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WO1997047655A1 - Procedes de detection d'un recepteur mutant d'hemochromatose aux fins de diagnostic - Google Patents

Procedes de detection d'un recepteur mutant d'hemochromatose aux fins de diagnostic Download PDF

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Publication number
WO1997047655A1
WO1997047655A1 PCT/US1997/010276 US9710276W WO9747655A1 WO 1997047655 A1 WO1997047655 A1 WO 1997047655A1 US 9710276 W US9710276 W US 9710276W WO 9747655 A1 WO9747655 A1 WO 9747655A1
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hemochromatosis
receptor
antibody
ligand
sample
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PCT/US1997/010276
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English (en)
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WO1997047655A9 (fr
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Barry E. Rothenberg
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Rothenberg Barry E
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Priority to AU35703/97A priority Critical patent/AU3570397A/en
Publication of WO1997047655A1 publication Critical patent/WO1997047655A1/fr
Publication of WO1997047655A9 publication Critical patent/WO1997047655A9/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology

Definitions

  • This invention relates generally to the field of diagnosis of genetic diseases and, more specifically the diagnosis of hemochromatosis.
  • Hemochromatosis represents a group of diseases that result from enhanced absorption of iron from the GI tract by active transport. Hemochromatosis is presently recognized as the most common genetic malady in humans, far exceeding cystic fibrosis, phenylketonuria and muscular dystrophy combined. One explanation for the high incidence of this genetic disease may be that mutations in multiple linked genes produce a similar phenotype. Hemochromatosis occurs most frequently in populations of European origin with a frequency in homozygotes and heterozygotes of approximately 0.3 and 13%, respectively. Genetic analysis of families indicate that the genes responsible for hemochromatosis are located to the major histocompatibility complex region, however, no specific genes have yet to be identified.
  • Hemochromatosis is not usually brought to clinical attention until symptoms develop, and simple removal of the iron after the development of tissue damage does not necessarily improve the organ function. Hemochromatosis is an under diagnosed and under treated disease that would benefit greatly from early diagnosis and an effective treatment if the gene or genes causing the underlying metabolic defect were known. Thus, there exists a need to identify the genes involved in hemochromatosis and to develop a diagnostic test which can identify individuals genetically predisposed to hemochromatosis or aid in the diagnosis of this disease.
  • the present invention provides an antibody that specifically binds to the hemochromatosis receptor.
  • the invention provides an antibody that specifically binds to the non-mutated form of the hemochromatosis receptor.
  • the present invention also provides methods to diagnose hemochromatosis or identify an individual genetically prediposed to this disease by detecting the hemochromatosis receptor in a sample from the individual.
  • the method involves contacting the receptor with an antibody specific for the receptor.
  • the present invention further provides methods to detect the hemochromatosis receptor by using a first and second antibody that bind to different antigenic determinants that are specific for the hemochromatosis receptor.
  • the invention also provides methods for purifying a hemochromatosis ligand that binds to iron and to the hemochromatosis receptor. The method involves isolating the ligand from a sample by contacting the sample with an iron matix support. The method further includes isolation of the ligand by binding to a solid support containing the hemochromatosis receptor.
  • the present invention discloses methods to diagnose a genetic predisposition to hemochromatosis by detecting a mutant form of the gene or genes involved in this disease.
  • hemochromatosis there are multiple genes involved in hemochromatosis which fall into one of two functional classes.
  • One class of genes encodes a protein ligand that binds iron and is involved in transporting the iron from the gastrointestinal tract into the body.
  • the term "hemochromatosis ligand” refers to the genetically encoded iron-binding ligand that is involved in iron uptake through the gastrointestinal tract.
  • the hemochromatosis ligand is composed of both protein and carbohydrate.
  • the second class of genes involved in hemochromatosis encode a recognition receptor that normally binds to the hemochromatosis ligand, assisting in transport of iron into the body.
  • the receptor is mutated resulting in abnormal accumulation of iron in the body-iron stores.
  • the term "hemochromatosis receptor” refers to the mutated form of the receptor that is found in individuals with hemochromatosis.
  • the hemochromatosis receptor can recognize the carbohydrate portion of the hemochromatosis ligand and, assist in transport of iron into the body through the gastrointestinal tract.
  • the phrase "non-mutated form of the hemochromatosis receptor” refers to the wildtype form of the receptor that binds to the hemochromatosis ligand.
  • the hemochromatosis receptor can also have the ability to chemically modify the carbohydrate associated with the hemochromatosis ligand, either by catalyzing further addition of carbohydrate or by catalyzing chemical modification of one or more of the existing carbohydrate residues.
  • the hemochromatosis ligand is both recognized and then enzymatically modified by the hemochromatosis receptor.
  • Those hemochromatosis receptors that can both bind and modify carbohydrate have the activity known as a leczyme.
  • Both the mutated and non-mutated forms of the hemochromatosis receptor are structurally related to the class I molecule encoded by the major histocompatibility complex.
  • Class I MHC molecules contain an approximately 45 kDa polymorphic heavy chain or ⁇ chain associated noncovalently with a small nonpolymorphic protein called ⁇ 2 microglobulin ( ⁇ 2 M) .
  • ⁇ 2 M small nonpolymorphic protein
  • the normal form of the hemochromatosis receptor also associates with ⁇ 2 M.
  • the heavy chain is an MHC-encoded gene product located on chromosmome 6 in humans, near or within the A, B or C regions of the human HLA complex and within or near the K or D/L regions of the mouse H-2 complex.
  • other class I-like heavy chains have been mapped to other chromosomes.
  • ⁇ 2 M is encoded by a gene located outside the MHC and on a different chromosome
  • the heavy chain of the class I molecule is encoded by a gene located within the MHC, thereby including a class I molecule within the definition of an. MHC-encoded gene product.
  • the non-mutated form of the hemochromatosis receptor or the hemochromatosis receptor can be expressed on the cell-surface by virtue of having a transmembrane region and cytoplasmic tail, as in the case of the classical transplantation antigens.
  • Leczymes also can be linked to the cell-surface in a manner similar to some class I-like molecules.
  • many of the class I-like molecules such as Qa and Tla molecules are linked to the cell-surface by a phosphatidylinositol (PI) linkage, and the product of the Q10 gene appears to be secreted (Devlin et al., EMBO J. 4:369-374 (1985)).
  • Qa and Tla antigens lack the classical class I cytoplasmic exons including the phosphorylation site in exon seven (Thor et al., J. Immunol.. 151:211-224 (1993)), although the transmembrane domain and the seventh exon is present in Ql and Q2 gene products.
  • the hemochromatosis receptor heavy chain like the MHC class I heavy chain is organized into three external domains ( ⁇ l, ⁇ 2 and ⁇ 3) , each containing about 90 amino acids each, a transmembrane domain of about 30 amino acids and a cytoplasmic anchor segment of about 30 amino acids.
  • ⁇ 2 M is similar in size and in organization to the external ⁇ 3 domain of the heavy chain.
  • X-ray crystallographic analysis of the extracellular portion of MHC class I molecules shows that the c*l and ⁇ 2 domains interact with each other, while all three heavy chain domains interact with ⁇ 2 M.
  • the interacting ⁇ l and ⁇ 2 domains form a platform containing a deep groove or cleft located on the top surface of the molecule.
  • the non-mutated form of the hemochromatosis receptor and the hemochromatosis receptor exhibit limited tissue distribution in comparison with the classical class I MHC molecules. Expression is primarily found in cells of the gastrointestinal tract, kidney and in lymphocytes. This type of limited distribution is similar to the Qa and Tla antigens, the products of the Q and T genes, that are expressed on subpopulations of lymphocytes (for review, see Flaherty et al. Critical Reviews in Immunology. 10:131-175 (1990)).
  • Hemochromatosis receptors that have leczyme activity can exist with a variety of enzymatic activities.
  • a leczyme can have a glycosyl transferase enzymatic activity that results in the catalytic transfer of a glycosyl group (mono or oligosaccharide) from a glycosylnucleotide to an acceptor molecule such as a protein, carbohydrate or lipid.
  • Other activities include sulfation, phoshorylation or carboxylation.
  • not all glycosyl transferases are leczymes.
  • the non-mutated and mutated hemochromatosis receptor can be obtained from cells using classical methods of protein or glycoprotein purification that have been applied to purify the related class I molecule.
  • the gene encoding the hemochromatosis receptor can be cloned and expressed to yield the non- mutated and mutated hemochromatosis receptor heavy chain gene product in a cell, which can then be used for as a source of starting material for purification of the receptor.
  • the genes encoding the non-mutated or mutated hemochromatosis receptor can be cloned by PCR using oligonucleotides are identified to be specific for the receptor.
  • oligonucleotides a fragment located within the hemochromatosis receptor is first synthesized by PCR.
  • oligonucleotides homologous to highly conserved sequences occurring in the class I-like heavy chain of the receptor are synthesized and used for PCR.
  • the conserved sequences near to cysteines involved in disulfide bridging or involved in contact with ⁇ 2 M are places where the oligonuceotide sequences are determined.
  • oligonucleotides common to the sequences shared by class I-like genes including A, B, C, E, F, G, H and MICA include, for example, the following:
  • a template providing source of the hemochromatosis receptor is obtained from a series of YACs (yeast artificial chromosomes) that contain a portion of chromosome 6.
  • YACs yeast artificial chromosomes
  • Such YAC map to the hemochromatosis gene.
  • YACs include 430A1, 8F2, 152G3, 368D7, 225B1, 168E5, 912H11, 950H11, 731E1 and 755G10, all of which are commercially available (Genome Systems Inc. St Louis MO) .
  • PCR is performed using combinations of 5' and 3' oligos. For example, CSE-1 with CSF-2, CSE-2 with CSF-2, CSE-1 with CSF-2 and CSE-2 with CSF-1.
  • the resulting PCR product of about 180 base pairs is cloned into a holding vector and sequenced.
  • the PCR can be performed using as template, a cDNA library from the human gastrointestinal tract or kidney, rather than a YAC.
  • total RNA or mRNA from the human gastrointestinal tract or kidney can be first transcribed into cDNA and then the PCR performed.
  • These alternative methods will also produce a 180 base pair product when performing PCR using the above specified oligos.
  • These methods have been successful in isolating a PCR fragment of the hemochromatosis receptor having the predicted molecular weight (for the above mentioned oligos the predicted fragment length is approximately 180 base pairs) .
  • the 180 base pair fragments are sequenced to identify unique class I-like sequences that are absent from known class I genes.
  • New oligonucleotides corresponding to these unique sequences are used in a PCR reaction as a primer together with a vector primer and the cDNA library template derived from' human gastrointestinal tract or kidney. The resulting product is sequenced to identify unique sequences at the 5' and 3' ends of exon 2, 3 and 4 of the class I-like genes. Oligonucleotides corresponding to these regions are used in a PCR using as a template genomic DNA isolated from patients that are known to be homozygous for hemochromatosis. These fragments are sequenced to identify the mutations that effect binding of the heavy chain of the hemochromatosis receptor to ⁇ 2 M.
  • the gene encoding the normal form of the hemochromatosis receptor is cloned from a cosmid library by screening the library using any of the sequences that are unique to this class I-like heavy chain. The same approach can be used for cloning the hemochromatosis receptor from a genomic library derived from patients that are known to be homozygous for hemochromatosis.
  • the gene sequences from the cosmids are subcloned into an expression vector and then introduced into a host cell to produce the receptors.
  • Vectors are well known in the art and include, for example, cloning vectors and expression vectors, as well as plasmids or viral vectors (see, for example, Goedell, Methods in Enzymolo ⁇ v. vol.
  • a baculovirus vector is an example of a vector that can be used to express a leczyme in insect cells and result in expression of new carbohydrate ligands on the cell.
  • a vector comprising a nucleic acid molecule encoding a leczyme also can contain a promoter or enhancer element, which can be constitutive or inducible and, if desired, can be tissue specific.
  • Host cells also are known in the art and an appropriate host cell can be selected for the particular vector to be used.
  • a baculovirus transfer vector can be used with baculovirus DNA to infect insect cell lines such as SF21 cells. Cloning of such transformed cells to produce a stable cell line can provide a source of the expressed hemochromatosis receptor.
  • the gene encoding the non-mutated or mutated hemochromatosis receptor can be expressed as a fusion protein to assist in purification or in use as an immun ⁇ gen to raise an antibody.
  • the non- mutated or mutated hemochromatosis receptor can be produced as a chimeric protein fused to the CH2 or CH3 domain that constitutes the Fc binding region of an immunoglobulin molecule, as was performed previously for expressing the CD22S lectin (Stamenkovic et al. Cell. 66:1133-1144 (1991)).
  • the use of Protein A from Staphylococcus aureus bound to a solid support which is readily available from commercial sources, can be used to purify the Fc containing chimeric receptor.
  • the present invention provides a method for diagnosing a predisposition for hemochromatosis or for diagnosing hemochromatosis by detecting a mutation in the hemochromatosis receptor.
  • a mutation in the hemochromatosis receptor an individual who does not have an iron storage disease, but is suspected of inheriting a mutation that can predispose the individual to develop an iron storage disease later in life can also benefit from testing for mutations by the methods disclosed herein.
  • the method relies on detecting either the mutation or the effect of the mutation that occurs in the heavy chain of a class I-like MHC molecule of the hemochromatosis receptor.
  • a mutation that is diagnostic for the disease is one that results in a reduced affinity of the heavy chain for human ⁇ 2 M.
  • Mutations in the class I-like heavy chain of the hemochromatosis receptor that affect its ability to bind to ⁇ 2 M result from any mutation that affect the nature of the residues in the heavy chain responsible for directly contacting ⁇ 2 M.
  • Such positions occur in the ⁇ l domain approximately at amino acid positions 6-12, 25-36, in the ⁇ 2 domain at approximately 93-100, 113-121 and in the ⁇ 3 domain at approximately amino acid position 186-192, 200-208, 231-238, 239-245.
  • a mutation that occurs in the cysteine residues involved in formation of disulfide bridges are examples of a mutation in hemochromatosis.
  • Such residues include those cysteines that occur in the ⁇ 2 domain at 5
  • a mutation that is diagnostic for hemochromatosis also can occur in a region of the heavy chain that is near to a phosphorylation site. Such a mutation can reduce the ability of the heavy chain to associate with ⁇ 2 M if the phosphate group added to this site cannot be removed in a cell.
  • Methods to detect a mutation in the class I- like heavy chain of the hemochromatosis receptor can be based either on analysis of the protein or the nucleic acid encoding the protein.
  • the class I-like heavy chain can be purified from a source of cells or body fluids of an individual and the heavy chain can be isolated from ⁇ 2 M. Methods to purify a class I MHC molecule and isolate the heavy chain from ⁇ jM are well known in the art. The isolated heavy chain can then be subjected to amino acid sequencing, peptide mapping, detection by antibody or other such protein analyses to determine if their is a mutation. Such methods for protein determination are well known to those in the art.
  • the present invention provides antibodies that are specifically reactive with the mutated and non- mutated hemochromatosis receptor.
  • antibody is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as polypeptide fragments of antibodies that retain a specific binding affinity for hemochromatosis receptor of at least about 1 X 10 s M' 1 .
  • antibody fragments such as Fab, F(ab') 2 and Fv fragments can retain specific binding activity for their target antigen and, thus, are included within the definition of an antibody to the hemochromatosis receptor.
  • antibody as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies such as domain-deleted antibodies (Morrison and Oi, WO 89/07142, Aug. 10, 1989, which is incorporated herein by reference) or single chain Fv (Ladner and Bird, U.S. Patent NO. 5,250,203, Nov. 9, 1993, which is incorporated herein by reference).
  • non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al. , Science 246:1275-1281 (1989), which is incorporated herein by reference.
  • Non- naturally occurring antibodies within the present invention include bispecific antibodies, in which the antibody contains at least two different binding specificities that can be univalent or multi-valent for each particular binding specificity.
  • Methods for producing bispecific antibodies by chemical crosslinking or by heterohybridoma formation are well known in the art (for trivalent antibodies, see, for example, Ahlem and Huang, U.S. Patent No. 5,273,743, Dec. 28, 1993), which is incorporated herein by reference) .
  • An antibody specific for the non-mutated form of the hemochromatosis receptor or the hemochromatosis receptor can be prepared using the purified heavy chain of the receptor which can be obtained from natural sources or produced by recombinant DNA methods or chemical synthesis.
  • Recombinant DNA methods can be used to express the heavy chain of the receptor alone or as a fusion protein, for example fused to glutathionine-S- transferase, which can facilitate purification of the antigen and enhance its immunogenicity.
  • peptide containing the mutated sequence of the hemochromatosis can be used as an immunogen. If not sufficiently immunogenic, such fragments or peptides can be made immunogenic by expressing the hapten as a fusion protein.
  • Recombinant DNA methods can also be used to express the heavy chain in a cell line that produces ⁇ 2 M.
  • the hemochromatosis receptor can also be made immunogenic by coupling it to a immunogenic carrier molecule such as bovine serum albumin or keyhole limpet hemocyanin (KLH) .
  • a immunogenic carrier molecule such as bovine serum albumin or keyhole limpet hemocyanin (KLH) .
  • KLH keyhole limpet hemocyanin
  • Various other carrier molecules and methods for coupling a non-immunogenic peptide to a carrier molecule are well known in the art (see, for example, Harlow and Lane, Antibodies: A laboratory Manual Cold Spring Harbor Laboratory Press, (1988) , which is incorporated herein by reference) . Methods for raising an antibody are routine and described, for example, by Harlow and Lane (supra, 1988) .
  • An antiserum containing polyclonal antibodies specific for the hemochromatosis receptor can be raised in rabbits, goats or other animals.
  • the resulting antiserum can be processed by purification of an IgG antibody fraction using protein A Sepharose chromatography and, if desired, can be further purified by affinity chromatography using, for example, Sepharose conjugated with a peptide antigen (see Example V) .
  • the ability of polyclonal antibodies to specifically bind to a given molecule can be manipulated,, for example, by dilution or by adsorption to remove cross reacting antibodies to the non-mutated form of ' the hemochromatosis receptor or to an unrelated molecule. Methods to manipulate the specificity of polyclonal antibodies are well known to those in the art (See Harlow and Lane, supra, 1988) .
  • a monoclonal antibody specific for the non- mutated form of the hemochromatosis receptor or the hemochromatosis receptor can be produced using known methods (Harlow and Lane, supra, 1988) .
  • spleen cells from a animal immunized with receptor or fragment containing the mutation can be fused to an appropriate myeloma cell line such as SP2/0 myeloma cells to produce hybridoma cells.
  • Cloned hybridoma cell lines can be screened using a labeled hemochromatosis receptor to identify clones that secrete an appropriate monoclonal antibody.
  • a hybridoma that expresses an antibody having a desirable specificity and affinity can be isolated and utilized as a continuous source of monoclonal antibodies. Methods for identifying an antibody having an appropriate specificity and affinity for the hemochromatosis receptor and, therefore, useful in the invention are known in the 55
  • 16 art and include, for example, enzyme-linked immunoabsorbance assays, radioimmunoassays, precipitin assays and immunohistochemical analyses (see for example, Harlow and Lane, supra, 1988; chap. 14) .
  • an antibody specific for the hemochromatosis receptor can be used to identify the presence of the receptor in a sample from a person suspected of carrying the gene encoding the mutation.
  • the antibody can be labeled with a detectable moiety such as a radioisotope, an enzyme, a fluorochrome or biotin using methods well known in the art (see, for example, Harlow and Lane, supra, (1988); chap. 9).
  • a detectable moiety such as a radioisotope, an enzyme, a fluorochrome or biotin using methods well known in the art (see, for example, Harlow and Lane, supra, (1988); chap. 9).
  • a detectable moiety such as a radioisotope, an enzyme, a fluorochrome or biotin using methods well known in the art (see, for example, Harlow and Lane, supra, (1988); chap. 9).
  • an antibody specific for the non-mutated form of the hemochromatosis receptor can be used to diagnose hemochromatosis by
  • a labeled second antibody also can be used to identify specific binding of an unlabeled antibody to the hemochromatosis receptor.
  • a second antibody generally will be specific for the particular class of the first antibody. For example, if the hemochromatosis receptor antibody is of the IgG class, a second antibody will be an anti-IgG antibody. Such second antibodies are readily available from commercial sources.
  • the second antibody can be labeled using a detectable moiety as described above.
  • a labeled second antibody can be one that reacts with a chemical moiety, for example biotin or a hapten that has been conjugated to the first antibody (see for example, Harlow and Lane, supra (1988) ; chapter 9) .
  • the presence of the hemochromatosis receptor in a sample can be determined by contacting the sample suspected of containing the receptor with the specific antibody under suitable conditions, which allow the antibody to specifically bind to the receptor. Suitable conditions can be achieved using well known methods and can be optimized, for example, by varying the concentration of reactants or the temperature of the reaction. After the antibody specifically binds to the hemochromatosis receptor in the sample, the presence of the receptor can be determined by detecting specific binding of the antibody.
  • detectable label means a molecule whose presence can be detected due to a physical, chemical or biological characteristic of the molecule. Detectable labels include, for example, radioisotopes, fluorescent molecules, enzyme/substrate systems, or visually detectable molecules. Methods to produce a probe for detecting a protein are well known in the art (see, for example, Harlow and Lane, supra, (1988)) and include, for example labeling the agent with a radioisotope, fluorescence molecule or histochemically useful enzyme or visible particle or colloid.
  • An antibody often can bind to a limited but detectable level to non-target substances such as the assay container or to a similar epitope in a different molecule and can result in background binding.
  • a sample represents the presence of the hemochromatosis receptor
  • the level of background binding of an antibody can be determined using a control sample, which is similar in composition to the sample being tested but which does not contain the hemochromatosis receptor or contains defined amounts of the non-mutated form of the hemochromatosis receptor.
  • An antibody useful for detecting the hemochromatosis receptor does not require that it directly detect the mutated sequence. It is useful provided it can detect any portion of the molecule that is effected by the mutation.
  • Another antibody useful for detecting the presence of the hemochromatosis receptor is one that is specific for a heavy chain epitope that is exposed when the heavy chain is not in association with ⁇ 2 M. Such an antibody is useful for detecting the mutated receptor heavy chain but not the normal receptor since the latter properly associates with ⁇ 2 M.
  • a mutation in a nucleic acid sequence can be detected by various methods to analyze nucleic acids such as by nucleic acid sequencing, polymerase chain reaction or hybridization. Such methods are well known to those in the art (see, for example, Sambrook et al, supra, 1989; Hames and Higgins Nucleic Acid Hybridization: a practical approach (IRL Press, New York, 1985) , both of which are incorporated herein by reference) .
  • Methods to detect decreased binding of a mutated heavy chain with ⁇ 2 M can be used for diagnosing an iron storage disease such as hemochromatosis.
  • the heavy chain of an class I MHC molecule is isolated from an individual and contacted with ⁇ 2 M under conditions suitable for a non-mutated such heavy chain to associate with ⁇ 2 M.
  • a control reaction which contains a non-mutant form of the same or similar class I heavy chain to the one being tested for a mutation is performed in parallel.
  • the reaction is incubated under suitable conditions, including, for example, an appropriate buffer concentration and pH and time and temperature, which is sufficient for the control heavy chain to associate with ⁇ 2 M.
  • the heavy chain being tested from the individual is considered to have a mutation diagnostic for an iron storage disease when the fraction of this heavy chain that associates with ⁇ jM is significantly less than the fraction of control heavy chain that associates with ⁇ 2 M.
  • the association of a class I heavy chain with ⁇ 2 M can be detected, for example, by attaching one of the molecules to a solid support and attaching a detectable label such as a radionucleotide or a fluorescent label to the other molecule and measuring the amount of detectable label that is associated with the solid support, wherein the amount of label detected indicates the amount of association of the heavy chain with . ⁇ 2 M.
  • An method to diagnose hemochromatosis can involve use of a specific antibody to the receptor and the hemochromatosis ligand.
  • the ligand can be used to capture the receptor or can be used to detect the receptor that is first bound by the specific antibody.
  • the disclosed methods for detecting a mutation in the hemochromatosis receptor that is involved in iron uptake is generally applicable to the detection of diseases associated with iron metabolism.
  • one form of anemia involves mutations in the normal form of the hemochromatosis receptor or in its associated ⁇ 2 M.
  • the mutation in the receptor is different from that in hemochromatosis.
  • anemia can result from a mutation that occurs in the promotor region such as the GATA box or the DREs that regulate expression of the normal form of the hemochromatosis receptor (see Rothenberg and Voland,
  • the present invention provides a method to purify the hemochromatosis ligand.
  • a substantially purified source of the hemochromatosis ligand is useful as pharmaceutical to modulate iron absorption in the body.
  • the purified hemochromatosis ligand can be used to isolate other receptors that bind to the ligand and are involved in ion metabolism.
  • the purified hemochromatosis ligand can be used as a probe to detect the presence of the hemochromatosis receptor in a sample as an approach to diagnose hemochromatosis.
  • substantially purified means the hemochromatosis ligand is relatively free from other contaminating molecules such as lipids, proteins, nucleic acids, carbohydrates or other molecules which are not normally associated with the hemochromatosis ligand in a cell.
  • a sample containing a source of the hemochromatosis ligand suitable for purification can be obtained, for example, from the cells or the secretion of the cells of the human or other mammalian gastrointestinal tract.
  • Such cells may be obtained from a sample of tissue or may be obtained as cultured gastrointestinal cells obtained from cell repositories (see the American Type Culture Collection, Rockville MD)
  • the hemochromatosis ligand can be extracted from the cells or tissues by standard procedures such as by mechanical disruption with non-ionic detergents.
  • the sample containing the hemochromatosis ligand can be initially isolated from most of the other molecules in the sample by virtue of the ligand's ability to bind to iron.
  • the sample is contacted with an iron chelate affinity matrix at suitable conditions such as low pH in a loading buffer that supports binding of the iron to the hemochromatosis ligand.
  • suitable conditions include, for example, an appropriate buffer concentration and pH and time and temperature that permits binding of the hemochromatosis ligand to the metal.
  • Iron chelate affinity matrixes' are available from commercial sources (see Pharmacia, Sweden) .
  • the matrix After binding the hemochromatosis ligand to the matrix, the matrix is washed with the loading buffer and the bound ligand eluted by competition with excess iron or other divalent cations. Excess iron can be removed from the eluted hemochromatosis ligand by dialysis or size or charge chromatography.
  • An alternative method for eluting the the hemochromatosis ligand from the support that avoids excess iron involves the chelating agent such as ethylenediametetracetic acid.
  • Another approach for isolating the hemochromatosis ligand by binding to iron involves first contacting the sample containing the ligand with iron at low pH in solution. The iron binds to the hemochromatosis ligand to form a hemochromatosis ligand- iron complex. The complex is then separated from the remainder of the material by attracting the complex to a magnetic source. The hemochromatosis ligand is then obtained by removal of the magnetic source. The hemochromatosis ligand isolated after binding to iron can be further purified by affinity chromatography over a second column containing the non- mutated form of the hemochromatosis receptor or the hemochromatosis receptor itself.
  • the solid support is prepared by attaching the receptor to a poly (vinyalchohol) or poly (acrylate/acrylamide) .
  • the hemochromatosis ligand is contacted ..with the receptor support under suitable conditions that allow for binding of the ligand to the receptor.
  • the support is then washed to remove unbound material and the hemochromatosis ligand is obtained by adding an excess of a monosaccharide such as mannose, fucose or sialic acid.
  • the hemochromatosis ligand can be dissociated from the receptor using well known treatments such as extremes in pH, high salt concentration or chaotropic agents (see, for example, Harlow and Lane, supra, 1988) , which is incorporated herein by reference and Varki, supra, 1994) .
  • the substantially purified hemochromatosis ligand obtained by the disclosed methods is suitable for structural analysis as described above, in order to enable future production of the ligand by chemical or enzymatic synthesis.
  • An alternative method for isolating a substantially purified hemochromatosis ligand involves first isolating the receptor using the a solid support containing the non-mutated form of the hemochromatosis receptor and then further purification by binding to iron as described above.
  • substantially purified hemochromatosis ligand may be obtained using only one of the two disclosed affinity chromatography approaches disclosed herein.
  • the hemochromatosis ligand isolated by the disclosed affinity chromatography approaches can be subjected to further purification using general methods well known in the art for purification of glycoconjugates(see, for example, Ausabal et al, In Current Protocols in Molecular Biology Vol. 2, chapter 17, (Green Publishing Associates and Wiley Interscience, New York, 1994) ; Fukuda and Kobata, Glycobiolo ⁇ y: A practical Approach. (IRL Press, New York, 1993), both of which are incorporated herein by reference) .
  • these methods are useful for structural characterization, including sequencing of the hemochromatosis ligand. Elucidation of the structure of the hemochromatosis ligand purified from a tissue or a cell can enable future production of the ligand by direct chemical synthesis or enzymatic synthesis or purification from a natural source.
  • hemochromatosis can be purified by binding to iron provides a general approach to isolating any metal binding ligand that is bound by a leczyme.

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Abstract

La présente invention concerne un procédé de diagnostic de l'hémochromatose ou de détection d'une prédisposition à l'hémochromatose, consistant à détecter la présence d'une mutation dans la chaîne lourde de type classe I d'un récepteur d'hémochromatose. Ladite méthode comprend la détection du récepteur d'hémochromatose au moyen d'anticorps réagissant à des déterminants antigéniques propres au récepteur. La présente invention concerne également la purification du ligand de l'hémochromatose, qui se fixe au récepteur d'hémochromatise ainsi qu'au fer. Le processus de purification comprend en outre l'isolation du ligand par fixation de la forme mutée ou non mutée du récepteur d'hémochromatose contenu dans un support solide.
PCT/US1997/010276 1996-06-14 1997-06-16 Procedes de detection d'un recepteur mutant d'hemochromatose aux fins de diagnostic WO1997047655A1 (fr)

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Application Number Priority Date Filing Date Title
AU35703/97A AU3570397A (en) 1996-06-14 1997-06-16 Methods for the detection of a mutant receptor diagnostic for hemochromatosis

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US66519496A 1996-06-14 1996-06-14
US08/665,194 1996-06-14

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WO1997047655A1 true WO1997047655A1 (fr) 1997-12-18
WO1997047655A9 WO1997047655A9 (fr) 1998-05-28

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4434156A (en) * 1981-10-26 1984-02-28 The Salk Institute For Biological Studies Monoclonal antibodies specific for the human transferrin receptor glycoprotein
US5385888A (en) * 1987-03-20 1995-01-31 The Regents Of The University Of California Class I MHC modulation or surface receptor activity
WO1996017870A1 (fr) * 1994-12-06 1996-06-13 Rothenberg Barry E Identification, purification et utilisation de leczymes et de ligands contenant des hydrates de carbone

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4434156A (en) * 1981-10-26 1984-02-28 The Salk Institute For Biological Studies Monoclonal antibodies specific for the human transferrin receptor glycoprotein
US5385888A (en) * 1987-03-20 1995-01-31 The Regents Of The University Of California Class I MHC modulation or surface receptor activity
WO1996017870A1 (fr) * 1994-12-06 1996-06-13 Rothenberg Barry E Identification, purification et utilisation de leczymes et de ligands contenant des hydrates de carbone

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
EMBO JOURNAL, June 1989, Vol. 8, No. 6, WATTS et al., "Organization and Structure of the Qa Genes of the Major Histocompatibility Complex of the C3H Mouse: Implications for Qa Function and Class I Evolution", pages 1749-1759. *
HUM. GENET., 1994, Vol. 94, ZHENG et al., "Exclusion of Ferritins and Iron-Responsive Element (IRE)-Binding Proteins as Candidates for the Hemochromatosis Gene", pages 159-164. *
IMMUNOGENETICS, 1993, Vol. 38, WANG et al., "Expression of the Q2 Gene of the MHC in Thymus and Intestinal Epithelial Cells", pages 370-372. *
J. EXP. MED., June 1993, Vol. 177, CULLEN et al., "Identification of a Recombinogenic Major Histocompatibility Complex Q Gene with Diverse Alleles", pages 1803-1807. *

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