WO1994028161A1 - Anticorps specifiques du produit genique dcc - Google Patents

Anticorps specifiques du produit genique dcc Download PDF

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Publication number
WO1994028161A1
WO1994028161A1 PCT/US1994/005277 US9405277W WO9428161A1 WO 1994028161 A1 WO1994028161 A1 WO 1994028161A1 US 9405277 W US9405277 W US 9405277W WO 9428161 A1 WO9428161 A1 WO 9428161A1
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dcc
ser
antibody
pro
leu
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PCT/US1994/005277
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English (en)
Inventor
Arthur Bruskin
David E. Jarosz
Karen Johnson
Kenneth W. Kinzler
Bert Vogelstein
James R. Zabrecky
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The Johns Hopkins University
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Priority to JP7500697A priority Critical patent/JPH09501045A/ja
Priority to EP94919126A priority patent/EP0700446A1/fr
Publication of WO1994028161A1 publication Critical patent/WO1994028161A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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

Definitions

  • This invention relates to the area of cancer diagnostics.
  • it relates to immunological methods and tools for determining expression of the tumor suppressor gene DCC.
  • Colorectal tumorigenesis provides an excellent model system to test this multi-step hypothesis at the molecular level (Vogelstein, et al., New Eng. J. Med. 319:525 (1988)). Colorectal tumors progress through a continuum of histopathologically identifiable states, proceeding from normal epithelium, through benign tumors called adenomas, to the malignant stage known as carcinomas (Sugarbaker, Colorectal cancer: Principles and practices of Oncology (ed. V.T. Devita, S. Hellman, and S.A.
  • the tumor suppressor gene DCC (deleted in colorectal carcinoma), located on the long arm of chromosome 18, encodes a cell surface protein.
  • pl5-65 was used as a starting point for a bidirectional chromosomal walk which eventually encompassed 370 kb of contiguous genomic DNA.
  • the walk generated 117 Eco RI fragments which were used as probes in cross-species Southern blot analysis. Twenty-four of the fragments hybridized to at least one of the species tested. Rat and human homologues from seven of the evolutionarily conserved fragments were cloned and sequenced. The sequences were examined for the presence of open reading frames as well as for 5' and 3' splice donor and acceptor sites, which would indicate possible exons.
  • a sensitive expression assay for mRNA was developed using the polymerase chain reaction to amplify cDNA.
  • DCC for deleted in colorectal carcinoma
  • the cDNA was cloned and the nucleic acid sequence of DCC was found to predict a 1447 amino acid transmembrane protein with a unique cytoplasmic domain and an extracellular portion with significant homology to neural cell adhesion molecules and other cell surface glycoproteins ( Figure 1).
  • Somatic mutations of putative tumor suppressor genes in human tumors have been used as evidence for their importance in the development of the tumors analyzed (Baker, et al., Science 244:211 (1989)).
  • the DCC gene has been shown to be somatically altered in colorectal carcinomas by a variety of mechanisms, including deletions, insertions and point mutations (Fearon, et al., Science 247:49 (1990)).
  • the search for additional mutations is ongoing. There is a need in the art for additional tools and methods for assessing somatic alterations of the DCC gene.
  • an antibody preparation which consists essentially of antibodies which: (1) are specifically immunoreactive with an epitope on the extracellular domain or on the cytoplasmic domain of DCC protein and (2) do not cross-react with neural cell adhesion molecules.
  • a method for detecting mutations in a human DCC gene comprises the steps of: extracting proteins from a sample selected from the group consisting of a tissue and a body fluid; separating said extracted proteins on a polyacrylamide gel; blotting said separated proteins onto a filter; contacting said filter with antibodies which: (1) are specifically immunoreactive with DCC protein and (2) do not cross-react with neural cell adhesion molecules, to bind said antibodies to proteins blotted on said filter; detecting the antibodies which bind to said proteins, wherein the absence of binding of antibodies to a protein having the size of DCC indicates the presence of a DCC mutation in the sample.
  • a method for determining the presence in a human of DCC mutations comprises the steps of: contacting a body sample of a human with a first antibody which: (1) is specifically immunoreactive with an epitope of DCC and (2) does not cross-react with neural cell adhesion molecules to bind components of said body sample to said antibody; determining the amount of antibody which is bound to said body sample, wherein an absence of binding of said antibody to said body sample indicates the presence of a DCC mutation in the human.
  • a solid support for use in performing enzyme-linked immunosorbent assays is provided.
  • the solid supports are coated with an antibody: (1) which is specifically immunoreactive with an epitope contained within the extracellular domain of DCC and (2) does not cross-react with neural cell adhesion molecules.
  • hybridoma cell secretes an antibody: (1) which is specifically immunoreactive with an epitope contained within the extracellular or the intracellular domain of DCC and (2) does not cross-react with neural cell adhesion molecules.
  • the present invention thus provides the art with immunological methods and reagents for assessing the mutational status of DCC in human tissues.
  • DCC immunologically specific for DCC protein.
  • DCC was known to have substantial homology to NCAMs (neural cell adhesion molecules); nonetheless antibodies have been found which react with DCC but not with NCAMs.
  • NCAMs neural cell adhesion molecules
  • Such antibodies can be used diagnostically. It has been determined that DCC is expressed in one type of differentiated cells in normal colonic epithelium, the goblet cells. However, DCC expression is lost during the progression of a subset of colorectal tumors. (The non-mucinous carcinomas and late stage adenomas lose DCC expression.) The ability of antibodies to detect DCC expression in normal colonic cells is unexpected, since no DCC specific mRNA could be detected in such cells by Northern blotting.
  • the antibodies of the present invention can be used to test colonic samples for expression of DCC. Lack of DCC expression is associated with a poorer prognosis, as well as being indicative of the stage of the colorectal tumor.
  • the 5 '-end of the open reading frame (ORF) of DCC has an initial hydrophobic sequence of 25 amino acids, suggestive of a signal sequence found in membrane-bound proteins, followed by 725 amino acids with significant homology to neural cell adhesion molecules (NCAMs) and other related cell surface glycoproteins.
  • the DCC protein also contains an 1100 amino acid extracellular domain of four immunoglobulin-like C2 domains and six fibronectin type III domains, and an intracellular (cytoplasmic) domain of 324 amino acids.
  • the cytoplasmic domain has no significant homology to any sequence in the
  • Polyclonal antibodies have been generated against bacterial fusion proteins containing various parts of the DCC cDNA sequence. Iodination of cell surface proteins by the lactoperoxidase method, followed by immunoprecipitation with anti-DCC antibodies has documented cell surface labeling of DCC. Immunocytochemistry of transfected cell lines expressing high levels of DCC demonstrates a diffuse membrane staining pattern with areas of cell-cell contact showing intense staining. This suggests that higher protein concentrations exist locally at these cellular contact sites.
  • antibodies are provided. Two types of antibodies have been prepared which are useful diagnostically. One type is specifically immunoreactive with an epitope on the extracellular domain of DCC protein, i.e., amino acids 26-1126 (SEQ ID NO:2), but does not cross-react with neural cell adhesion molecules. The other type is specifically immunoreactive with an epitope contained within the intracellular domain of DCC, i.e., amino acids 1123-1447 (SEQ ID NO:2) and also does not cross-react with neural cell adhesion molecules.
  • One portion of the DCC gene which encodes part of the extracellular domain consists of base pairs 1195-2854 (SEQ ID NO:1). Another portion comprises base pairs 1-3189 (SEQ ID NO:1).
  • Chimeric genes can be constructed which contain these or other portions of DCC.
  • the fusion proteins can be expressed and purified according to known means.
  • the fusion proteins can be used to immunize animals to raise either polyclonal or monoclonal antibodies.
  • Two monoclonal antibodies according to the present invention are made by the hybridoma cell lines AF5 and AF1, which have been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 (ATCC) as HB 11289 and 11299, respectively.
  • Other cell lines may be isolated which secrete antibodies which have the same epitopic specificity as these cell lines. These may be antibodies which are isotype switch variants, anti-anti-idiotypic antibodies, or independently derived antibodies.
  • a number of different immunological assays can be performed.
  • One such assay is a Western Blot in which proteins are extracted from a test sample.
  • the test sample may be either a tissue, cancerous or normal, or a body fluid, such as serum, blood, urine, sputum, saliva, feces etc. Any antibody which is specifically immunoreactive with DCC protein can be used. If a protein having the size of DCC is not present on the Western blot, then a DCC mutation is indicated in the human sample donor. Although DCC has a predicted molecular weight of 153 kd, a variety of larger bands have been observed, such as a 180 kd and a 190 kd protein. This variation in size is attributed to N-glycosylation.
  • a body sample is tested for immunoreactivity with an antibody which is specifically immunoreactive with an epitope of DCC and which does not cross-react with neural cell adhesion molecules.
  • the body sample can comprise a fixed cell sample, such as peripheral blood mononuclear cells, a fixed or frozen tissue section, an extract of cells or tissues, a body fluid, such as serum, blood, urine, sputum, saliva, feces etc.
  • the body sample and the antibody are contacted under conditions where antibodyantigen complexes form and are stable. Such conditions are known in the art.
  • an antibody which binds to an epitope which is contained within the extracellular domain of DCC it may be desirable to use an antibody which binds to an epitope which is contained within the extracellular domain of DCC.
  • an antibody which binds to an epitope which is contained within the intracellular domain of DCC it appears that the latter type of antibody is preferred for immunohistochemistry on tissue sections.
  • adjacent, normal tissue to the tissue sample to be tested be collected and tested. Thus the absence of binding in the test tissue sample can be confirmed as representing a DCC mutation, if the adjacent normal tissue can be shown to bind to anti-DCC antibodies.
  • either the antibody or the body sample may be attached to the solid support on which the assay is performed.
  • solid supports which are typically microtiter dishes or dipsticks, are coated with an antibody which is specifically immunoreactive with DCC.
  • the solid support may be any material which is typically used in immunological assays, including plastics, glass, or other polymeric substances.
  • the form of the support may be sheets, wells, tubes, beads, fabrics, etc.
  • This example describes the preparation of polyclonal anti-DCC antibodies.
  • Two rabbit polyclonal antibodies were raised against bacterial fusion proteins containing non-overlapping epitopes of DCC, using selected portions of the DCC cDNA.
  • One antibody was raised to a 1.65 kb portion of the extracellular domain (nucleotides 591-1239, SEQ ID NO:1) and the other was raised to the entire cytoplasmic domain (nucleotides 3309-4453, SEQ ID NO: 1).
  • the bacterial fusion proteins were constructed in pATH vectors (Kinzler, et al., Molecular and Cellular Biology, 10: 649-642, 1990) with the non-overlapping portions of the DCC cDNA.
  • the fusion proteins were isolated from SDS-polyacrylamide gels and injected subcutaneously in rabbits.
  • the antisera were affinity purified using bacterial fusion proteins from constructs in pGEMEX expression vectors.
  • This example describes the screening of cell lines and tissues for those which express detectable levels of the DCC gene product and also describes the determination of its cellular localization.
  • Tissue culture cell lines were metabolically labelled with 35 S-methionine for four hours at 37°C.
  • the cells were lysed in RIPA buffer (10 mM Tris(pH 7.5) and 0.5% SDS) under denaturing conditions. Approximately 200 ⁇ 10 6 counts were immunoprecipitated with affinity purified anti-DCC antibodies and Protein A sepharose beads. The samples were analyzed by SDS-PAGE.
  • Frozen tissue was cryostat sectioned at 7 ⁇ m thickness for immunohistochemistry. The sections were fixed for 30 minutes at room temperature in 10% buffered formalin. Endogenous peroxidase activity was blocked at incubation in 0.3% H 2 O 2 and signal intensity was increased using the antigen retrieval system from Biogenex. The sections were blocked with goat serum followed by incubation with 3 ng/ ⁇ l of anti-DCC antibody. A secondary goat anti-rabbit biotinylated antibody, in conjunction with the Vectastain Elite reagent from Vector Laboratories, was used for detection.
  • DCC expression was detected in only one cell line, 577MF, a non-seminomatous testicular germ cell tumor. Both antibodies precipitated a protein of approximately 180 kd from metabolically labelled 577MF cells, providing strong evidence that it represents the DCC gene product.
  • the sequence of the full length DCC cDNA predicts an unmodified transmembrane molecule of approximately 153 kd. The sequence also suggests that DCC could be N-glycosylated (Fearon, et al., Science 247:49 (1990)), possibly explaining the larger apparent molecular weight observed in 577MF. All of the colorectal carcinoma cell lines tested by immunoprecipitation were negative, correlating with previous RNA expression assays in which DCC RNA was not detectable by RNase protection, but was detected by reverse-transcription polymerase chain reaction.
  • Tissue distribution of DCC expression was examined by Western blot analysis.
  • Expression studies on normal tissues by reverse transcription polymerase chain reaction revealed that the highest level of expression was in brain.
  • Western blot analysis a faint, but detectable, band was present at approximately 180 kd in human brain tissue lysates; however, DCC was not detected in any other tissues.
  • DCC expression indicates that expression of DCC in cell lines and in tissues is extremely low. However, the previous analyses were performed with total cellular mRNA or total cellular protein. If DCC expression were locally expressed at high concentrations at either intra- or inter-cellular locations it might be more readily detected by in situ methods. We tested this hypothesis on tissue from the central nervous system, based on the earlier mRNA expression data. Staining of frozen human spinal cord sections demonstrated strong staining in axons at several levels of the spinal cord. DCC expression appeared to be present in both myelinated and non-myelinated nerve cells. Subsequent examination of brain revealed a similar specificity, with pronounced staining limited to nerve tracks predominantly in the white matter.
  • DCC Puri ⁇ nje cells in the cerebellum.
  • the expression of DCC in Purkinje cells was verified by in situ hybridization using DCC antisense RNA probes. This technique was then applied to tissues. Although faint staining of occasional cells was observed in several tissues (including bladder and endometrium) the most pronounced staining was found in colon and skin. In the skin, only the more differentiated keritinocytes stained.
  • colonic epithelial cells In the colon, immunohistochemical staining localized detectable DCC expression to a small subset of colonic epithelial cells, specifically the goblet cell population.
  • Normal colonic mucosa is composed of epithelial gland like structures called crypts, surrounded by the supporting lamina basement which contains fibroblasts and inflammatory cells.
  • the colonic crypts contain two major differentiated cell types: absorptive cells, called enterocytes, responsible for fluid and electrolyte transport; and secretory cells, known as goblet cells, which produce and secrete mucin.
  • the goblet cells are the most conspicuous cell type because of their large apical intracellular mucin accumulation. However, they only account for approximately 20-30% of the conic crypt epithelium (Hafez, Cell Growth and Differentiation 1:611 (1990)).
  • the expression pattern of DCC within the goblet cells is specific.
  • the signal is localized to the basolateral aspect of the cell and in the supranuclear cytoplasmic space, just below the apical mucin (data not shown). It was not possible to determine with certainty whether DCC was localized to the cell membrane in these sections, but the expression pattern is consistent with that possibility.
  • the supranuclear staining may represent nascent protein in the golgi apparatus where nascent protein is known to be localized (Bloom and Fawcett, A Textbook of Histology, pg. 662, W.B. Saunders, Philadelphia (1975)).
  • Pre-immune serum did not stain any of the reactive cells described above.
  • the analyses were performed in the presence of soluble fusion proteins as competitors. DCC fusion protein at the same concentration as the DCC antibody completely blocked the signal detected by the antibody in the goblet cells, whereas an irrelevant fusion protein, even at five-fold the antibody concentration, failed to reduce the signal.
  • This example demonstrates the status of expression of the DCC gene product in tissue sections from colorectal tumors at different stages during its progression. Immunohistochemistry was then used to investigate DCC protein expression in various tumors of the colorectum.
  • hyperplastic polyps These tumors, as their name implies, consist of hyperproliferating cells that are not neoplastic. They retain a relatively normal architectural pattern and features indicative of cellular differentiation. Although epidemiological studies have suggested an association of hyperplastic polyps with colorectal cancer, there is little evidence that they are precursors to cancer. Relatively intense staining was observed in these polyps with the anti-DCC antibody in all of the epithelial cells within these polyps.
  • the staining within each cell had a different pattern than that observed in the goblet cells of the normal colonic epithelium.
  • the staining was not confined to the basolateral regions but appeared to be dispersed over the entire cell.
  • Normal epithelial cells adjacent to the polyps showed the expected distribution of DCC staining confined to the goblet cells.
  • DCC expression was increased rather than decreased in these non-neoplastic but proliferative and differentiating cells.
  • adenomatous polyps generally considered to be the direct precursors of colorectal cancers.
  • Early and intermediate stage adenomas retained expression of DCC in a largely normal pattern, limited to goblet cells.
  • Two late adenomas with significant dysplasia revealed undetectable levels of DCC protein, with complete absence of staining in the neoplastic epithelium.
  • Six invasive colorectal carcinomas were next examined. Four of the carcinomas completely lacked detectable DCC staining. However, two of the carcinomas stained intensely for DCC.
  • This example demonstrates the production of monoclonal antibodies which are specifically immunoreactive with the extracellular portion of DCC.
  • An expression construct was generated by first introducing a translation termination codon into a full length DCC cDNA contained in a plasmid vector for in vivo recombination into vaccinia virus, such that the open reading frame was interrupted at codon 1063.
  • a recombinant virus vAbT 492 which expresses amino acids 1-1063 (the entire extracellular domain except for the C-terminal 34 amino acids) of DCC with the addition of a single serine residue at the carboxy terminus. Lacking its membrane anchor but still possessing the signal sequence, the truncated DCC gene product is secreted.
  • a 4.5 kb Xho I - Eco RI fragment containing the entire DCC coding region, was cloned into a HindIII M insertion vector for in vivo recombination into vaccinia virus.
  • a universal translational termination oligonucleotide (Pharmacia) was inserted into the Bal I site at nucleotide 3211. Insertion into the correct Bal I site was confirmed by restriction analysis with Ase I.
  • the resulting plasmid encodes the sequence of amino acids 1-1063 of DCC whose expression is regulated by the vaccinia virus 40k promoter.
  • Vero cells were infected with vAbT 492 at a MOI of 10 in serum-free media. After 24 hr, the media was collected, clarified by centrifugation and analyzed by SDS-PAGE. Vero cells infected with NYCBH (Lyons et al., Infect, and Immun. 58:4089-4098 (1990); ATCC VR-325) served as the control. Comparison of the conditioned media revealed the presence of an additional protein of apparent molecular mass of 150 kDa in the media from vAbT 492 infected cells.
  • Lectin affinity chromatography was attempted as a first step in purification of gp150 DCC because the structurally related glycoprotein, NCAM, has been shown to bind to lectins. Con A and wheat germ lectins were tested for their ability to bind gp150 DCC . Although gp150 DCC bound to both affinity matrices, binding was more efficient and specific to Con A.
  • gp150 DCC from 100-fold-concentrated, conditioned media from vAbT 492 infected Vero cells was efficiently bound to and eluted from a Con A agarose column. Binding of gp150 DCC appeared to be complete after the first pass through the column. Elution with 0.5 M methyl-D-mannopyranoside resulted in substantial enrichment of gp150 DCC with only a few contaminating proteins of lower apparent molecular weight. Since gp150 DCC eluted slowly over several fractions the elution was followed with a wash of one column volume of 8 M urea to determine if any gp150 DCC remained bound to the column.
  • gp150 DCC In order to separate gp150 DCC from the major contaminants of lower apparent molecular weight, individual fractions from the Con A column were further purified by HPLC size exclusion chromatography. SDS-PAGE demonstrated that the peak between 15 and 17 min represented purified gp150 DCC and that the lower molecular weight contaminants were well resolved. The gp150 DCC preparation was substantially purified and appeared to contain only a single minor contaminating protein.
  • the purified protein was subjected to N-terminal sequence analysis to verify that it was the extracellular domain of the DCC gene product.
  • the sequence generated from the first 15 cycles exactly match the sequence predicted from the cDNA starting at Phe-32. This specifies the N-terminus of the processed DCC gene product in Vero cells and confirms the presence of a signal sequence of 31 amino acids. The result is in agreement with the predicted N-terminus based on hydrophobicity analysis and genomic structure.
  • mice were immunized with the purified, secreted DCC protein fragment and their spleen cells fused with myeloma cells, according to known techniques.
  • the initial hybridomas were screened by ELISA on the Con A-purified DCC. All positives were transferred to 48-well plates, re-grown, and re-screened by ELISA on Con A purified DCC that had been further purified by HPLC size exclusion chromatography. All positives were single cell cloned (primary cloning) and single clones were screened on HPLC-purified DCC. Positive primary clones went through a secondary cloning and screening. Three such clones were AF5,
  • Monoclonal antibodies were characterized by western blot and immunoprecipitation on lysates of SW 480 cells transfected with full length or extracellular DCC, CHO Kl cells transfected with extracellular DCC, and BSC-40 cells infected with recombinant vaccinia virus expressing full length DCC.
  • 577 MF is a human cell line which expresses a 190 kD, endogenous form of DCC. A lysate of these cells was immunoprecipitated with 10 ⁇ g of each purified antibody. The precipitations were then separated by SDS-PAGE and transferred to nitrocellulose. The blot was probed with 10 ug/ml of purified antibody. In this format, all 3 monoclonal antibodies are able to precipitate the 190 kD DCC produced by 577 MF.
  • AGT GGA ATG TAT ACC TGT GTT GTC ACA TAT AAA
  • AAT GAG AAT ATT AGT 960 Ser Gly Met Tyr Thr Cys Val Val Thr Tyr Lys Asn Glu Asn Ile Ser
  • AGT ACT TGG AGC ATG ACT GCA CAT GCC ACC ACG TAT GAA GCA GCC CCC 2832 Ser Thr Trp Ser Met Thr Ala His Ala Thr Thr Tyr Glu Ala Ala Pro
  • AGC ACC CTA AAT GAG CCG CCA ATT GGA CAA ATG CAC CCC CCG CAT GGC 3264 Ser Thr Leu Asn Glu Pro Pro Ile Gly Gln Met His Pro Pro His Gly
  • AGT GCT GGC AAA AGG AAG GGC AGC CAG AAG GAC CTC CGA CCC CCT GAT 3456 Ser Ala Gly Lys Arg Lys Gly Ser Gln Lys Asp Leu Arg Pro Pro Asp
  • AAA AGC ACC TCT CAT TCA GGT CAA GAC ACT GAG GAA GCA GGG AGC TCT 3648 Lye Ser Thr Ser Hie Ser Gly Gln Asp Thr Glu Glu Ala Gly Ser Ser

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Abstract

L'invention concerne des anticorps capables de détecter la protéine DCC dans des échantillons biologiques en dépit du niveau extrêment bas d'expression de DCC. Des procédés immunologiques de détection des modifications de DCC sont également décrits. Ces réactifs et ces procédés permettent de détecter les mutations de DCC sans avoir besoin d'analyser ce gène très vaste dans des échantillons individuels.
PCT/US1994/005277 1993-05-26 1994-05-18 Anticorps specifiques du produit genique dcc WO1994028161A1 (fr)

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JP7500697A JPH09501045A (ja) 1993-05-26 1994-05-18 Ｄｃｃ遺伝子産物特異的抗体
EP94919126A EP0700446A1 (fr) 1993-05-26 1994-05-18 Anticorps specifiques du produit genique dcc

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009964A1 (fr) * 1990-01-04 1991-07-11 The Johns Hopkins University Suppression d'un gene dans le cancer colo-rectal des humains
WO1992016656A1 (fr) * 1991-03-13 1992-10-01 The Johns Hopkins University Gene a mutation dans le cancer colo-rectal chez les humains
EP0577028A2 (fr) * 1992-06-25 1994-01-05 Kyowa Hakko Kogyo Co., Ltd. Des anticorps monoclonaux spécifiques pour le produit du gène DCC

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009964A1 (fr) * 1990-01-04 1991-07-11 The Johns Hopkins University Suppression d'un gene dans le cancer colo-rectal des humains
WO1992016656A1 (fr) * 1991-03-13 1992-10-01 The Johns Hopkins University Gene a mutation dans le cancer colo-rectal chez les humains
EP0577028A2 (fr) * 1992-06-25 1994-01-05 Kyowa Hakko Kogyo Co., Ltd. Des anticorps monoclonaux spécifiques pour le produit du gène DCC

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FEARON ET AL: "IDENTIFICATION OF A CHROMOSOME 18Q GENE THAT IS ALTERED IN COLORECTAL CANCERS", SCIENCE, vol. 247, 1990, WASHINGTON D.C.,USA, pages 49 - 56 *

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