  • PCR primers complementary to the nucleic acid encoding a polypeptide of the present invention can be used to identify and analyze mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis.
  • DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985)). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Si protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.
  • restriction enzymes e.g., Restriction Fragment Length Polymorphisms (RFLP)
  • RFLP Restriction Fragment Length Polymorphisms
  • mutations can also be detected by in situ analysis.
  • the present invention also relates to a diagnostic assay for detecting altered levels of SCD proteins in various tissues since an over-expression or under-expression of the proteins compared to normal control tissue samples may detect the presence of abnormal surfactant production, which in turn may indicate the presence of certain lung diseases.
  • Assays used to detect levels of protein in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western Blot analysis, ELISA assays and "sandwich” type assays.
  • An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initially comprises preparing an antibody specific to an antigen to the polypeptides of the present invention, preferably a monoclonal antibody.
  • a reporter antibody is prepared against the monoclonal antibody.
  • a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme.
  • a sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein like bovine serum albumen.
  • the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any polypeptides of the present invention attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer.
  • the reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to the protein of interest. Unattached reporter antibody is then washed out.
  • Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of a polypeptide of the present invention present in a given volume of patient sample when compared against a standard curve.
  • a competition assay may be employed wherein antibodies specific to a polypeptide of the present invention are attached to a solid support and labeled SCD and a sample derived from the host are passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of a polypeptide of the present invention in the sample.
  • a “sandwich” assay is similar to an ELISA assay.
  • a polypeptide of the present invention is passed over a solid support and binds to antibody attached to a solid support.
  • a second antibody is then bound to the polypeptide of interest.
  • a third antibody which is labeled and specific to the second antibody is then passed over the solid support and binds to the second antibody and an amount can then be quantified.
  • sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • DNAs to chromosomes is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sub-localization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow- sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20kb).
  • the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an i munogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chirneric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. See generally Monoclonal Antibodies, a Laboratory Manual, Harlow and Lane, eds. . 1988 Examples include the hybridoma technique (Kohler and Milstein, Nature, 256:495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 4:72 (1983)), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Techniques described for the production of single chain antibodies (U.S. Pat. No.
  • 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.
  • tiansgenic mice maybe used to express humanized antibodies to immunogenic polypeptide products of this invention.
  • Humanized antibodies may also be produced by methods described in U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; and 5,693,762, incorporated herein by reference.
  • Example 1 Differential gene expression by rat alveolar type II cells using Affymetrix
  • EHS Engelbreth-Holm-Swarm
  • RNA was then isolated from the total RNA using FastTrack 2.0 Kit (cat. No. Kl 593-02) from Invitrogen (Carlsbad, CA) according to the manufacturers protocol which utilizes an oligo dT cellulose extraction purification procedure.
  • RNA was used to generate first and second strand cDNA (complimentary DNA) using the Superscript Choice kit (cat. No. 18090-019) from Gibco BRL/Life Technologies according to the manufacturers instructions.
  • the double stranded cDNA was then purified using a Phase Lock Gel kit (cat. No. pl-188233) from Eppendorf-5 Prime, Inc.(Boulder, CO).
  • Purified double-stranded cDNA was then used to generate biotin-labeled cRNA (complimentary RNA) by in vitro transcription using a BioArray High Yield RNA Transcript Labeling kit (cat. No. 900182) from Enzo Biochem (New York, NY) according to the instructions of the manufacturer.
  • RNAeasy Mini Kit Cat. No. 74104
  • Qiagen Valencia, CA
  • Hybridization Method Array hybridization was performed using a GeneChip Eukaryotic Hybrization Contiol Kit
  • the hybridization cocktail containing fragmented cRNA, control oligonucleotides, herring sperm DNA, acetylated bovine serum albumin, and MES hybridization buffer was loaded onto the GeneChip and incubated at 45 °C for 16 hours followed by washing at high stringency.
  • the quality of the cRNA was first evaluated by contiol hybridization to Affymetrix Test Chips containing probes built to match the 5', middle, and 3' sequences of housekeeping genes. Fluorescently labeled cRNA samples from cell preparations were then hybridized to Affymetrix rat U34 oligonucleotide arrays containing approximately 7000 full length genes and 17000 ESTs.
  • KGF short term KGF (20 ng/ml) treatment of cells increased SCD expression by over 2 fold, specifically at lhour of treatment (Figure 3A). After seven days of treatment, KGF (10 ng/ml) treatment of cells also increased SCD expression, an effect that may have been enhanced by the addition of dexamethasone, but not serum (FIG. 3B).

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Abstract

L'invention porte sur des procédés de modulation de la production d'agents de surface utilisant des polypeptides SCD et des agonistes ou des antagonistes capables de moduler l'activité de SCD. L'invention porte plus particulièrement sur l'utilisation de polypeptide SCD ou de polypeptides agonistes ou antagonistes pour le traitement d'une maladie ou d'un état clinique lorsque la production d'agents de surface est pertinente dans la relation de causalité ou le traitement de la maladie ou de l'état clinique, notamment de maladies pulmonaires, y compris le syndrome de détresse respiratoire chez l'adulte/enfant, les lésions pulmonaires aiguës, les maladies pulmonaires obstructives chroniques, l'emphysème et la bronchite chronique, l'asthme, la protéinose alvéolaire et les pathologies pulmonaires associées. L'invention porte également sur les compositions pharmaceutiques renfermant les polypeptides SCD ou les polypeptides agonistes ou antagonistes et l'application de ces compositions pharmaceutiques dans le traitement des maladies ou des états cliniques précités.
PCT/US2002/002661 2001-02-02 2002-01-30 Procedes de modulation de la production d'agents de surface WO2002074786A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012157736A1 (fr) * 2011-05-19 2012-11-22 東レ株式会社 Agent d'induction d'immunité

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012157736A1 (fr) * 2011-05-19 2012-11-22 東レ株式会社 Agent d'induction d'immunité
CN103547278A (zh) * 2011-05-19 2014-01-29 东丽株式会社 免疫诱导剂
JP5954175B2 (ja) * 2011-05-19 2016-07-20 東レ株式会社 免疫誘導剤
US10537623B2 (en) 2011-05-19 2020-01-21 Toray Industries, Inc. Immunity induction agent

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