WO1990012892A1 - Mucine intestinale humaine - Google Patents

Mucine intestinale humaine Download PDF

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Publication number
WO1990012892A1
WO1990012892A1 PCT/US1989/003206 US8903206W WO9012892A1 WO 1990012892 A1 WO1990012892 A1 WO 1990012892A1 US 8903206 W US8903206 W US 8903206W WO 9012892 A1 WO9012892 A1 WO 9012892A1
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nucleic acid
mucin
intestinal mucin
human intestinal
polypeptide
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PCT/US1989/003206
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English (en)
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Young S. Kim
James R. Gum, Jr.
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The Regents Of The University Of California
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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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates generally to polypeptides and nucleic acid sequences characteristic of the human intestinal mucin proteins and, more specifically, to their uses in preparing reagents for diagnosis or treatment of particular biological disorders.
  • Human intestinal mucus is a viscous gel that lubricates and protects the delicate epithelium of the digestive tract. This substance derives its characteristic fluid mechanical properties from a content of mucins, which are large glycoproteins (Mr > 250,000 daltons) consisting of ⁇ 75% carbohydrate, -20% protein, and trace quantities of other compounds.
  • mucins large glycoproteins (Mr > 250,000 daltons) consisting of ⁇ 75% carbohydrate, -20% protein, and trace quantities of other compounds.
  • the oligosaccharides that account for most of the mass of mucins are heterogeneous and frequently branched, consisting of as many as 20 individual sugar residues per chain. Mucin oligosaccharides are bound to serine and threonine residues in the protein backbone by a terminal N-Acetyl-Galactosamine residue.
  • the protein backbone itself appears to be covered with these O-linked oligosaccharides, since only about 10% is susceptible to proteolysis.
  • mucin and its properties see, generally, Neutra, M. , and Forstner, J., in "Physiology of the Digestive Tract” (Johnson, L.F., Ed.) Second ed. , pp. 975-1009, Raven Press, New York, which is incorporated herein by reference.
  • the oligosaccharide moieties of normal colonic mucin have been recently characterized, little is known about the structure and amino acid sequence of the protein core of this high molecular weight conjugate. Indeed, intestinal mucin protein has been difficult to characterize because of the heavy glycosylation. Removal of the carbohydrate portions either significantly deteriorates or destroys the polypeptide backbone, rendering the modified
  • cystic fibrosis Several human diseases have been observed to be directly associated with alterations in intestinal mucin production, including cystic fibrosis, familial polyposis 0 coli, ulcerative colitis and colon cancer. Patients with cystic fibrosis produce excessive amounts of mucin in their gastrointestinal, respiratory and reproductive tracts. Patients with familial polyposis ⁇ oli, ulcerative colitis and colon cancer produce mucins that are abnormally glycosylated. 5
  • obtaining polypeptide or nucleic acid information for intestinal mucin has been a goal of researchers for several years. Unfortunately, efforts have been generally unsuccessful, despite significant advances in protein chemistry and, of course, recombinant DNA 0 technology. This lack of sequence information has significantly hindered research on intestinal mucin, limiting diagnostic analyses and medical treatments.
  • the present invention provides, for the first time, novel purified and isolated nucleic acid sequences encoding polypeptide products having at least part of the primary structural conformation (i.e., continuous sequence of amino
  • polypeptides are economically produced in large quantities by procaryotic or eucaryotic host expression (e.g., by bacterial, yeast and mammalian cells in culture) of exogenous DNA sequences obtained by genomic or cDNA cloning or by gene 5 synthesis. Products of microbial cellular or vertebrate
  • polypeptides of the invention may be glycosylated naturally or with mammalian or other eucaryotic 15 carbohydrates. Alternatively, the polypeptides may be nonglycosylated.
  • Novel aspects of the invention include those polypeptides having a primary structural conformation sufficiently duplicative of that of a naturally-occurring (e.g., human) intestinal mucin to allow possession of one or 20 more of the biological properties thereof and having an average carbohydrate composition which differs from that of the naturally-occurring protein.
  • a naturally-occurring e.g., human
  • other proteins which share these sequence properties including invertebrate proteins and intestinal 25 mucins from other organisms, usually vertebrates. These vertebrates will include mice, monkeys, pigs and other mammals.
  • nucleic acids normally DNA fragments, and polypeptide sequences suitably deduced therefrom which represent, respectively, the primary structural conformation of human intestinal mucin.
  • the nucleic acid may be a DNA, RNA, mRNA or even the complementary strand of an encoding strand.
  • portions previously hidden by glycosylation have been elucidated.
  • novel biologically functional viral and circular plasmid DNA vectors incorporating DNA sequences of the invention and microbial (e.g., bacterial, yeast and mammalian cell) host organisms stably transformed or transfected with such vectors.
  • microbial e.g., bacterial, yeast and mammalian cell
  • novel methods for the production of useful polypeptides comprising cultured growth of such transformed or transfected microbial hosts under conditions facilitative of large scale expression of the exogenous, vector-borne DNA sequences and isolation of the desired polypeptides from the growth medium, cellular lysates or cellular membrane fractions.
  • Isolation and purification of recombinantly expressed polypeptides may be by conventional means including, e.g., preparative chromatographic separations and immunological separations involving monoclonal and/or polyclonal antibody preparations.
  • the present invention provides for the total and/or partial manufacture of corresponding DNA sequences and including such advantageous characteristics as incorporation of codons "preferred" for expression by selected non-mammalian hosts, provision of site for cleavage by restriction endonuclease enzymes and provision of additional initial, terminal or intermediate DNA sequences which facilitate construction of readily expressed vectors.
  • the present invention provides for manufacture (and development by site specific mutagenesis of cDNA and genomic DNA) of DNA sequences coding for microbial
  • polypeptide analogs or derivatives which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (i.e., deletion analogs containing less than all of the residues specified for human intestinal mucin and/or 0 substitution analogs, wherein one or more residues specified are replaced by other residues and/or addition analogs, wherein one or more amino acid residues is added to a terminal or medial portion of the polypeptide) ; and which share some or all of the properties of naturally-occurring
  • I 5 forms e.g., one or more epitopes.
  • references to nucleic acid sequences are intended to include their complementary strands.
  • compositions comprising polypeptide
  • labelled by covalent association with a detectable marker substance (e.g., radiolabelled with 125 I) to provide reagents useful in detection markers (such as radiolabels and non- isotopic labels such as biotin) and employed in DNA hybridization processes to ascertain expression levels or isolate any related gene family in human and other mammalian species. They can also be used for identifying the gene disorders at the DNA level and be used as gene markers for identifying neighboring genes and their disorders. 35 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 —
  • SMUC 40 Reactivity of SMUC 40 plaques with antibodies prepared to deglycosylated and native mucin.
  • SMUC 40 200 p.f.u/100-mm plate
  • Ej_ coli strain Y1090(r-) was plated on Ej_ coli strain Y1090(r-) in soft agar and incubated at 37"C for 2.5 h.
  • the four plates were then overlayed with isopropyl 0D-thiogalactopyranoside- saturated nitrocellulose membranes, and incubation was continued for 3 h more.
  • the membranes were then assayed for the presence of antibody-reactive plaques using 1:50 dilutions of antisera to HFA, HFB, and native (non- deglycosylated) mucin.
  • the control was serum from a nonimmunized rabbit.
  • SMUC 40-42 Lysates containing 100 ⁇ g of lysogen protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose. Immunoblot analysis was conducted using a 1:50 dilution of anti-HFB. Lanes 40, 41, and 42 are from lysogens of SMUC 40-42, respectively. Lane C is a nonrecombinant ⁇ gtll lysogen control.
  • FIG. 3 Sequencing strategy for clones SMUC 40-42.
  • the arrows represent the length and direction of individual sequencing reactions.
  • the terminal regions of SMUC 40-42 were ⁇ equenced using templates derived from vectors containing each clone in its entirety.
  • Most of the sequencing of the interior regions of SMUC 40 and 41 was performed using exonuclease Ill-deleted clones.
  • restriction fragments obtained from TaqI and Mspl digests of SMUC 40 and 42 were force cloned into AccI- and EcoRI-digested M13mpl8 and used to generate templates. Sequencing done using this latter method is indicated using dashed arrows.
  • Figure 4 Sequencing strategy for clones SMUC 40-42.
  • the arrows represent the length and direction of individual sequencing reactions.
  • the terminal regions of SMUC 40-42 were ⁇ equenced using templates derived from vectors containing each clone in its entirety.
  • Nucleotide and deduced amino acid sequence of intestinal mucin cDNA clones Nucleotide position is indicated by the numbers at the right and amino acid position by those at the left. Asterisks appear every 10 nucleotides except for 4E where they appear every 5 nucleotides. The repeat units are indicated by the arrows and two putative N- glycosylation sites are underlined. All clones are flanked by EcoRI sites generated using the synthetic linker GGAATTCC
  • amino acids are not numbered relative to a start point.
  • the longer 24 amino acid segment has an extra amino acid, methionine, at the indicated position.
  • the shorter 22 amino acid segment has a blank at the appropriate position.
  • Figure 6 immunoblots using anti-HFB and anti-MRP. Blots were prepared containing 5 ⁇ g/lane of HFA, HFB, BSA, or MRP- conjugated BSA and probed using anti-HFB (panel A) or anti- MRP (panel B) . MRP was conjugated to BSA using glutaraldehyde as described by Avrameus (Avrameus, S., (1969) Immunochemistry 6:43-52) .
  • RNA blot analysis Poly(A) * RNA samples (0.5 ⁇ g) were analyzed as described under "Experimental" using cDNA clone SMUC 41 as a probe.
  • LS-G is a substrain of colon cancer cell line LS174T which contains high levels of butyrate-inducible alkaline phosphates (Gum, J.R, et al., (1987) J. Biol. Chem. 262:1092-1097).
  • LM-12 and HM-7 are LS174T cell variants which contain a low and high content of mucin, respectively (Kuan, S.F., et al., (1987) Cancer Res. 47:5715-5724).
  • SW1116 is a colon cancer cell line which, like LS-G, is uncharacterized in terms of its mucin content (Leibovitz, A., et al., (1976) Cancer Res. 36:4562-4569).
  • H498 is a recently isolated colon cancer cell line which produces and secretes high levels of mucin (Park, J.G., et al., (1987) Cancer Res. 47:6710-6718).
  • the placenta, small intestine, and colon samples used here were from normal individuals.
  • S.J. tumor and colon were from tissue surgically removed from a patient with colon cancer.
  • the last lane contained poly (A) + RNA isolated from a thyroid tumor.
  • the 28 S and 18 S rRNA subunits were used as size markers (5400 and 2100 bases, respectively) .
  • Genomic DNA Blots Genomic DNA Blots. Genomic DNA blots were prepared and analyzed using SMUC 41 as a probe. Panel A, DNA prepared from the lymphocytes of two healthy donors (PS and VE) and from four colon cancer cell lines was digested with EcoRI prior to electrophoresis. Panels B and C, DNA prepared from colon cancer cell lines was digested with Hinfl and Sau3A, respectively. Eight ⁇ g of DNA was used per lane. Figure 10 —
  • RNA and DNA blots probed using 5' and 3' segments of SMUC 41 were probed with 3'-466 as discussed in the text and autoradiography was conducted. The probe was then removed by twice incubating the blots at 65 ⁇ C in 10 mM sodium phosphate, pH 6.5, and 50% formamide for 1 h. Following a rinse in 2 x SSC, and 0.1% sodium dodecyl sulfate the blots were probed again using 5'- 370.
  • Panel A blot was prepared using 0.5 ⁇ g of poly(A) + RNA from human small intestine and colon.
  • Panel B 8 ⁇ g of DNA samples digested with the indicated restriction enzymes were analyzed.
  • nucleic acid sequences encoding portions of the polypeptide sequence of human intestinal mucin have been isolated and characterized. Further, the isolated DNA segments have been expressed in providing isolatable quantities of polypeptides displaying biological (e.g., immunological) properties of naturally-occurring human intestinal mucin, as well as both in vivo and in vitro biological activities of human intestinal mucin.
  • biological e.g., immunological
  • a human small intestine ⁇ gtll cDNA library was screened using antisera prepared against the deglycosylated protein backbone of human colon cancer xenograft mucin.
  • Three cDNAs were isolated from this screening, designated SMUC 40-42. These cDNAs were all found to contain tandem repeats of 69 nucleotides which encoded a threonine- and proline-rich protein consensus sequence of
  • RNA blots probed with one of these cDNAs, SMUC 41 exhibited large, polydisperse hybridization bands at -7600 bases. Band intensities were strongest when human small intestine, colon, and colon cancer poly(A) + RNA was used. I ⁇ vitro translation of poly(A) + RNA from human small intestine, colon, and colon cancer cells produced a 162,000-dalton peptide that was immunoprecipitated with antibodies to deglycosylated mucin. SMUC 41 was also used to prCbe DNA blots, which indicated the presence of restriction fragment length polymorphisms in the intestinal mucin gene.
  • methods for identifying cancers in human patients rely on detection of the non-glycosylated expression product of the human intestinal mucin gene in a biological specimen, typically body fluids, tissue, or stool samples.
  • detection is accomplished by immunological techniques, such as immunohistochemical staining of a cell sample employing either monoclonal or polyclonal antibodies specific for the human intestinal mucin protein.
  • Detection of the human intestinal mucin protein is particularly useful in distinguishing specific cancers, such as epithelial cancers, from morphologically similar cancers and for the early detection and differential diagnosis of cancers, such as gastric, colon, rectal and pancreatic cancers.
  • a normal glycosylation of the human intestinal mucin protein has been associated with the pathogenesis of a number of human tumors, particularly epithelial cell tumors.
  • the expression product of the human intestinal mucin gene is a highly glycosylated protein including heterogeneous carbohydrate comprising -75% of the glycoprotein.
  • the polypeptide portion has an estimated molecular weight of 162 kd. This molecular weight, of course, is only approximate and subject to experimental error in the measurement techniques reported in the Experimental section hereinafter.
  • neoplastic transformation of certain epithelial cells is characterized by detectable expression of the polypeptide epitopes normally hidden by extensive glycosylation.
  • the polypeptides of the present invention will be either haptenic or antigenic, typically including at least 6 amino acids, usually at least 9 amino acids, and more usually 12 or more amino acids found contiguously within the natural human intestinal mucin protein.
  • the contiguous amino acids may be located within any region of the polypeptide and will correspond to at least one epitopic site which is characteristic of the protein.
  • characteristic it is meant that the epitopic site will allow immunologic detection of the exposed polypeptide segment in a cell sample with reasonable assurance, in most cases allowing human intestinal mucin to be immunologically distinguished from other related • proteins, such as human mammary mucin.
  • sequences of these polypeptides are as follows: a) TTTTVTPTPTPT b) PTTTPIT.TTTTVTPTPTPT.GTQT. Sequence (a) , which is extremely highly conserved, is contained within sequence (b) between the .'s.
  • Sequence (b) itself is quite highly conserved and is often tandemly repeated without intervening amino acids.
  • Sequence (a) corresponds to the nucleic acid sequences:
  • Substantial homology or identity of a nucleic acid sequence indicates either that: a) there is greater than about 65% homology or identity, more typically greater than about 75%, and optimally greater than 90%; or ) the homologous nucleic acid sequence will hybridize to the consensus sequence or its complementary strand under stringent conditions of temperature and salt concentration. These stringent conditions will generally be at temperatures greater than about 22 ⁇ C, more usually greater than about 30*C and preferably greater than about 45 • C. Preferred salt concentrations are less than about 1 M, more usually less than about 500 mM and optimally less than about 200 mM.
  • the combined conditions will be more important than either the salt concentration or the temperature alone.
  • Other parameters which are used to define stringency include GC content of the sequence, extent of complementarity of the sequences and length of segments involved in the hybridization, besides composition of buffer solutions used in the hybridization mixture.
  • Synthetic polypeptides which are immunologically cross-reactive with the natural intestinal mucin protein may be produced by either of two general approaches.
  • polypeptides having fewer than about 50 amino acids, more usually fewer than about 20 amino acids may be synthesized by the well-known Merrifield solid-phase synthesis method where amino acids are sequentially added to a growing chain (Merrifield (1963) J. Am. Chem. Soc. 85:2149-2156).
  • the amino acid sequences of such synthetic polypeptides may be based on the consensus or especially homologous sequence of Fig. 5 described in the Experimental section hereinafter, or on the sequence for the entire intestinal mucin gene.
  • the second and preferred method for synthesizing the polypeptides of the present invention involves the expression in cultured cells of recombinant DNA molecules encoding a desired portion of an intestinal mucin gene, most likely the tandemly repeated segments.
  • the gene may itself be natural or synthetic.
  • the natural gene is obtainable from cDNA or genomic libraries using available probes, such as
  • SMUC 40-42 probes may be synthesized based on the DNA sequences reported in Fig. 4, as described in the Experimental section hereinafter. Suitable cDNA and genomic libraries may be obtained from human cell lines known to contain the intestinal mucin gene, such as intestinal tumor cells. Alternatively, polynucleotides may be synthesized by other well-known techniques. For example, short single- stranded DNA fragments may be prepared by the phosphoramidite method described by Beaucage and Carruthers (1981) Tett. Letters 22:1859-1862.
  • a double-stranded fragment may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Particular DNA sequences may be based on Fig. 4 herein.
  • DNA constructs capable of introduction to and expression in an in vitro cell culture will be incorporated in DNA constructs capable of introduction to and expression in an in vitro cell culture.
  • the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction and integration within the genome of cultured mammalian or other eukaryotic cell lines.
  • DNA constructs prepared for introduction into bacteria or yeast will include a replication system recognized by the host, the intestinal mucin DNA fragment encoding the desired polypeptide product, transcriptional and translational initiation regulatory sequences joined to the 5'-end of the intestinal mucin DNA
  • transcriptional regulatory sequences will include a heterologous promoter which is recognized by the host.
  • available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with an insertion site for the intestinal mucin DNA sequence may be employed. To be useful in the detection methods of the
  • the polypeptides are obtained in substantially pure form, that is, typically about 50% w/w or more purity, substantially free of interfering proteins and contaminants, especially carbohydrate containing compounds.
  • the intestinal mucin polypeptides are isolated or
  • polypeptide 15 synthesized in a purity of at least about 80% w/w and, more preferably, in at least about 95% w/w purity.
  • homogeneous polypeptides of at least 99% w/w can be obtained.
  • the proteins may be purified by use of the
  • affinity chromatography is performed by first linking the antibodies to the solid support and then contacting the linked antibodies with the source of the intestinal mucin proteins, e.g., lysates of
  • in vitro techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides, while in vivo techniques require the injection of the polypeptides into a wide variety of vertebrates. Suitable vertebrates are
  • Polypeptides having more than about 30 amino acids,, particularly more than about 50 amino acids, may serve directly as immunogens. If the polypeptide is smaller than about 10 kD, particularly less than about 6 kD, it may be necessary to join the polypeptide to a larger molecule to elicit the desired immune response.
  • the immunogens are then injected into the animal according to the predetermined schedule, and the animals are bled periodically with successive bleeds generally having improved titer and specificity.
  • the injections may be made intramuscularly, intraperitoneally, subcutaneously, or the like, and usually an adjuvant, such as incomplete Freund's adjuvant, will be employed.
  • monoclonal antibodies can be obtained by preparing immortalized cell lines capable of producing antibodies having the desired specificity.
  • immortalized cell lines may be produced in a variety of ways. Conveniently, a small vertebrate, such as a mouse, is hyperimmunized with the desired antigen by the method just described. The vertebrate is then killed, usually several days after the final immunization, the spleens removed, and the spleen cells immortalized. The manner of immortalization is not critical. Presently, the most common technique is fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519. Other techniques include EBV transformation, transformation with bare DNA, e.g., oncogenes, retroviruses, etc., or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies.
  • the manner of fusion is not critical and various techniques may be employed.
  • the spleen cells and myeloma cells are combined in the presence of a nonionic detergent, usually polyethylene glycol, and other additives such as Dulbecco's Modified Eagle's Medium, for a few minutes.
  • a nonionic detergent usually polyethylene glycol
  • other additives such as Dulbecco's Modified Eagle's Medium
  • the nonionic detergent is rapidly removed by washing the cells.
  • the fused cells are promptly dispensed in small culture wells (usually in a microtiter plate) at relatively low density, ranging from about l-5xl0 5 per well, in a selective medium chosen to support growth of the hybrid cells while being lethal to the myeloma cells.
  • the myeloma cell line has been mutated to be sensitive to a lethal agent, typically being HAT sensitive.
  • the cell line can be maintained as viable cultures and/or by lyophilization or frozen storage.
  • hybridomas may be furtherscreened. Hybridomas providing high titers are desirable. Furthermore, cytotoxic antibodies, e.g., IgG2a, IgG2b, IgG3 and IgM, may be selected for use in therapeutic treatment of colorectal cancers. For use in immunodiagnostic assays, antibodies having very high specificity for the antigenic site are desirable.
  • monoclonal antibodies may be isolated from the supernatants of the growing colonies. The yield of antibodies obtained, however, is usually low. The yield may be enhanced by various techniques, such as injection of the hybridoma cell line into the peritoneal cavity of a vertebrate host which will accept the cells.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Proteinaceous and other contaminants will usually be removed from the monoclonal antibodies prior to use by conventional technique, e.g., chromatography, gel filtration, precipitation, extraction, or the like.
  • polypeptides used as the im unogen antibodies having high specificity and affinity for the intestinal mucin protein can be obtained.
  • the polypeptide selected should represent one or more epitopic sites which are unique to the intestinal mucin protein and which can distinguish intestinal mucin from closely related proteins such as mammary mucin. Such unique epitopes are found on polypeptides expressed by cells containing sequences from SMUC 40-42 or synthetic MRP's.
  • polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labelled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • Antibodies and polypeptides prepared as described above can be used in various immunological techniques for detecting intestinal mucin protein in biological specimens, particularly cell samples such as biopsy tissue samples and body fluid samples, including blood, plasma, serum, urine, stool and the like. Depending on the nature of the sample, both liquid phase assays and solid-phase immunohistochemical staining techniques will find use. Conveniently, immunohistochemical staining techniques may be used with cell samples including tissue samples, sputum, and lung lavage samples. For example, a tissue sample may be fixed in formalin, B-5, or other standard histological preservative, dehydrated and embedded in paraffin as is routine in any hospital pathology laboratory. Sections may then be cut from the paraffinized tissue block and mounted on glass slides.
  • the intestinal mucin protein may then be detected in the cytoplasm or extracellular space by exposure with labelled anti-intestinal mucin antibody or exposure to unlabelled anti-intestinal mucin antibody and a labelled secondary antibody.
  • Sputum and lavage samples are typically prepared in a similar manner where the sample is first dehydrated by exposure to a dehydrating agent, typically a low molecular weight alcohol.
  • a dehydrating agent typically a low molecular weight alcohol.
  • Liquid phase immunoassays or Western Blot analysis will also find use in the detection of the intestinal mucin protein particularly in body fluids when the protein is shed into such fluids, e.g., blood or stool.
  • Solid tissue and sputum samples may also be assayed in liquid phase systems by 5 lysing the cellular sample in order to release the intracellular protein. Once the protein is released, the sample will be placed in a suitable buffer, the sample buffer subjected to a suitable immunoassay. Numerous competitive and non-competitive immunoas
  • anti-intestinal mucin antibodies may be coupled to toxins, such as diphtheria toxin and the ricin A chain, and administered to patients suffering from epithilial cell cancers.
  • toxins such as diphtheria toxin and the ricin A chain
  • the use of , antibody conjugated toxins in cancer therapy is described generally in U.S. Patent Nos. 4,093,607; 4,340,535; 4,379,145; and 4,450,154.
  • Antibodies alone may also find use in treatment, particularly by blocking or interrupting some function activity of the intestinal mucin protein which contributes to the neoplastic phenotype.
  • the products of gene expression are also useful as a ready substrate for assay of glycosyltransferase activity (e.g., UDP-N-Acetylgalactosamine transferase) , and other glycosylation enzymes.
  • glycosyltransferase activity e.g., UDP-N-Acetylgalactosamine transferase
  • the resultant partially glycosylated products may serve as substrates for other enzymes.
  • HFA and HFB preparations of LS174T xenograft mucin deglycosylated with hydrogen fluoride as described herein
  • MRP a synthetic peptide with the mucin repeat sequence
  • BSA bovine serum albumin
  • bp base pairs
  • kb kilobase pairs
  • SSC standard sodium citrate buffer.
  • Mucin was purified from LS174T human colon cancer cell tumors (grown in nude mice) using gel filtration and CsCl density gradient centrifugation. This mucin had an amino acid composition that was 29% threonine, 14% serine, and 15% proline, similar to that found previously for human intestinal mucin (Mantle, M. , et al., (1984) Biochem. J. 217:159-167; Mantle, M., et al., (1984) Biochem. J. 224:345- 354; Wesley, A., et al., (1985) J. Biol. Chem. 260:7955- 7959) .
  • Antibodies were prepared in New Zealand White rabbits against HFA, HFB, or native mucin using three or four subcutaneous injections of 50-100 ⁇ g of antigen. Enzyme-linked immunosorbent assays indicated that all immunogens elicited antibodies ( Figure 11) .
  • a human jejunal cDNA library constructed in the ⁇ gtll expression vector was obtained from Dr. Yvonne Edwards (Medical Research Council, Human Biochemical Genetics Unit, University College London, London, United Kingdom) (Green, F., et al., (1987) Gene (Amst.) 57:101-110).
  • This library was plated in soft agar at a density of 25,000 plaques/150-mm plate as described (Huynh, T., et al., (1985) in "DNA Cloning Techniques: A Practical Approach" (Glover, D. , Ed.) pp. 49- 78, IRL Press, Oxford).
  • the plates were incubated at 37 ⁇ C until plaques began to appear and were then overlayed with isopropyl 0-D-thiogalactopyranoside-saturated nitrocellulose membranes and incubated for an additional 3 h.
  • the membranes were then removed and immunoscreened using anti-HFB serum at a 1:50 dilution and horseradish peroxidase-conjugated goat anti-rabbit IgG (Tago) using previously described methods (Young, R.A. , and Davis, R.W. (1985) in "Genetic Engineering” (Setlow, J., and Hollaender, A., Eds.) Vol. 7, pp. 29-41, Plenum Publishing Corp., New York).
  • RNA purification and poly(A) + RNA isolation, gel electrophoresis, transfer to nylon membranes, and hybridization probe analysis was conducted as described (Gum, J.R., et al., (1987) J. Biol. Chem. 262:1092-1097). Protein immunoblots were performed using a 1:50 dilution of antibody (Gum, J.R., ibid.). High molecular weight DNA was prepared .
  • a peptide with the sequence KYPTTTPISTTTMVTPTPTPTGTQT was prepared using an Applied Biosystems model 430A peptide synthesizer by Joel Boymel of the National Jewish Center for Immunology and Respiratory Medicine, Denver, CO.
  • the final 23 residues of this peptide represent the sequence of the first repeat of SMUC 40; the initial K and Y residues were added to allow glutaraldehyde conjugation and radioiodination (for future studies) , respectively.
  • 1 mg of peptide was emulsified in complete Freund's adjuvant and injected intrader ally at multiple sites into a female New Zealand rabbit (Vaitukaitus, J.L. (1981) Methods Enzymol. 73:46-52).
  • Three weeks later a second set of injections using 0.5 mg of peptide in incomplete adjuvant was administered, and the rabbit was bled 12 days later and serum prepared.
  • Antibodies prepared against HFB were used to screen the intestinal cDNA library and three positives were obtained from a screening of 230,000 recombinant plaques. These clones, which were designated SMUC 40, SMUC 41, and SMUC 42, were purified and tested for antigenicity using anti-HFA, anti-HFB, and anti-native mucin as shown in Fig. 1. Only antisera against the completely deglycosylated HFB produced positive plaques in this experiment.
  • Fig. 2 shows immunoblot analysis of the 0-galactosidase fusion proteins produced by lysogens of these recombinants. Anti-HFB reacts strongly with the fusion proteins produced by SMUC 40-42.
  • the recombinant phage DNA was digested with EcoRI, and each clone was found to contain a single, unique insert. Sequence analysis of the terminal regions of these clones indicated that they all contained repetitive sequences. Exonuclease III was then used to generate partially deleted clones for sequence analysis of the interior regions of these _ ⁇
  • SMUC 87 The sequence of this clone, SMUC 87, is shown in Figure 4E from base 442 to its 3' -end.
  • This clone contains 4 tandem repeats.
  • the first two tandem repeats are of the typical 23 amino acid variety, and thus 69 nucleotides long.
  • the third and fourth however, contain 24 and 22 amino acids respectively, corresponding to 72 and 66 nucleotides each.
  • the extra amino acid in the third repeat is a methionine residue (underlined) while a threonine is absent from the fourth repeat (indicated by a ⁇ ) .
  • Sequences upstream of base 442 in clone SMUC 87 are questionable as they contain multiple stop codons in all three reading frames. They may represent an unspliced intron, as there is a possible 3' -splice junction at base 518 (indicated by an arrow) , or they may be some artifact of cloning.
  • Clone 87 was isolated from the same cDNA library as were the rest of the clones. It was identified however, by hybridization probe screening using SMUC 41 as a probe, whereas SMUC 40-42 were identified using antibody probes. In this procedure, the library was screened following its transfer to nylon membrane using hybridization and wash conditions described in the publication disclosing Northern and Southern blotting.
  • SMUC 42 and the 3'-terminal 471 nucleotides of SMUC 41 can be clearly identified as not consisting of these repeat units.
  • anti-HFB serum appears to be strongly immunoreactive with the protein encoded by the 69-bp repetitive element.
  • the amino acid sequences deduced for each of these tandem repeats are shown in Fig. 5.
  • the 23-amino acid consensus sequence of these repeat units contains 14 threonine and 5 proline residues, including a group of five consecutive threonines and a stretch containing three threonine-proline direct repeats.
  • the 14 repetitive units contained in the three partial cDNA clones isolated in this study have 90% overall sequence identity with the consensus sequence shown in Fig. 5. Even more conserved is the 12-amino acid stretch enclosed in the box in Fig. 5, which exhibits 98% overall sequence identity with the consensus sequence. Only 11 serine residues are found dispersed among these 14 tandem repeats and nine of them occur as substitutions for threonine in the consensus sequence. On the other hand, the carboxylterminal 157-amino acid region deduced from the 3'- terminal 471 nucleotides of clone SMUC 41 (which does not consist of the tandem repeats) contains 25 serine residues.
  • SMUC 41 contains the only cysteine, present as a cyscys dipeptide, and most of the aromatic amino acids.
  • Two potential N- glycosylation recognition sites are encoded in the sequences presented here, one in the last repeat unit of SMUC 40 and one near the 3'-terminal of SMUC 41.
  • RNA from a number of human cell lines and tissues was subjected to RNA blot analysis using SMUC 41 cDNA as a probe (Fig. 7) .
  • the messages that hybridized to SMUC 41 were large and polydisperse, averaging 7600 bases in length. In addition, a distinct but faint band at 1850 bases was sometimes detectable (Fig. 10) .
  • the strongest hybridization signals observed in these experiments were expressed by colon, colon tumor, and small intestine RNA.
  • LM-12 a low mucin-producing variant of LS174T cells (Kuan, S.F., et al., (1987) Cancer Res. 47:5715-5724), exhibits only a faint hybridization signal as does RNA from LS-G and SW1116 cells. No detectable signal was obtained with either placenta or the thyroid tumor poly(A) + RNA used here.
  • Genomic DNA was isolated from the lymphocytes of two human donors and five colon cancer cell lines, restriction endonuclease-digested, and subjected to electrophoresis and hybridization blot analysis using the SMUC 41 probe (Fig. 9) .
  • Six of these DNA samples were cleaved with EcoRI and all exhibited a single hybridization band that was larger than the 23.1 kb standard (Fig. 9A) .
  • As a control for restriction endonuclease cleavage these same blots were examined using a probe for carcinoembryonic antigen, and the resulting band pattern was similar to previously published results (Thompson, J.A. , et al., (1987) Proc. Natl. Acad.
  • RNA and genomic DNA blots were probed with segments of SMUC . 41 representing the tandem repeats and the 3*-terminal region.
  • SMUC 41 was digested with Apal which cleaves after base 370, five nucleotides downstream from the end of the last repeat.
  • probe 3'-466 hybridizes to every tested restriction fragment that hybridizes to probe 5'-370 suggests that sequences similar to those in the 3'-terminal portion of SMUC 41 are repeated elsewhere in the intestinal mucin gene, presumably upstream of the 69-bp tandem repeats. This latter conclusion must be considered tentative at present, however, as exons, pseudogenes, related genes, and multiple alleles of the intestinal mucin gene could all contribute to the complex and band pattern observed.

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Abstract

Des séquences d'ADN, codant pour une partie de la séquence polypeptidique de la mucine intestinale humaine (MIH), ont été isolées et caractérisées. Ces séquences peuvent être exprimées en procaryotes, pour produire des polypeptides de MIH non glycosilés ou en eucaryotes, pour produire des versions glycosilées, tous pouvant servir pour dresser des anticorps ou pour d'autres usages. En outre, l'ADN a été utilisé pour caractériser l'expression et l'organisation génomique de la famille des gènes de la MIH. Des réactifs, fondés sur ces découvertes, fournissent des moyens de diagnostic et de traitement de divers troubles biologiques, tels que les fibroses cystiques et les cancers du côlon.
PCT/US1989/003206 1989-04-14 1989-07-25 Mucine intestinale humaine WO1990012892A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997043643A1 (fr) * 1996-05-15 1997-11-20 The Regents Of The University Of California Inhibiteurs de la production de mucine provoquee par pseudomonas aeruginosa et traitements des patients souffrant de mucoviscidose

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4132600A (en) * 1977-06-01 1979-01-02 Massachusetts Institute Of Technology Enzymatic noninvasive method for detecting cancer
US4818682A (en) * 1984-06-25 1989-04-04 Mucan Diagnostics Pty., Ltd. In vitro detection of gastrointestinal cancer
US4851357A (en) * 1986-03-14 1989-07-25 Ikuo Yamashina Plates for assay of sugar chaindirected antibody and production thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4132600A (en) * 1977-06-01 1979-01-02 Massachusetts Institute Of Technology Enzymatic noninvasive method for detecting cancer
US4818682A (en) * 1984-06-25 1989-04-04 Mucan Diagnostics Pty., Ltd. In vitro detection of gastrointestinal cancer
US4851357A (en) * 1986-03-14 1989-07-25 Ikuo Yamashina Plates for assay of sugar chaindirected antibody and production thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL JOURNAL, Volume 217, published in 1984, (Biochemical Society, London, England), M. MANTLE et al., "Antigenic and structural features of goblet-cell mucin of human small intestine", pages 159-167, see especially the Abstract. *
CANCER RESEARCH, Volume 38, published in 1978, (Waverly Press Inc., Baltimore, Maryland, USA), D.V. GOLD et al., "Comparison of Human Colonic Mucoprotein Antigen from Normal and Neoplastic Mucosa", pages 3204-3211. *
CANCER RESEARCH, Volume 46, published in 1986, (Waverly Press Inc., Baltimore, Maryland, USA), J. BARA et al., "Monoclonal Antibodies against Oncofetal Mucin M1 Antigens Associated with Precancerous Colonic Mucosae", pages 3983-3989, see especially the Abstract. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 263, Number 26, published in 1988, (American Society for Biochemistry and Molecular Biology Inc., Baltimore, Maryland, USA), S. GENDLER et al., Communication: "A Highly Immunogenic Region of a Human Polymorphic Epithelial Mucin Expressed by Carcinomas is Made up of Tandem Repeats", pages 12820-12823. *
NATURE, Volume 328, published in 1987, (Macmillan Magazines Ltd., New York, New York), D. SWALLOW et al., "The human tumour-associated epithelial mucins are coded by an expressed hypervariable gene locus PUM", pages 82-84, see especially the Abstract. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA), Volume 84, published in 1987, (The National Academy of Sciences, Washington D.C.), S.J. GENDLER et al., "Cloning of partial cDNA encoding differentiation and tumor-associated mucin glycoproteins expressed by human mammary epithelium", pages 6060-6064, see especially the abstract. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997043643A1 (fr) * 1996-05-15 1997-11-20 The Regents Of The University Of California Inhibiteurs de la production de mucine provoquee par pseudomonas aeruginosa et traitements des patients souffrant de mucoviscidose

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