WO1991008217A1 - Mucines intestinales humaines - Google Patents

Mucines intestinales humaines Download PDF

Info

Publication number
WO1991008217A1
WO1991008217A1 PCT/US1990/007087 US9007087W WO9108217A1 WO 1991008217 A1 WO1991008217 A1 WO 1991008217A1 US 9007087 W US9007087 W US 9007087W WO 9108217 A1 WO9108217 A1 WO 9108217A1
Authority
WO
WIPO (PCT)
Prior art keywords
mucin
nucleic acid
sequence
smuc
polypeptide
Prior art date
Application number
PCT/US1990/007087
Other languages
English (en)
Inventor
Young S. Kim
James R. Gum, Jr.
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Publication of WO1991008217A1 publication Critical patent/WO1991008217A1/fr

Links

Classifications

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

Definitions

  • This invention relates generally to polypeptides and nucleic acid sequences characteristic of 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.
  • 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.
  • cystic fibrosis Several human diseases have been observed to be directly associated with alterations in intestinal mucin production, including cystic fibrosis, familial polyposis 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 coli, ulcerative colitis and colon cancer produce mucins that are abnormally glycosylated.
  • the present invention provides 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 acid residues), and one or more of the biological properties (e.g., immunological properties and in vivo and jjn vitro biological activity) , of naturally-occurring human intestinal mucins, including allelic variants thereof.
  • At least two distinct families of the protein have been detected, one similar to and exemplified by the SMUC forms and a second similar to and exemplified by SIB forms.
  • These 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 synthesis.
  • Products of microbial or vertebrate (e.g., mammalian and avian) cellular expression may be further characterized by freedom from association with human proteins or other contaminants which may be associated with human intestinal mucins in the natural cellular environment or in extracellular fluids.
  • the products of typical yeast (e.g., Saccharomyces cerevisiae) or procaryote (e.g., E_j_ coli) host cells are free of association with any mammalian proteins.
  • polypeptides of the invention may be glycosylated naturally or with carbohydrate patterns characteristic of mammalian or other eucaryotic cells. 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 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 some of these sequence properties including invertebrate proteins and intestinal mucins from other organisms, usually vertebrates. These vertebrates will include mice, monkeys, pigs and other mammals.
  • synthetic polypeptides wholly or partially duplicative of continuous sequences of human intestinal mucin amino acid residues which are herein disclosed.
  • sequences by virtue of sharing primary, secondary or tertiary structural and conformational characteristics with a naturally-occurring human intestinal mucin protein may possess biological activity and/or immunological properties uncommon with the naturally-occurring product such that they may be employed as biologically active or immunological substitutes for human intestinal mucin in therapeutic and immunological processes.
  • monoclonal and polyclonal antibodies generated by standard means which are immunoreactive with such polypeptides and, preferably, also immunoreactive with naturally-occurring human intestinal mucin.
  • nucleic acids normally DNA fragments, and polypeptide sequences suitably deduced therefrom which represent, respectively, the primary structural conformation of different human intestinal mucin proteins.
  • 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 or vertebrate cell 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 or 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 expression of polypeptide analogs or derivatives (including gene fusions) , 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 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 forms (e.g., one or more epitopes) .
  • one or more amino acid residues i.e., deletion analogs containing less than all of the residues specified for human intestinal mucin and/or substitution analogs, wherein one or more residues specified are replaced by other residues and/or addition analogs, wherein one
  • references to nucleic acid sequences are intended to include their complementary strands. This, of course, includes operable-linkage of a mucin sequence to a desired endogenous or exogenous controllable element (e.g., promoter).
  • diagnostic and pharmaceutical compositions comprising polypeptide products of the invention together with suitable diluents, adjuvants and/or carriers which allow for diagnosis or therapy, especially in the treatment of various disease states. Diagnostic kits may also be produced using the novel compositions of this invention.
  • Polypeptide products of the invention may be "labelled" by covalent association with a detectable marker substance (e.g., radiolabelled with 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.
  • a detectable marker substance e.g., radiolabelled with I
  • detection markers such as radiolabels and non- isotopic labels such as biotin
  • 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 sequenced 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. In a few cases, restriction fragments obtained from Taql and Mspl digests of SMUC 40 and 42 were force cloned into Accl- and EcoRI-digested M13mpl8 and used to generate templates. Sequencing done using this latter method is indicated using dashed arrows. •-- *- ⁇ "• ⁇ ⁇ -
  • Nucleotide and deduced amino acid sequence of various SMUC-type 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 2E 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 (not shown) .
  • Figure 5 Expression of SMUC-type MRP and HFB antigens in human normal and cancerous colon and gastric tissues using immunohistochemical methods. The technique described in Itzkowitz et al. , (1989) Cancer Res. 49.?197*-2G4, was used.
  • the arrows represent the length and direction of individual sequencing reactions.
  • Nucleotide and deduced amino acid sequence of various SIB-type intestinal mucin cDNA clones Nucleotide position is indicated by the numbers at the right and amino acid position by those at the left.
  • the repeat units for SIB 124 and 139 are indicated by the arrows.
  • a six amino acid insert in SIB 124 is indicated by braces.
  • nucleic acid sequences encoding portions of the polypeptide sequence of distinct forms of human intestinal mucins have been isolated and characterized. Further, the isolated DNA segments of the SMUC-type mucins have been expressed in providing isolatable quantities of polypeptides displaying biological (e.g., immunological) properties of naturally- occurring SMUC-type human intestinal mucins, as well as both in vivo and .in vitro biological activities of the SMUC-type human intestinal mucins.
  • the isolated DNA segments of the SIB-type mucins have similarities characterized in a consensus sequence.
  • the consensus sequence has 5 serines and 7 threonines, in contrast to the SMUC consensus sequence which has 0 and 14 respectively (compare Figs. 8 and 3) .
  • the SIB consensus sequence contains 1 proline in contrast to 5 for the SMUC. Consequently, the SIB clones are more similar in composition to the neutral fraction of intestinal mucin than the acidic fraction described by Wesley et al.. (see Fig. 9) .
  • the serines and threonines in the SIB consensus sequence appear in clusters of two or three.
  • a human small intestine ⁇ gtll cDNA library was screened using antisera prepared against the deglycosylated SMUC-type 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 PTTTPITTTTTVTPTPTPTGTQT.
  • RNA blots probed with one of these cDNAs, SMUC 41 exhibited large, polydisperse hybridization bands at -7600 bases.
  • the SMUC 87 gene is notable in possessing homology segments of 24 amino acids and of 22 amino acids, demonstrating that these conserved segments may be of variable length, having an amino acid insertion or deletion.
  • the SIB-type sequences were hypothesized after reports of isolation of separable species of a human mucin preparation from small intestinal samples. See Wesley et al.. J. Biol. Chem. 260:7955-59 (1985), who demonstrated that human small intestinal mucin could be separated using ion exchange chromatography into two fractions with different amino acid contents. Total human small intestinal mucin was deglycosylated and antibodies were prepared. Clones of human intestinal mucin nucleic acid sequences were isolated with the resulting antibody.
  • SIB-type sequences are distinct from the SMUC-type sequences and seem not to immunologically cross react. According to another aspect of the present invention, methods for identifying cancers in human patients rely on detection of the non-glycosylated expression product of the human intestinal mucin genes 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 one or a combination of the distinct human intestinal mucin proteins.
  • Detection of 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.
  • Abnormal glycosylation of the human intestinal mucin proteins has been associated with the pathogenesis of a number of human tumors, particularly epithelial cell tumors.
  • the expression products of the human intestinal mucin genes are highly glycosylated proteins including heterogeneous carbohydrate comprising -75% of the glycoprotein.
  • the polypeptide portion of the SMUC-type protein 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 on one or more of the distinctive mucin proteins.
  • the polypeptides of the present invention will be either haptenic or antigenic, typically including at least about 6 amino acids, usually at least about 9 amino acids. and more usually about 12 or more amino acids found contiguously within a 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 particular mucin protein. By 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 typically tandemly repeated without intervening amino acids within or between repeat segments.
  • the SIB-type sequence is characterized by a particular repetitive amino acid segment as follows:
  • Sequence (a) corresponds to the consensus nucleic acid sequence: C) ACC ACC ACT ACG GTG ACC CCA ACC CCA ACA CCC ACC, which is contained within the consensus nucleic acid sequence encoding sequence (b) : d) CCA ACC ACG ACA CCC ATC ACC.ACC ACC ACT ACG
  • the sequence (x) corresponds to the consensus nucleic acid sequence: y) GAG ACC ACC TCA CAC AGT ACT CCC AGC TTC ACT TCT TCA ATC ACC ACC.
  • Substantial homology or substantial identity of a nucleic acid sequence indicates either that: a) there is greater than about 65%, more typically greater than about 75%, and optimally greater than 90% homology with a disclosed segment; or b) 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. As is well known, 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 a natural intestinal mucin protein may be produced by either of at least two general approaches.
  • polypeptides having fewer - than about 50 amino acids, more usually fewer than -abou 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. 3 (for the SMUC-type mucin) or of Fig. 7 (for the SIB-type mucin) described below or on the sequence for the entire intestinal mucin gene.
  • a 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, 53 or 87 or SIB 121, 124, 134, 136 and 139. Using such segments or genes, additional homologous gene sequences might be isolated from other individuals Such genes might represent, among other possibilities, other alleles of or phantom genes of related polypeptides.
  • probes may be synthesized based on the DNA sequences reported in Fig. 2 (for the SMUC-type mucins) or in Fig. 7 (for the SIB-type mucins) , as described in the Experimental section hereinafter.
  • Suitable cDNA and genomic libraries may be obtained from human cell lines known to contain the appropriate intestinal mucin gene, such as intestinal tumor cells.
  • polynucleotides may be synthesized by other well-known techniques.
  • 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--isomplementary strand using DNA poly erase with an appropriate primer sequence.
  • Particular DNA sequences may be based on those reported in Fig. 2 (for the SMUC-type mucins) or in Fig. 7 (for the SIB- type mucins) 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 eucaryotic cell lines.
  • DNA constructs prepared for introduction into bacteria or yeast will include a replication system recognized by the host, the appropriate 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 sequence, and transcriptional and translational termination regulatory sequences joined to the 3'-end of the intestinal mucin sequence.
  • the transcriptional regulatory sequences will typically 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.
  • a single or multiple vectors under individual or coordinate control may be used to produce a desirable combination of various forms or types of human intestinal mucin. These may also be coupled with other accompanying enzymes or proteins, e.g., glycosylation, modification or other desired activities.
  • 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 synthesized in a purity of at least about 80% w/w and, more preferably, in at least about 95% w/w purity.
  • polypeptides of at least 99% w/w can be obtained.
  • the proteins may be purified by use of the antibodies described hereinafter using immunoadsorbent affinity chromatography.
  • affinity chromatography is performed by first linking antibodies or appropriate affinity reagents to the solid support and then contacting the linked antibodies or affinity reagents with the source of the intestinal mucin proteins, e.g., lysates of cells which naturally produce intestinal mucin or which produce intestinal mucin as a result of introduction of a recombinant intestinal mucin DNA molecule.
  • polyclonal antibodies specific for the intestinal mucin protein may be produced by in vitro or in vivo techniques.
  • In vitro techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or fragments, while in vivo techniques require the injection of the polypeptides or fragments into any of a wide variety of target immune systems, such as vertebrates. Suitable vertebrates are typically non-human, including mice, rats, rabbits, sheep, goats, and the like.
  • Polypeptides having more than about 5 to 30 amino acids, particularly more than about 50 to 100 amino acids, may serve directly as immunogens.
  • 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 a 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, ⁇ an . usually an adjuvant, such as incomplete Freu ⁇ d'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 depending upon the target immune system.
  • a small vertebrate such as a mouse
  • the vertebrate is then killed, usually several days after the final immunization, the spleen removed, and the spleen cells immortalized.
  • the manner of immortalization is not critical.
  • 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 1-5x10 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. After sufficient time, usually from one to two weeks, colonies of hybrids are observed and plates containing hybrid positive wells are identified. The plates and wells having only one colony per well are selected, and supernatants from these wells are tested for binding activity against the desired intestinal mucin protein or the isolated antigen. Once positive hybridomas are identified, the cell line can be maintained as viable cultures and/or by lyophilization or frozen storage.
  • Hybridomas providing high titers are desirable.
  • cytotoxic antibodies e.g., IgG 2 _, IgG- ⁇ , IgG 3 and IgM, may be selected for use in therapeutic treatment of colorectal cancers.
  • 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 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 immunogen antibodies having high specificity and affinity for the desired type of intestinal mucin protein can be obtained.
  • the polypeptide selected should represent one or more epitopic sites which are unique to the desired 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 SMUC- type MRP's or SIB-type mucins or their synthetic SIB-type MRP's.
  • polypeptides and antibodies of the present invention may be used with or without modification.
  • 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, which are incorporated herein by reference.
  • Antibodies and polypeptides prepared as described above can be used in various immunological techniques for detecting intestinal mucin proteins 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 proteins may then be detected in the cytoplasm or extracellular space by exposure with labelled anti-(type-specific) 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.
  • Liquid phase immunoassays or Western Blot analysis will also find use in the detection of the intestinal mucin proteins particularly in body fluids when the proteins are shed into such fluids, e.g., blood or stool.
  • Solid tissue and sputum samples may also be assayed in liquid phase systems by 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.
  • a suitable buffer the sample buffer subjected to a suitable immunoassay.
  • 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 functional activity of intestinal mucin protein which contributes to the neoplastic phenotype.
  • a vaccination for inducing a cellular immune response *• can be prepared by standard procedures ⁇ . See* e.g., Rosenberg, S. (1990) Scientific American 262:62-69; Brown et al. (1989) Vaccines 89: Modern Approaches to New Vaccines Including Prevention of AIDS. Cold Spring Harbor Press, New York; Murray, K. et al. (1988) "Application of recombinant DNA technology in the development of viral vaccines," Vaccine 6:164- ; Mitchel, G. et al. (1988) "The Way Ahead for
  • Vaccines and Vaccination Symposium Summary," Vaccine 6:200; and Paul, W. (1989) Fundamental Immunology (2d Ed.), Raven Press; each of which are hereby incorporated herein by reference.
  • the products of gene expression are also useful as ready substrates for assay of glycosyltransferase activity (e.g., UDP-N-Acetylgalactosamine transferase) , and other glycosylation enzymes.
  • 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
  • SMUC-type MRP a synthetic peptide with the SMUC-type mucin repeat sequence
  • SIB-type MRP a synthetic peptide with SIB-type 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 muci -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.
  • 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
  • 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. 262:1092-10971. Protein immunoblots were performed using a 1:50 dilution of antibody (Gum, J.R. , ibid.). High molecular weight DNA was prepared using proteinase K, RNase A, and phenol as described by Blin and Stafford (Blin, N. , and Stafford, D.W. (1976) Nucleic Acids Res. 3:2303-2315).
  • 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 intradermally 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 anti-SMUC-type serum prepared.
  • Antibodies prepared against HFB were used to screen the intestinal cDNA library and three positive plaques 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 SMUC-type native mucin. Only antisera against the completely deglycosylated HFB produced positive plaques in this experiment. Immunoblot analysis of the ⁇ -galactosidase fusion proteins produced by lysogens of these recombinants was performed. Anti-HFB reacts strongly with the fusion proteins produced by SMUC 40-42.
  • Clones SMUC 53 and 87 were isolated from the same cDNA library as were the rest of these clones. They were 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 87 Each of these clones was found to contain tandem repeats, usually of 69 nucleotides (Fig. 2) . Examples of shorter and of longer nucleotide stretches have been found in SMUC 87 (see Fig. 2E) . The sequence of this clone, SMUC 87, is shown in
  • Fig. 2E 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 ar 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.
  • 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. 3. Even more conserved is the 12-amino acid stretch enclosed in the box in Fig. 3, 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 serine residues in the SMUC-type intestinal mucin are clustered in regions other than the tandem repeats.
  • the 3'-terminal region of SMUC 41 also contains the only cysteine, present as a cys-cys 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.
  • the broad smear of antibody reactive protein in the HFB sample is indicative of the cleavage of the mucin backbone that occurs during deglycosylation.
  • SMUC-type MRP conjugated BSA exhibits polydispersity, on the other hand, due to irregular conjugation of BSA with itself and the peptide.
  • Antibodies against the SMUC-type MRP had a specifity similar to anti-i-HFB. Again, reactivity was apparent with HFB- and SMUC-type MRP-conjugated BSA but not with HFA- or unconjugated BSA.
  • RNA from a number of human cell lines and tissues was subjected to RNA blot analysis using SMUC 41 cDNA as a probe) .
  • 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.
  • 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 th 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. 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. 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. Sci. USA 98:2965-2969).
  • 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. The 5*-terminal 370-base fragment and the 3'-terminal 466- base fragment were purified by gel electrophoresis (Maniatis, T., et al., (1982) Molecular Cloning A Laboratory Manual.
  • the SIB-type sequences were discovered by isolating clones screened using an antibody prepared against a deglycoslyated preparation of a human small intestinal mucin polypeptides.
  • the antiserum recognized polypeptides of both the SMUC-type and a second type of human intestinal mucin, thus selecting out clones encoding two different types.
  • the SMUC-type clones were identified using a SMUC-type gene hybridization assay leaving the five clones reported, designated SIB-type mucins.
  • the SIB-type sequences are distinct from the SMUC-type sequences and appear not to immunologically cross-react with one another.
  • the SIB-type sequences are characterized by a consensus sequence reported in Fig. 8.
  • the consensus SIB- sequence has 5 serine residues and 7 threonine residues, in contrast to the SMUC-type consensus sequence, containing 0 and 14 respectively.
  • the SIB-type consensus sequence contains only 1 proline residue as compared with 5 for the SMUC-type consensus sequence.
  • the SIB clones are more similar in composition to the neutral fraction of intestinal mucin than the acidic fraction described by Wesley et al. (Fig. 9) .
  • the SIB-type consensus sequence has its serine residues and threonine residues clustered in groups of two or three. Both the SMUC- and SIB-type consensus repeats have two hydrophobic resides per repeat.
  • the SIB- and SMUC-type segments are further compared in Figs. 10 and 11.
  • SIB-type immunogens were performed essentially as described for the SMUC-type immunogen.
  • THe SIB-type mucin is not produced by the LS174T cell tumors used to isolate the SMUC-type mucin, though the immunogen used to produce the antibodies reactive against the SIB-type mucin also contained SMUC-type immunogen.
  • the clones selected from the SIB screening were also screened with the SMUC-type gene segments. Clones reactive with the mixture of SMUC- and SIB-type antibodies but which were not reactive with the SMUC-type gene segments were characterized, disclosing the newly designated SIB-type sequences.
  • the SIB- type apomucin and SMUC-type apomucin do not share significant epitopes.
  • SIB 139 There are three potential N-glycosylation sites in SIB 139. These are on the asparagine residues that start repeats number 1, 4 and 5 (see Fig. 7B) .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

On a isolé et caractérisé des séquences d'ADN codant une partie des séquences polypeptidiques des mucines intestinales humaines (HIM). Ces séquences peuvent être exprimées dans des procaryotes afin de produire des polypeptides non glycosylés de HIM, ou dans des eucaryotes afin d'en produire des versions glycosylées, dont la totalité est adaptée à la croissance des anticorps et à d'autres utilisations. Par ailleurs, on a utilisé l'ADN pour caractériser l'expression et l'organisation génomique de la famille des gènes de HIM. On peut utiliser des réactifs à base de ces découvertes pour diagnostiquer et traiter divers troubles biologiques, tels que la mucoviscidose et le cancer du côlon.
PCT/US1990/007087 1989-12-05 1990-12-04 Mucines intestinales humaines WO1991008217A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US44714089A 1989-12-05 1989-12-05
US447,140 1989-12-05

Publications (1)

Publication Number Publication Date
WO1991008217A1 true WO1991008217A1 (fr) 1991-06-13

Family

ID=23775141

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1990/007087 WO1991008217A1 (fr) 1989-12-05 1990-12-04 Mucines intestinales humaines

Country Status (1)

Country Link
WO (1) WO1991008217A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0804231A1 (fr) * 1993-07-30 1997-11-05 Olivera J. Finn Peptides synthetiques a repetitions en tandem multiples, a base de mucine et d'analogues, et utilisations
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
WO1999065924A2 (fr) * 1998-06-19 1999-12-23 Genzyme Corporation Preparation et utilisation de vaccins superieurs
US6262249B1 (en) * 1998-06-23 2001-07-17 Chiron Corporation Pancreatic cancer genes
WO2003008446A1 (fr) * 2001-07-19 2003-01-30 Mitsubishi Pharma Corporation Polypeptides se rapportant au transfert de signaux de recepteur de produits terminaux a glycation avancee

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
US4863854A (en) * 1985-08-12 1989-09-05 Sloan-Kettering Institute For Cancer Research Monoclonal antibodies to mucin-like human differentiation antigens

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
US4863854A (en) * 1985-08-12 1989-09-05 Sloan-Kettering Institute For Cancer Research Monoclonal antibodies to mucin-like human differentiation antigens

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 46, issued August 1986, BANA et al., "Monoclonal Antibodies Against Oncofetal Mucin M1 Antigens Associated with Precancerous Colonic Mucosae", pages 3983-3989. *
NATURE, Volume 328, issued 02 July 1987, SWALLOW et al., "The Human Tumor-Associated Epithelial Mucins are Coded by an Express Hypervariable Gen Locus PUM", pages 82-84. *
PROCEEDING OF THE NATIONAL ACADEMY OF SCIENCE, Volume 84, issued September 1987, GENDLER et al., "Cloning of Partial cDNA Encoding Differentiation and Tumor Associated Mucin Glycoproteins Expressed by Human Mammary Epithelium", pages 6060-6064. *
THE BIOCHEMICAL JOURNAL, Volume 224, issued 1984, MANTLE et al., "Biochemical Characterization of the Component Parts of Intestinal Mucin from Patients with Cystic Fibrosis", pages 345-354. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 260, No. 13, issued 05 July 1985, WESLEY et al., "Neutral and Acidic Species of Human Intestinal Mucin", pages 7955-7959. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 263, No. 26, issued 15 September 1988, GENDLER et al., "A Highly Immunogenic Region of a Human Polymorphic Epithelial Mucin Expressed by Cancinomas is Made up of Tandem Repeats", pages 12820-12823. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 264, No. 11, issued 15 April 1989, GUM et al., "Molecular Cloning of Human Intestinal Mucin cDNAs", pages 6480-6487. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0804231A1 (fr) * 1993-07-30 1997-11-05 Olivera J. Finn Peptides synthetiques a repetitions en tandem multiples, a base de mucine et d'analogues, et utilisations
EP0804231A4 (fr) * 1993-07-30 1999-08-18 Olivera J Finn Peptides synthetiques a repetitions en tandem multiples, a base de mucine et d'analogues, et utilisations
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
WO1999065924A2 (fr) * 1998-06-19 1999-12-23 Genzyme Corporation Preparation et utilisation de vaccins superieurs
WO1999065924A3 (fr) * 1998-06-19 2000-04-13 Genzyme Corp Preparation et utilisation de vaccins superieurs
US7666992B2 (en) 1998-06-23 2010-02-23 Novartis Vaccines And Diagnostics, Inc. Pancreatic cancer genes
US6664054B2 (en) 1998-06-23 2003-12-16 Chiron Corporation Pancreatic cancer genes
US7541142B2 (en) 1998-06-23 2009-06-02 Novartis Vaccines And Diagnostics, Inc. Pancreatic cancer genes
US6262249B1 (en) * 1998-06-23 2001-07-17 Chiron Corporation Pancreatic cancer genes
US7863423B2 (en) 1998-06-23 2011-01-04 Novartis Vaccines And Diagnostics, Inc. Pancreatic cancer genes
US8030455B2 (en) 1998-06-23 2011-10-04 Novartis Vaccines And Diagnostics, Inc. Pancreatic cancer genes
US8460873B2 (en) 1998-06-23 2013-06-11 Novartis Vaccines And Diagnostics, Inc. Pancreatic cancer genes
WO2003008446A1 (fr) * 2001-07-19 2003-01-30 Mitsubishi Pharma Corporation Polypeptides se rapportant au transfert de signaux de recepteur de produits terminaux a glycation avancee

Similar Documents

Publication Publication Date Title
AU698823B2 (en) DNA sequence and encoded mammary-specific breast cancer protein
US6054438A (en) Nucleic acid fragments encoding portions of the core protein of the human mammary epithelial mucin
US4918162A (en) Assays and antibodies for N-MYC proteins
EP0562123B1 (fr) Nouvelle substance physiologiquement active appelee l'epimorphine, gene codant pour cette substance, et anticorps de l'epimorphine
US5705341A (en) MDC proteins and DNAs encoding the same
WO1991008217A1 (fr) Mucines intestinales humaines
Van den Ouweland et al. Characteristics of a multicopy gene family predominantly consisting of processed pseudogenes
US7067635B2 (en) Nucleotide and deduced amino acid sequences of tumor gene Int6
EP0974652A1 (fr) Gene associe a la metastase cancereuse
US5837239A (en) Physiologically active substance designated as epimorphin genes encoding the same and antibodies thereto
WO1987006940A1 (fr) ANALYSES ET ANTICORPS DES PROTEINES N-myc
WO1990012892A1 (fr) Mucine intestinale humaine
US5171850A (en) Intestinal oncofetal gene
JP3670030B2 (ja) Ｍｄｃ蛋白質およびそれをコードするｄｎａ
JPH06293800A (ja) 新規生理活性物質エピモルフィン、それをコードする 遺伝子及びエピモルフィンに対する抗体
EP0871493A1 (fr) Facteur de migration derive des cellules des muscles lisses
WO1994028161A1 (fr) Anticorps specifiques du produit genique dcc
MXPA97009364A (en) Sequence of desoxirribonucleico acid and specific codified breast protein to detect m cancer

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

NENP Non-entry into the national phase

Ref country code: CA