WO1994006829A1 - Proteines granulaires basophiles - Google Patents

Proteines granulaires basophiles Download PDF

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Publication number
WO1994006829A1
WO1994006829A1 PCT/US1993/008511 US9308511W WO9406829A1 WO 1994006829 A1 WO1994006829 A1 WO 1994006829A1 US 9308511 W US9308511 W US 9308511W WO 9406829 A1 WO9406829 A1 WO 9406829A1
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pro
bgp
asp
val
gly
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PCT/US1993/008511
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Randy W. Scott
Gerald J. Gleich
Craig G. Wilde
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Incyte Pharmaceuticals, Inc.
Mayo Foundation For Medical Education And Research
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Priority to AU48542/93A priority Critical patent/AU4854293A/en
Priority to EP93921456A priority patent/EP0662091A4/fr
Publication of WO1994006829A1 publication Critical patent/WO1994006829A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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 is related to novel proteins, pharmaceutical compositions containing such, therapeutics and human immunology. More specifically, it relates to proteins found in the cytoplasmic granules of human basophils, to the genes which encode them, to the antibodies which recognize them, and to the use of these proteins, oligonucleotides, and antibodies in the diagnosis and treatment of disease.
  • the basophil along with the mast cell, contains cytoplasmic granules with an affinity for basic dyes.
  • the basophil is produced by the bone marrow and circulates in the blood. Basophils are associated with helminthic parasitic infections and allergic reactions and they possess a high affinity receptor for IgE antibodies. Little is known however about the proteins which comprise the granule, in part because, under normal conditions, basophils constitute less than 1% of peripheral blood cells and it is therefore difficult to obtain an adequate amount of purified material for study. While some researchers have proposed that basophils are the precursors of mast cells, recent data suggests that basophils represent terminally differentiated leukocytes, possibly more closely related to eosinophils (Galli, S.J. and Lichtenstein, L.M., in Allergy:Principles and Practice. Middleton et al (Eds.), 3rd Ed, Vol. 1 (1988), pp 106-134).
  • Basophils appear to participate in many inflammatory, immunological and pathological reactions. For a general review see Galli et al, Pro ⁇ Allercry (1984) 4.:1. The most striking tissue infiltrates of basophils occur in cutaneous basophil hypersensitivity reactions (Galli and Askenase, in The Reticuloendothelial System:A Comprehensive Treatise. Abramoff et al (Eds.) pg 321, Plenum Press 1986) . Recent studies suggest that basophils are essential for expression of immunity to the feeding of larval Amblyomma americanum ticks.
  • basophils may collaborate with eosinophils in the expression of immunity by acting to attract eosinophils into tissues where the eosinophils subsequently release toxic cationic proteins (Brown, S.J. et al, J Immunol (1982) 129:790) .
  • Basophils are also elevated during helminthic infections, suggesting that they might participate in host defense to these parasites (Ogilvie, B.M. et al, Immunol (1980) H:385; Lindor, L.J., Parasite Immunol (1983) 1:13; Juhlin, L. and Michaelsson, G. , Lancet (1977) 1:1233) .
  • glycosaminoglycans of these proteins showed a mixture of GAGs including chondroitin sulfate, dermatin sulfate, and small amounts of heparin sulfate (Orenstein, N.S. et al, J Immunol (1978) 121:586) .
  • mast cell tryptase can be identified in human basophils at about 40 pg/cell, a level roughly 500-fold lower than in human mast cells (Castells, M.C. et al, J Immunol (1987) 138:2184) .
  • bradykinin generating activity has been ascribed to basophils by virtue of the release of this enzyme from peripheral white blood cells by IgE dependent stimulation (Newball, H.H. et al, J Clin Invest (1979) £4:466) .
  • the present invention was facilitated by a patient that presented with basophilic leukemia. Leukocyte counts were over 10 5 cell/ ⁇ l and contained 78% basophils. On two occasions this patient underwent cytophoresis for removal of leukocytes and a total of l. ⁇ xlO 11 basophils were obtained. Examination of the granule proteins of these basophils have revealed a number of novel proteins with unique N-terminal amino acid sequences.
  • BGPs basophil granule proteins
  • One aspect of the invention is directed to BGPs, which include proteins found in the granules of basophils, and fragments, mutations and modifications of these natural proteins which retain their respective BGP biological characteristics.
  • the polypeptides can be recombinantly produced by cells in culture, produced by chemical synthesis or isolated and purified from basophils.
  • aspects of the invention are an expression system comprising DNAs which encode these BGPs; host cells transformed with these expression systems; and methods to produce BGPs which utilize host cells transformed with said expression systems.
  • Still other aspects include antibodies, both monoclonal and polyclonal, which are specific for BGPs.
  • Additional aspects include methods of diagnosis and treatment of diseases characterized by abnormal expression or release of BGPs by basophils or other cells, or by genetic abnormalities within genes encoding BGPs. Further aspects include methods of treating diseases by the administration of the BGPs, antibodies, and DNA described herein.
  • Figure 1 shows a HPLC chromatogram of human basophil granule proteins obtained per Example 1
  • Figure 2 includes HPLC chromatograms 2-1 to 2-8 of human basophil granule proteins obtained per Example 2; and
  • Figure 3 includes HPLC chromatograms 3-1 to 3-3 of human basophil granule proteins obtained per Example 3.
  • BGP refers to purified forms of any and/or all of the novel proteins that may be purified from basophil granules as disclosed here as well as fragments and variants thereof, which retain a useful biological characteristic of natural BGP which BGP may be obtained by extraction, synthetically produced or produced using recombinant DNA methodology. It is recognized that basophil granules contain certain proteins that have been demonstrated from other sources. The invention allows the description of additional novel proteins that are discoverable from basophil granules.
  • a fragment of a BGP is a polypeptide having a primary amino acid sequence identical to any part of a naturally occurring BGP and retaining a useful biological characteristic of the BGP.
  • a variant of a BGP is any naturally occurring (allelic variant) , recombinantly engineered or chemically synthesized peptide or protein resulting from changes in the primary amino acid sequence or posttranslational processing of the BGP described, but retaining useful biological characteristics of the BGP isolated from the cytoplasmic granule of human basophils.
  • the variant forms of natural BGP include those wherein one or more instances of amino acid deletions, substitutions or insertions occur.
  • the variant forms of natural BGP also include those wherein altered patterns of glycosylation or lipidation occur.
  • Variants also include BGP made synthetically wherein substitutions by amino acids which are not encoded by the gene are made.
  • amino acids include but are not limited to norleucine, citrulline, ornithine, hydroxyproline, and cysteic acid.
  • the biological "characteristics" refer to the structural and/or biochemical properties of a BGP and include its specific antigenicity or immunogenicity and its ability to mediate inflammatory and immunological responses in vivo.
  • a “mutated” protein is a protein with an altered primary structure (relative to the commonly occurring protein) resulting from changes in the nucleotide sequence of the DNA which encodes it. These mutations include allelic variants.
  • a “modified” protein differs from the commonly occurring protein as a result of post-translational events which change the glycosylation or lipidation pattern, or the primary, secondary, or tertiary structure of the protein. Changes in the primary structure of a protein can also result from deletions, additions or substitutions.
  • a “deletion” is defined as a polypeptide in which one or more internal amino acid residues are absent.
  • An “addition” is defined as a polypeptide which has one or more additional intrernal amino acid residues as compared to the wild type.
  • a “substitution” results from the replacement of one or more amino acid residues by other residues.
  • a protein "fragment” is a polypeptide consisting of a primary amino acid sequence which is identical to a portion of the primary sequence of the protein to which the polypeptide is related.
  • Preferred altered forms of "natural" BGP described above are those which have at least 80% homology with natural BGP. At least 90% homology is more preferred, especially those including conservative substitutions.
  • the altered forms of natural BGP include those wherein one or more of the residues of the native sequence is deleted, substituted for, or inserted by a different amino acid or acids.
  • Preferred substitutions are those which are conservative, i.e., wherein a residue is replaced by another of the same general type.
  • naturally occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar.
  • three of the encoded amino acids are aromatic. It is generally preferred that peptides differing from the natural BGP contain substitutions which are from the same group as that of the amino acid replaced.
  • the basic amino acids Lys, Arg, and His are interchangeable; the acidic amino acids aspartic and glutamic are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gin, and Asn are interchangeable; the nonpolar aliphatic acids Gly, Ala, Val, lie, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, lie and Leu are more closely related) , and the aromatic amino acids Phe, Trp, and Tyr are interchangeable. While proline is a nonpolar neutral amino acid, it presents difficulties because of its effects on conformation, * and substitutions by or for proline are not preferred, except when the same or similar conformational results can be obtained.
  • Polar amino acids which represent conservative charge include Ser, Thr, Gin, Asn; and to a lesser extent, Met.
  • Ala, Gly, and Ser seem to be interchangeable, and Cys additionally fits into this group, or may be classified with the polar neutral amino acids.
  • Phenyl glycine for example, can be substituted for Trp, Tyr, or Phe as aromatic neutral amino acids; citrulline (Cit) and methionine sulfoxide (MSO) are polar but neutral, cyclohexyl alanine (Cha) is neutral and nonpolar, cysteic acid (Cya) is acidic, and ornithine (Orn) is basic.
  • citrulline (Cit) and methionine sulfoxide (MSO) are polar but neutral
  • cyclohexyl alanine (Cha) is neutral and nonpolar
  • cysteic acid (Cya) is acidic
  • Orn ornithine
  • the conformation conferring properties of the proline residues may be retained if one or more of these is substituted by hydroxyproline (Hyp) .
  • N-terminal methionine residue may be retained in the finished product. Cleavage of the N-terminal methionine to liberate the native sequence may or may not be complete.
  • the biological "characteristics" of a protein refer to the structural or biochemical function of the protein in the normal biological processes of the organism in which the protein naturally occurs.
  • biological characteristics of a BGP include its specific antigenicity or immunogenicity, its anti- helminthic activity when this is associated with a particular protein, and/or its ability to mediate inflammatory and immunological responses in vivo.
  • a host cell "expresses” a gene or DNA when the gene or DNA is transcribed.
  • a protein or polypeptide is “expressed” when the protein or polypeptide has been produced.
  • Recombinant host cell means a procaryotic or eucaryotic cell which contains an expression vector comprising heterologous structural DNA and is capable of expressing the polypeptides encoded by the heterologous DNA.
  • purified basophils are washed with PBS and contaminating erythrocytes are lysed by exposure to Tris-ammonium chloride for 5 minutes.
  • the cell suspension is centrifuged at about 400g, washed with Hank's BSA-EDTA and suspended in cold 0.25M sucrose containing DNAase and heparin using a
  • the cell suspension is next centrifuged at 400g for 10 minutes and the sediment is again suspended in 0.25M sucrose containing 2mg DNAase per 15ml cell suspension. After 1-2 minutes, heparin (20 IU) dissolved in 2ml 0.24M sucrose is added and the preparation is subjected to a shearing force by repeated passage (15 times) through a 20 gauge needle. The suspension is centrifuged at 400g to remove any remaining intact cells, and the granules are then purified by centrifugation through a cushion of 40% sucrose.
  • the proteins of the isolated basophil granules are solubilized by exposure to 0.C5M borate buffer at pH 9 in the presence of 5mM diisopropylfluorophosphate, 1x10 "7 M pepstatin A, and lO M EDTA to inhibit protease activity.
  • solubilized proteins of human basophilic granules in the same solvent described supra. are separated by reverse phase HPLC using a Brownlee BU-300 C4 column.
  • the mobile phase is 0.1% trifluoroacetic acid (TFA) containing 0-70% acetonitrile. Fractions are collected across the acetonitrile gradient as shown in Figure 1, where absorbance at 214 nm is shown on the ordinate. The relative homogeneity of each fraction is determined by SDS-PAGE electrophoresis.
  • ion exchange chromatography can also be employed.
  • a Mono-Q column (Pharmacia) is used under conditions as would be understood in the art whereby most typical proteins would bind to the column (e.g. 20 mM Tris, pH9.0) . The proteins are then eluted in a gradient from 0 to 2 M NaCl.
  • Purified proteins recovered from reverse phase HPLC are sequenced by subjecting up to 100 pmoles (estimated from chromatographic peak height and staining intensity on acrylamide gels) to automated Edman degradation.
  • N-terminal sequencing may not be adequate to support efforts to clone the cDNAs.
  • some granule proteins may be blocked or modified at the N-terminal or alternatively, the N-terminal sequences may not show favorable regions for generation of oligonucleotide probes.
  • Such proteins are digested with trypsin and the tryptic peptides are purified and sequenced in order to generate additional information.
  • the protein is concentrated to a 5 ⁇ l volume by vacuum centrifugation, and is then digested by incubation (4 hours, 37 C) with 1/40 (w:w) TPCK-trypsin in 1 ml of 50mM ammonium bicarbonate, pH 8.0. Tryptic fragments are purified for sequencing by reverse phase HPLC using a Brownlee RP 18 narrow bore column and an Applied Biosysterns 130A liquid chromatograph or other suitable device for HPLC - designed specifically for purification of pmole samples.
  • Sequence data thus obtained are compared to known protein sequences by computerized searches of the Protein Identification Resource of the NBRF, and/or of the Swiss protein database, in order to determine their novelty or relationship to other protein sequences.
  • Lag is a well known phenomenon in sequencing, where signal deriving from cycle n is also present in cycle n+1.
  • Lag presents a special problem when Cys is present at cycle n+1 since the amino acid present at cycle n may mistakenly be assigned at cycle n+1 due to the lag phenomenon. Because of such characteristic uncertainties, data from an analysis may support more than one interpretation. Nevertheless, examination of the sequence data allows one skilled in the art to judge whether two sequences derive from the same protein, even when some discrepancies exist. In accordance with the considerations, the sequences determined are submitted as a description rather than a definition of the proteins which have been isolated.
  • Basophils mature in cultures of human umbilical cord blood cells. Thus these cultures can be used to prepare a cDNA library which is then screened for particular DNA sequences that encode proteins unique to human basophil granules (BGP) (Saito, H. et al, Proc Nat Acad Sci (1988) 11:2288) .
  • BGP basophil granules
  • BGPs Other candidate cDNA libraries which may express BGPs include unstimulated HL-60 cells, or HL-60 cells driven to basophilic differentiation by culturing in a protein free medium (Muroi, K. et al, Leukemia Res (1989) H:157) or in the presence of sodium butyrate (Hutt-Taylor, S.R. et al., Blood (1988) 71:209). Although basophils and mast cells appear to be distinct in their lineages, granules of both cells contain mast cell tryptase (Castells, M.C. et al, J Immunol (1987) 138:2184) and these cells may therefore share other common proteins.
  • cDNA libraries made from human mast cells are another source of BGP encoding sequences.
  • the preparation of these cDNA libraries is described in detail in Maniatis, T. et al, Molecular Cloning. (1982) CSHL Press, and is well known to those skilled in the art.
  • a convenient approach is the insertion of cDNA fragments into a lambda phage vector e.g. lambda gtlO or lambda gtll as described by Maniatis, supra.
  • oligonucleotide probes are synthesized with a DNA synthesizer (380A: Applied Biosystems Inc. Foster City CA) by the phosphora idite method.
  • Oligonucleotides are purified on Sephadex G-50 columns and stored at -20°C.
  • the redundant probes are 5' -labeled with T- [ 3 P]ATP (E.I. du Pont de Nemours & Co., Inc., Boston, MA) using T4 polynucleotide kinase. Libraries are screened using up to 10° individual plaques per library, with the redundant oligonucleotide probes.
  • Duplicate nylon membranes containing phage are prepared and prehybridized in 5x SSPE (0.9M Nail, 50mM NH 2 P0 4 , 5mM EDTA, pH7.4), 0.2% SDS, and 0.005% denatured salmon sperm DNA for 2 hours at 50°C with 8 filters per 50 ml prehybridization fluid per bag.
  • Membranes are hybridized with approximately 1 ng of labeled probe per ml, in fresh hybridization fluid, overnight at the appropriate temperature for the redundant probe mixture.
  • Membranes are then washed at room temperature for 45 minutes in 1 liter of 5x SSPE per 40 filters, followed by a 1 minute wash in fresh buffer at 50°C, slightly air-dried, and exposed to Kodak XAR-5 film, with intensifying screens, for 72 hours at -70°C. After analysis, filters are stripped of hybridized label by incubation in 5x SSPE at 70°C for 10 minutes and subsequently hybridized with a second probe under the same conditions. This procedure is repeated for each probe. Recombinant clones which hybridize with probes will be selected from the library and plaque purified.
  • Recombinant phage DNA is then purified and digested with an appropriate restriction endonuclease to yield the amplified cDNA insert. Inserts are then ligated into M13mp series phage and sequenced using the dideoxy method described by Sanger (Biggin, M.D. et al, Proc Nat Acad Sci (1983) 10:3963) . Depending on the size of the cDNA, it may be necessary to restrict the clone, and subclone the fragments into M13. If the cDNA clones are not complete, a repeat screen of the library with the partial cDNA would be required. The complete sequence of the BGP cDNA is then compared against known sequences in the GenBank database.
  • DNAstar is used for nucleotide and polypeptide analyses and sequence comparisons. Selected cDNA inserts which encode a BGP can then be incorporated into an expression system.
  • the cDNA is operably linked to heterologous control sequences to form an expression vector.
  • the control sequences are chosen to be functionally compatible with the recombinant host cell into which the expression vector is introduced.
  • Expression can be in procaryotic or eucaryotic systems. Procaryotes most frequently are represented by various strains of E. coli. However, other microbial strains may also be used, such as bacilli (e.g. Bacillus subtilis) , various species of Pseudomonas. or other bacterial strains. In such procaryotic systems, plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used. For example, E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species by Bolivar et al., Gene (1977) 2 :95.
  • bacilli e.g. Bacillus subtilis
  • plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used.
  • E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species by Bol
  • procaryotic control sequences which are defined herein to include operons with promoters for transcriptional initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) promoter, lactose (lac) promoter systems (Chang et al., Nature (1977) 198:1056) . the tryptophan (trp) promoter system (Goeddel et al. , Nucleic Acids Res (1980) 1:4057) , the lambda-derived PL promoter and N-gene ribosome binding site (Shimatake et al. , Nature (1981) 292:128) . Any available promoter system compatible with procaryotes can be used.
  • promoters as the beta-lactamase (penicillinase) promoter, lactose (lac) promoter systems (Chang et al., Nature (1977) 198:1056) .
  • the expression systems useful in eucaryotic hosts comprise promoters derived from appropriate eucaryotic genes.
  • a class of promoters useful in yeast includes promoters for synthesis of glycolytic enzymes, including those for 3- phosph ⁇ glycerate kinase (Hitzeman et al., J Biol Chem (1980) 211:207) .
  • Other promoters include those from the enolase gene (Holland, M.J., et al. J Biol Chem (1981) 256:1385) or the Leu2 gene obtained from YEpl3 (Broach,, J., et al., Gene (1978) 1:121).
  • Suitable mammalian promoters include metallothionein, the early and late promoters from SV40 (Fiers et al., Nature (1978) 121:113), or other viral promoters such as those derived from polyoma, adenovirus II, bovine papilloma virus or retroviruses. Suitable viral and mammalian enhancers may also be used.
  • the nopaline synthesis promoter is appropriate (Depicker, A. , et al., J Mol APDI Gen (1982) 1:561).
  • the expression system is constructed from the foregoing control elements which are operably linked to the BGP sequences by employing standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and relegated in the forms desired.
  • Antisense oligonucleotides can be designed and produced based upon the nucleotide sequence of cDNA encoding a BGP.
  • the antisense oligonucleotide can be designed and used to regulate the translation within the cell of the specific mRNA to which it is complementary according to methods known in the art (Green et al., Ann Rev Biochem (1986) 55:569; Rossi et al., Pharmacol Ther (1991) 50:245).
  • the antisense reagent is made complementary to some portion of the mRNA encoding the BGP, preferably including a portion of the mRNA at or near the translation initiation site in the 5' region.
  • the antisense reagent may be R ⁇ A or preferably a modified R ⁇ A wherein the sugar phosphate backbone has been modified to increase resistance to R ⁇ ase activity or otherwise improve pharmacokinetic or pharmacodynamic properties.
  • the antisense oligonucleotide may be a D ⁇ A, which has been ligated to a promoter in an antisense orientation, such that transcription of the D ⁇ A produces a mR ⁇ A complementary to the mRNA encoding a BGP.
  • the antisense D ⁇ A may be incorporated into an expression vector for introduction into the body.
  • Triple helix oligonucleotides can also be designed and produced based upon the nucleotide sequence of cD ⁇ A encoding a BGP.
  • the triple helix oligonucleotide can be used to regulate the transcription within the cell of the specific D ⁇ A to which it is targeted and particularly to inhibit expression of a specific gene in individuals having diseases associated with expression of the gene.
  • Homopurine and homopyrimidine sequences which are appropriate for triple helix formation can be designed by methods known in the art (Griffen et al. Scfence (1989) 245:967).
  • triple helix oligonucleotide can be improved by synthesizing the reagents using the unnatural ⁇ -anomeric nucleotides to improve their nuc.lease resistance properties or by derivitizing the oligonucleotides with an intercalating agent such as ethidium bromide to stabilize the triple helix, once it has been formed.
  • an intercalating agent such as ethidium bromide
  • Antisense molecules are introduced into the body in one method by injecting the oligonucleotide either alone, encapsulated in a liposome or incorporated into a viral particle.
  • BGP-encoding genes are obtained from the genomic library of human fetal liver DNA in Charon 4A phage (ATCC 37333) .
  • the library contains 10 6 independent recombinants with an insert size of 15-20 kb and it is screened with cDNA essentially as previously described. Phage are sequentially adsorbed onto duplicate 8x8 cm nylon membrane filters. Filters are prehybridized in 5x SSPE, 50% formamide, 5x Denhardt's solution, 0.5% SDS and 0.005% denatured salmon sperm DNA for 2 hours at 42°C with 8 filters per 50 ml of prehybridization fluid.
  • Filters are hybridized with approximately 1.0 ng of labeled basophil protein cDNA per ml of fresh prehybridization fluid, containing 10% dextran sulphate and 2x Denhardt's solution, overnight at 42°C. BGP cDNA is labeled with ⁇ . 32 P dCTP and purified by Sephadex G-50 chromatography. Filters are then washed twice at room temperature for 15 minutes in 1 liter 2x SSPE and 0.2%
  • the amino acid sequence of BGPs as deduced from the gene is analyzed to determine regions of high immunogenicity.
  • the corresponding polypeptides are synthesized and are used in suitable immunization protocols to raise antibodies. Analysis to select appropriate epitopes is described by, for example, Ausubel, F.M. et al, in Current Protocols in Molecular Bi ⁇ logy. John Wiley __ Sons, Vol. 2, Sec. IV, ppll.14.1, 1989) .
  • the optimal selections are usually the C terminus, the N terminus and internal regions of the polypeptide, which are likely to be exposed to the external environment when the protein is in its natural conformation (this determination is based on the hydrophilicity of the sites) .
  • selected peptides are synthesized using an Applied Biosystems Peptide Synthesizer Model 431A using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma) by reaction with m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (See Ausubel et al. supra at pp 11.15.1) .
  • KLH keyhole limpet hemocyanin
  • MMS m- maleimidobenzoyl-N-hydroxysuccinimide ester
  • a cysteine is introduced at the N-terminus of the peptide to permit coupling to KLH.
  • Rabbits are immunized with the peptide- KLH complex in complete Freund's adjuvant and the resulting antisera tested for antipeptide activity, for example, by binding the peptide to plastic, blocking with 0.1% BSA, reacting with antisera, washing and reacting with radioiodinated affinity purified specific goat antirabbit IgG.
  • Hybridomas may be also be prepared and screened using standard techniques. Hybrids are screened using radioiodinated BGP to identify those producing monoclonal antibody.
  • prongs of plates FAST, Becton-Dickinson, Palo Alto, CA
  • affinity purified specific rabbit-antimouse (or suitable anti species Ig) antibodies at 10 ⁇ g/ml.
  • the coated prongs are blocked with 0.1% BSA, washed and exposed to supernatants from hybridomas. After incubation the prongs are exposed to radiolabeled protein, 1 ng/ l. Clones producing antibodies will bind a quantity of radioactivity which is detectable above background.
  • Such clones are expanded and subjected to 2 cycles of cloning at 0.3 cell/well.
  • Cloned hybridomas are injected into pristine treated mice to produce ascites, and monoclonal antibody is purified from the ascitic fluid by affinity chromatography on protein A.
  • Anti-BGPs are useful for the diagnosis of prepathologic conditions and as well as chronic and acute diseases which are characterized by abnormalities in the amount or distribution of BGPs.
  • the BGPs disclosed herein can be used to generate polyclonal and preferably monoclonal antibodies. These antibodies can be used to detect the BGPs in a sample such as a blood sample and determine the presence of and level of the BGPs in the sample. The type and/or amount of BGPs detected can be compared to a known standard -- an average for healthy individuals. Readings outside of the standard would be useful information in diagnosing abnormalities such as basophilic leukemia.
  • Immunoassay procedures are utilized to measure several major parameters in immunopathologic and prepathologic conditions which are characterized by BGP abnormalities - e.g. the increased or decreased production of BGPs by basophils, the aberrant production of BGPs by cells other than basophils, and the change in intracellular or extracellular distribution of BGPs during the genesis of disease.
  • peripheral blood mononuclear cells from normal individuals are prepared and analyzed as described by Ackerman et al for the localization of eosinophil granules MBP and Charcot- Leyden crystal protein to human basophils. (J Exp Med (1983) Hl:946; J EXP Med (1982) 111:1597).
  • freeze-thawed detergent extracts of cell suspensions enriched for basophils are analyzed by immunoassay, and the slope of the binding curves are then compared to comparable binding curves generated by the purified protein.
  • BGPs are also useful to remedy deficiencies in these proteins or to amplify immune-responses which are stimulated by these proteins.
  • BGPs can be administered to subjects exhibiting such conditions using standard formulations such as those set forth in Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton PA, Latest Ed.
  • compositions containing an effective amount of compounds of the present invention including the nontoxic addition of salts, amides and esters thereof, which may alone serve to provide the above-recited therapeutic benefits.
  • Such compositions can also be provided together with physiologically tolerable liquid, gel or solid diluents, adjuvants and excipients.
  • compositions can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents.
  • dosage required for therapeutic efficacy will range from about 0.01 to 10,000 mcg/kg, more usually 0.1 to 1000 mcg/kg of the host body weight.
  • dosages within these ranges can be administered by constant infusion over an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits have been obtained.
  • compositions are prepared as injectibles, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspended in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active ingredient is often mixed with diluents or excipients which are physiologically tolerable and compatible with the active ingredient. Suitable diluents or excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof.
  • the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like.
  • compositions are conventionally administered parenterally, by injection, for example, either subcutaneously or intravenously.
  • Additional formulations which are suitable for other modes of administration include suppositories, intranasal aerosols, and in some cases, oral formulations.
  • suppositories traditional binders and excipients may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders, and contain 10%-95% of active ingredient, preferably 25%-70%.
  • the peptide compounds may be formulated into the compositions as neutral or salt forms.
  • Pharmaceutically acceptable nontoxic salts include the acid addition salts (formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the core of the present invention relates to the novel proteins which are present in the cytoplasmic granules of human basophils, including a subset of those basophil granule proteins which are disclosed and described herein.
  • the proteins can be applied to various procedures for the diagnosis of diseases related to the abnormal (e.g., over or under) expression of the protein.
  • the proteins can be formulated into pharmaceutical compositions of various types and used for various types of treatments.
  • the genetic material which encodes the proteins is useful in producing the proteins.
  • the genetic material can be placed in plasmids and the plasmids used to transfect hosts each of which are part of the present invention.
  • the proteins can be used to produce monoclonal and polyclonal antibodies and these antibodies can be used in detection assays of the type described above.
  • the following procedures are used to purify human basophils, isolate the basophil granules, extract basophil granule proteins, fractionate and purify those proteins and determine their N-terminal amino acid sequence.
  • a patient with a form of chronic myelogenous leukemia underwent two treatments of cytophoresis from which l.SxlO 11 basophils were recovered.
  • the basophils were purified by centrifugation over a cushion of Ficoll-Hypaque from which 95% were recovered from the interface with greater than 90% purity. Subsequently, these basophils were lysed using modifications of the procedures described by Dvorak et al. Purified basophils were washed with PBS and erythrocytes were lysed by exposure to Tris-ammonium chloride for 5 minutes.
  • the cell suspension was centrifuged at 400 x g, washed with Hank's BSS-EDTA and suspended in cold 0.25M sucrose containing DNAase and heparin using a volume of 15 ml for 8 x 10 8 basophils.
  • the cell suspension was centrifuged at 400 x g for 10 minutes and the sediment was again suspended in 0.25M sucrose containing DNAase (2mg/15ml solution) . After 1-2 minutes, heparin (20 IU) dissolved in 2ml 0.24M sucrose was added and the preparation was subjected to a shearing force by repeated passage (15 times) through a 20 gauge needle.
  • the suspension was centrifuged at 400 x g to remove intact cells and granules were purified by centrifugation through a cushion of 40% sucrose. The granules were resuspended and stored in 30 aliquots at -70°C.
  • the borate extract was acidified by adjustment to 0.05-0.1% trifluoroacetic acid and was fractionated by reverse phase chromatography on a Brownlee BU300 column (2.1mm x 30mm) eluted with a 0-70% gradient of acetonitrile in 0.1% trifluoroacetic acid. Fractions judged to be appropriate for analysis were concentrated in a SpeedVac concentrator and sequenced by automated Edman degradation on the ABI 477 Protein Sequenator. The results are shown in Figure 1 and described as follows:
  • a cocktail of protease inhibitors was prepared containing diisopropylfluorophosphate, ethylenediaminetetraacetic acid and pepstatin A, and 0.50 ml of the mix was added to each of 2 aliquots of frozen granules. Four consecutive freeze/thaw cycles were carried out in order to lyse the granules. Proteins were extracted at 4°C by addition of 3 ml of 50mM sodium borate pH 9.0 to the pooled lysed granules. The extraction mix was vortexed occasionally over 1 hour to aid extraction. The insoluble material was removed by centrifugation for 15 minutes at 20000 x g, and the so ⁇ -uble extract was taken for chromatographic fractionation.
  • Reverse Phase HPLC Pools of fractions from size exclusion HPLC were acidified by adjustment to 0.05-0.1% trifluoroacetic acid and each pool was separately fractionated further by reverse phase chromatography on a Brownlee BU300 column (2.1mm x 30mm) eluted with a 0-70% gradient of acetonitrile in 0.1% trifluoroacetic acid. Fractions judged to be appropriate for analysis were concentrated in a SpeedVAc and sequenced by automated Edman degradation on the ABI 477 Protein Sequenator.
  • sequence 1.18 is homologous to a family of peptides previously described (Selsted, M.E., Harwig, S.L., Ganz, T., Schilling, J.W. , and Lehrer, R.I. (1985) J. Clin. Invest. 21,1436.), but differs from them by virtue of a deletion of Gly found in position 17 of those peptides.
  • sequence 4.12 is nearly identical to the sequence BGP 11 of U.S. parent application serial number 07/551,263 filed July 10, 1990, which is in turn very similar or identical to granulin A described by Bateman et al. (Bateman, A., Belcourt, D., Bennett, H. , Lazure, C. and Solomon, S. (1990) Biochem. Biophys. Res. Comm. 173. 1161), and bears sequence similarity to rat peptides termed epithelins, particularly epithelin l, described by Shoyab et al.
  • the above-listed 4.38 is homologous to human ⁇ l-antitrypsin (Long, G.L., Chandra, T. , Woo, S.L.C., Davie, E.W. and Kurachi, K. (1984) Biochemistry, 21, 4828) and may represent an allelic variant of ⁇ l- antitrypsin or may represent another member of a family of related molecules, the serpins, of which ⁇ l- antitrypsin is one.
  • a cocktail of protease inhibitors was prepared containing diisopropylfluorophosphate, ethylenediaminetetraacetic acid and pepstatin A, and 0.25 ml of the mix was added to each of 10 aliquots of frozen granules. Four consecutive freeze/thaw cycles were carried out in order to lyse the granules. Proteins were extracted at 4°C by addition of 5 ml of 50mM sodium borate pH 9.0 to the pooled lysed granules. The extraction mix was vortexed occasionally over a 1 hour period to aid extraction. The insoluble material was removed by centrifugation for 15 minutes at 20000 x g, and the soluble extract was taken for chromatographic fractionation.
  • Proteins present in other reverse phase fractions were prepared for sequencing of component proteins by Laemmli SDS polyacrylamide gel electrophoresis followed by electrophoretic transfer (2h at 500mA in lOmM CAPS ph 11.0, 10% methanol, 0.05% SDS) of proteins from the gel onto a polyvinylidinedifluoride membrane (ProBlott, ABI) . Such electroblotted bands were excised from the membrane and loaded directly onto the ABI 477 Protein Sequenator.
  • Val-Met-Ala 31rp Ile-Leu-Gly-Val-Phe-X-Val-Glu-Gln-X-Phe-Ser-Phe-X- Leu 37 Asp-Pro-Pro-Thr-Phe-Asn-Lys-Ile-Thr-Pro-Asn-Leu-Leu- Glu-Phe-Ala-Asp-Gly-Leu-Tyr-Lys-Gin-Glu 26bbl Ser-Glu-Leu-Thr-Lys-Met-Asn-Gln-Arg-Ser-Phe
  • rp indicates that the sequence was obtained following repurification of Fraction 31 of this example.
  • bbl indicates that the sequence was obtained following electroblotting of Fraction 26 of this example.
  • the above-listed sequence 37 has homology with respect to human ⁇ l-antitrypsin (Long, G.L., Chandra, T., Woo, S.L.C., Davie, E.W. and Kurachi, K. (1984) Biochemistry, 21,4828) and may represent an allelic variant of ⁇ .1-antitrypsin or may represent another member of a family of related molecules, the serpins, of which c ⁇ l-antitrypsin is one.
  • Each of the 29 remaining vials of basophil cells contains an estimated 200 ⁇ g of extractable protein.
  • Individual proteins recovered had yields ranging from 250 pmoles for peak 21 down to 25-50 pmoles for peaks 9 and 37 ( Figure 1) . Since 25 pmoles is usually sufficient for sequencing 20 or more residues at the N-terminus, the expenditure of more vials will enable rarer species of proteins to be sequenced and will also enable more residues to be sequenced from all proteins.
  • the instant invention has been shown and described herein and was considered to be the most practical, and preferred embodiments. It is recognized, however, that departures may be made therefrom which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art upon reading this disclosure.

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Abstract

L'invention se rapporte à plusieurs polypeptides naturels (protéines granulaires basophiles 'BGP') dérivés des granules cytoplasmiques des basophiles humains, ainsi qu'à des formes modifiées de ces polypeptides. Ceux-ci, l'ADN qui les code et les anticorps qui les reconnaissent peuvent être utilisés comme agents diagnostiques et thérapeutiques pour des pathologies impliquant des réactions inflammatoires et induites par IgE, des infections parasitaires et helminthiques, des réactions d'hypersensibilité ainsi que certains types de leucémies leucocytaires.
PCT/US1993/008511 1992-09-11 1993-09-10 Proteines granulaires basophiles WO1994006829A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU48542/93A AU4854293A (en) 1992-09-11 1993-09-10 Basophil granule proteins
EP93921456A EP0662091A4 (fr) 1992-09-11 1993-09-10 Proteines granulaires basophiles.

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US94381392A 1992-09-11 1992-09-11
US07/943,813 1992-09-11

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WO1994006829A1 true WO1994006829A1 (fr) 1994-03-31

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EP0651800B1 (fr) * 1990-07-10 1998-02-04 Incyte Pharmaceuticals, Inc. Proteines rencontrees dans des granulations basophiles

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Publication number Priority date Publication date Assignee Title
EP0651800B1 (fr) * 1990-07-10 1998-02-04 Incyte Pharmaceuticals, Inc. Proteines rencontrees dans des granulations basophiles

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Advances in Immunology, Volume 36, published 1984, R.A. LERNER, "Antibodies of Predetermined Specificity in Biology and Medicine", pages 1-44, especially pages 26-31. *
Biochemical and Biophysical Research Communications, Volume 173, No. 3, issued 31 December 1990, A. BATEMAN et al., "Granulins, a Novel Class of Peptide from Leukocytes", pages 1161-1168, especially table 2. *
Biochemistry, Volume 23, No. 21, issued 1984, G.L. LONG et al., "Complete Sequence of the cDNA for the Human Alpha1-Antitrypsin and the Gene for the S Variant", pages 4828-4837, figure 1. *
Journal of Biological Chemistry, Volume 264, No. 7, issued 05 March 1989, M.E. SELSTED et al., "Determination of the Disulfide Array in the Human Defensin HNP-2; a Covalently Cyclized Peptide", pages 4003-4007, figure 1. *
Proceedings of the National Academy of Sciences USA, Volume 85, issued October 1988, K.A. DAHER et al., "Isolation and Characterization of Human Defensin cDNA Clones", pages 7327-7331, figure 1. *
Proceedings of the National Academy of Sciences USA, Volume 89, issued March 1992, V. BHANDARI et al., "Isolation and Sequence of the Granulin Precursor cDNA from Human Bone Marrow Reveals Tandem Cysteine-Rich Granulin Domains", pages 1715-1719, figures 1 and 2. *
See also references of EP0662091A4 *

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AU4854293A (en) 1994-04-12

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