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WO1993010813A1 - Sequences de nucleotides et d'acides amines de l'antigene de pemphigus vulgaris et procede d'utilisation - Google Patents

Sequences de nucleotides et d'acides amines de l'antigene de pemphigus vulgaris et procede d'utilisation Download PDF

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Publication number
WO1993010813A1
WO1993010813A1 PCT/US1992/009933 US9209933W WO9310813A1 WO 1993010813 A1 WO1993010813 A1 WO 1993010813A1 US 9209933 W US9209933 W US 9209933W WO 9310813 A1 WO9310813 A1 WO 9310813A1
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Prior art keywords
pemphigus vulgaris
pva
cell
antigen
dna fragment
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PCT/US1992/009933
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English (en)
Inventor
John R. Stanley
Masayuki Amagai
Vera Klaus-Hovtun
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THE UNITED STAES OF AMERICA, represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES
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Publication of WO1993010813A1 publication Critical patent/WO1993010813A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the DNA sequence and clones can be used for diagrostic purposes.
  • pemphigus vulgaris antigen proteins have been made from the cDNA and these proteins have been used to raise antibodies. These proteins can also be used in ELISA assays for detection of
  • It is another object of the present invention to provide a method of detecting the presence of pemphigus vulgaris antigen in a sample comprising the steps of contacting the sample with the above-described antibody, and detecting the presence or absence of a complex formed between the pemphigus vulgaris antigen and the antibody.
  • A PV IgG affinity-purified on immunoblots of the 130-kD PVA.
  • B PV IgG affinity-purified by epitope selection on the fusion protein produced by clone MJ315.
  • C Control for B, epitope selection of PV serum by irrelevant clones.
  • D Rabbit antibodies raised against the MJ315 fusion protein.
  • Lane 4-NHEK positive control for PVA mRNA
  • Lane 5-human brain lane 6- human heart
  • lane 7-human lung lane 8-human liver
  • lane 9-human kidney lane 10-human placenta.
  • Lane 4-10 were exposed for 15 hr, and corresponding actin lanes were exposed for 2 hr. Even when exposed for 72 hr, lanes 5-10 did now show PVA mRNA) .
  • the putative signal sequence and transmembrane domain are marked by a dashed and double underline, respectively.
  • the p asumed recognition site for proteolytic cleavage is underlined.
  • the R-A-L sequence, which corresponds to the H-A-V sequence of typical cadherins is boxed.
  • Putative Ca 2+ -binding sites are shaded.
  • Horizontal arrows under the amino acid sequence show beginning of each domain.
  • the + ' s indicate the repetitive N-V/Y-X- V-T-E domains shared by PVA and DGI.
  • the identity and similarity of DGI and P-cadherin to PVA are shown for each domain to the right of the sequences. (NS indicates that similarity is not significant).
  • keratinocyte cells cause loss of cell-to-cell adhesion and blister formation.
  • the autoantibodies are specific to PVA, which has been characterized as a 130-kD glycoprotein linked by disulfide bonds to plakoglobin.
  • this invention relates to DNA sequences (including cDNA sequences) that encode PVA.
  • the invention further relates to DNA sequences that encode the entire amino acid sequence given in Figure 7 (the specific DNA sequence given in Figure 7 being only one example), or any portion comprising at least 12 base pairs thereof.
  • DNA sequences to which the invention relates also include those encoding proteins (or polypeptides) having substantially the same autoantibody binding characteristics of PVA (for example, allelic forms of the amino acid sequence of Figure 7).
  • extension clones contain one long continuous open reading frame encoding a protein of approximately the correct molecular weight and isoelectric point.
  • proteins or polypeptides having an amino acid sequence corresponding to any portion that is at least 4 amino acids of the protein depicted in Figure 7 (or allelic variations thereof).
  • the protein or polypeptides having an amino acid sequence corresponding to any portion that is at least 4 amino acids of the protein depicted in Figure 7 (or allelic variations thereof).
  • the protein or polypeptides having an amino acid sequence corresponding to any portion that is at least 4 amino acids of the protein depicted in Figure 7 (or allelic variations thereof).
  • the protein or
  • polypeptide can have an amino acid sequence corresponding to an epitope of the sequence of Figure 7 (or allelic variation thereof).
  • the protein can be used as an antigen, in protocols known in the art, to produce antibodies thereto, both monoclonal and polyclonal.
  • invention relates to a recombinant DNA molecule that includes a vector and a DNA sequence as described above (advantageously, a DNA sequence encoding the protein shown in Figure 7 or a protein having the autoantibody binding characteristics of that
  • the vector can take the form of a virus, a plasmid, or eukaryotic expression vector (for example, lambda gTII, pUEX, bacillovirus vectors and pcDNAIneo expression vectors).
  • the DNA sequence can be present in the vector operably linked to
  • the recombinant molecule can be suitable for transforming procaryotic or transfecting
  • eukaryotic cells advantageously, mammalian cells or insect cells.
  • pUEX plasmids are suitable for transforming bacterial cells
  • pcDNAIneo vector is suitable for eukaryotic
  • the present invention relates to host cells stably transformed or transfected with the above-described recombinant constructs.
  • the host cell can be prokaryotic (for example, bacterial), lower eukaryotic (for example, yeast or insect) or higher eukaryotic (for example, all mammals, including but not limited to mouse and human).
  • prokaryotic for example, bacterial
  • lower eukaryotic for example, yeast or insect
  • higher eukaryotic for example, all mammals, including but not limited to mouse and human.
  • transfections can be accomplished into Chinese hamster ovary cells (CHO) or COS-7 cells.
  • Transformation or transfection can be accomplished using protocols and materials well known in the art.
  • the transformed or transfected host cells can be used as a source of the DNA sequences described above (which sequence constitutes part of the recombinant construct).
  • the transformed or transfected cells can be used as a source for the above-described PVA protein.
  • an antibody can be raised against a peptide having the amino acid sequence of Figure 7 , or against a portion thereof of at least 4 amino acids in length.
  • Persons skilled in the art using standard methodology can raise monoclonal and polyclonal antibodies to the protein (or
  • polypeptide or a unique portion thereof.
  • the present invention relates to a method of detecting the presence of PVA or antibodies against PVA in a sample.
  • a diagnostic assay can be constructed by coating on a surface (i.e. a solid support) for example, a microtitration plate or a membrane (e.g. nitrocellulose membrane), all or a unique portion of the synthetic PVA protein described above, and contacting it with the serum of a person suspected of having PV.
  • the presence of a resulting complex formed between the PVA and antibodies specific therefor in the serum can be detected by any of the known methods common in the art, such as fluorescent antibody spectroscopy or colorimetry. This method of detection can be used, for example, for the diagnosis of PV.
  • a diagnostic kit which contains recombinantly produced PVA and ancillary reagents that are well known in the art and that are suitable for use in detecting the presence of antibodies to PVA in serum or a tissue sample.
  • Tissue samples contemplated can be monkey and human, or other mammals such as dog.
  • the present invention relates to a therapeutic method for the treatment of PV disease.
  • Plasmapheresis can be conducted on an individual having PV. Before reinfusion of the blood back into the individual, persons skilled in the art using standard
  • NHEK (Clonetics) were culture in keratinocyte grovth medium (Clonetics) nich has a Ca 2+ concentratic. of 0.15 mM. In some experiments the Ca 2+ concentration was raised to 2.55 mM for 24 hr before RNA extraction and for 48 hr before indirect immunofluorescence.
  • human foreskin epidermal cells were cultured on 3T3 cells as previously described (Rheinwald and Green, 1975; Fuchs and Green, 1981; Stanley et al., 1984), either with or without 2.5 ⁇ g/ml tunicamycin (Sigma), which was added for 1 hr before the
  • esophagus the standard substrate to detect PVA with patients' sera, or on cultured NHEK as previously described (Sabolinski et al., 1987; Stanley et al., 1981, 1982).
  • Proteins from cultured NHEK were extracted with sodium dodecyl sulfate (SDS) sample buffer with reduction, separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to
  • nitrocellulose membranes (Hashimoto et al., 1990; Towbin et al., 1979). Immunoblotting was performed with human sera or rabbit antisera and alkaline phosphatase labeled goat anti-human or anti-rabbit IgG (Stanley et al., 1984; Amagai et al., 1990).
  • affinity purification of PV IgG horizontal strips of nitrocellulose containing the 130-kD PVA were cut out, incubated wtih PV serum, washed, then bound antibodies were eluted with acid glycine buffer, neutralized, dialyzed against phosphate buffered saline, and concentrated as described (Mueller et al., 1989). Construction and screening of cDNA Library
  • cDNA was synthesized with random primers and the reverse transcriptase Superscript (Gibco-BRL) by the basic method of Gubler and
  • the cDNA library was screened at high stringency by hybridization with MJ315 labeled with 32 P by random primer labeling (Maniatis et al., 1982). From approximately 10 6 recombinant clones, E12 and E33 were isolated, and plaque purified.
  • Plaque lifts of nitrocellulose-bound fusion protein produced by MJ315 in ⁇ gt11 were used to affinity purify antibodies from the PV serum as described previously (Stanley et al., 1988).
  • the MJ315 cDNA insert was excised from its pGEM plasmid vector by amplification with polymerase chain reaction (PCR) with primers that annealed to both ends and that included either a BamHI or PstI site, so that the insert could be directionally subcloned, in frame, into the BamHI-PstI site of the expression plasmid vector pUEX 1 (Amersham).
  • PCR polymerase chain reaction
  • the crude ⁇ -galactosidase fusion protein produced by pUEX was isolated as previously described for fusion proteins produced in pEX (Tanaka et al., 1990).
  • the precipitated fusion protein was then partially purified by washing first with 0.5% Triton X-100 in 150 mM NaCl, 10 mM EDTA, 10 mM Tris-HCl pH 7.5, then with 2 M urea in 100 mM Tris-HCl pH 8. Rabbits were immunized subcutaneously with approximately 500 ⁇ g of this partially purified fusion protein mixed with complete (first immunization) or incomplete Freund's adjuvant, every 2 weeks for a total of 3 injections.
  • Poly(A) + RNA for Northern analysis was isolated from cultured NHEK and normal human
  • RNA was resolved in a 1% agarose/formaldehyde gel
  • Double stranded cDNA in pGEM or pBluescript was sequenced in both directions by the dideoxy chain termination method with Sequenase (United States Biochemical Corp.). Oligonucleotides, corresponding to vector or previously-determined sequence, were synthesized to use as primers.
  • PC/Gene software (Intelligenetics) was used to determine: a) statistical significance of amino acid identities and similarities between corresponding regions of PVA with DGI and P-cadherin, as well as between extracellular domains of PVA (PCOMPARE), and b) transmembrane regions and signal peptides.
  • N-linked complex carbohydrates did not reside in, or depend on, N- linked complex carbohydrates.
  • the affinity-purified anti-PVA antibodies were used to screen a ⁇ gt11 library constructed from poly(A) + RNA extracted from NHEK cultured in 2.55 mM Ca 2+ . Of 10 6 recombinant clones, one (cDNA insert designated MJ315), which strongly bound the
  • MJ315 encodes epitopes that specifically bind PV antibodies. However, not all PV sera are capable of recognizing the limited epitopes expressed on immunoblots by the MJ315 fusior protein.
  • MJ315 encodes PVA by immunizing rabbits with the MJ315 fusion protein made in pUEX 1.
  • These rabbit antibodies stained monkey esophagus in the same cell surface pattern as PV sera (Fig 2D), and bound the 130-kD PVA by immunoblotting (data not shown).
  • immunofluorescence is limited to stratified squamous epithelia (Beutner et al., 1968), we determined whether mRNA for PVA was expressed in cells and tissues of stratified squamous epithelia
  • mRNA for PVA was detected only in stratified squamous epithelia (Fig. 5).
  • DNA sequencing of the overlapping PVA cDNA clones indicated a total 3,336 bp cDNA with a 2,997 bp open reading frame (Fig 7). There are two tandem ATG potential translation initiation codons after an upstream in-frame stop codon. Either could be the initiation codon, however the bases surrounding the second ATG codon are more consistent with a
  • Hydrophobicity plots also identified a putative transmembrane region. There is a stop codon at bases 3081-3, and two more in frame stop codons within 10 codons after it. There is a 256 bp, incomplete, 3 ' non-coding region .
  • cadherin By homology with cadherins, it can be deduced that the mature PVA protein is probably cleaved from a precursor protein after a conserved sequence of basic amino acids with the sequence R- R-X-K-R (Shirayoshi et al., 1986; Gallin et al., 1987; Goodwin et al., 1990; Koch et al., 1990;
  • homology to typical cadherins can be divided into 5 domains of about equal size (Figs 7,8), EC1 to EC5, which, except for EC5, have homology with each other.
  • EC1 to EC5 which, except for EC5 have homology with each other.
  • the homology is greatest among EC1, EC2 and EC3, the most amino- terminal domains (Ringwald et al., 1987; Takeichi, 1991).
  • the extracellular regions of DGI and desmocollin have been divided into 5 domains, only the first four of which in DGI are homologous to typical cadherins (Koch et al., 1990; Nilles et al., 1991; Collins et al., 1991; Mechanic et al., 1991). All five extracellular regions of PVA show significant homology to corresponding domains in P- cadherin. However, in domains EC1, EC2, and EC3 the homology of PVA to DGI is much greater than to P- cadherin. Unlike DGI, which has a shortened EC5 region, the EC5 region of PVA is similar in size to that of P-cadherin.
  • the cytoplasmic domain of PVA (360 amino acids) is substantially longer than that of typical cadherins (approximately 160 residues) but shorter than that of DGI (480 residues) .
  • PVA and DGI each have 5
  • cytoplasmic region of PVA can be divided into 4 subdomains. (Koch et al., 1990; Nilles et al., 1991) (Figs 7,8). PVA is missing a fifth glycine rich C-terminal cytoplasmic domain found in DGI (Koch et al., 1990; Nilles et al., 1991).
  • the IA ("intracellular anchor") region of PVA is homologous to that of DGI, but unlike that of typical cadherins, which have basic amino acids just inside the membrane.
  • the Cl region of PVA is similar to DGI and typical cadherins, but as with EC1-EC3, the similarity is much greater with DGI. Finally, the C3 region of PVA has two of the five N-V-X-V-T- E repeats that are found in DGI (Nilles et al., 1991).
  • Cadherins are Ca 2+ -dependent cell-cell adhesion molecules that mediate homophilic binding (Takeichi, 1991, 1990). These molecules are thought to be important in establishing and maintaining epithelial and neural tissue integrity.
  • the typical cadherins, which were the first defined, are now well
  • cadherins are also crucial for homophilic binding (Nagafuchi and Takeichi, 1988) as well as for binding catenins and CAP 102, cadherin- associated proteins that may anchor cadherins to the actin cytoskeleton (Ozawa et al., 1989, 1990;
  • PVA shows significant homology to all cadherins, but most markedly to DGI. This homology extends across species, suggesting that the
  • PVA has a putative signal sequence and a well conserved sequence of basic amino acids that presumably serve as a signal for cleavage to a mature protein.
  • PVA like typical cadherins, can be divided into five extracellular domains, of which EC1 to EC4 show variable homology to each other.
  • EC5 shows minimal or no significant homology to the other extracellular domains.
  • PVA shows much greater similarity to corresponding domains of DGI than to those of typical cadherins.
  • PVA has an R- A-L site in EC1 that corresponds to the conserved H- A-V site in an equivalent position in typical cadherins. PVA also has several conserved putative Ca 2+ binding domains with all cadherins as well as two conserved N-glycosylation sites with DGI.
  • Desmoglein shows extensive homology to the cadherin family of cell adhesion molecules. Biochem. Biophys. Res. Commun. 173 , 1224-1230.
  • Neural cadherin role in selective cell-cell adhesion. Science. 245 , 631-635.
  • E-cadherin cDNA Nature. 329 , 341-343. Nagafuchi, A. and Takeichi, M. (1988). Cell binding function of E-cadherin is regulated by the
  • keratinocytes the formation of keratinizing
  • Cadherin cell adhesion molecules with distinct binding specificities share a common structure. EMBO. J. 5, 2485-2488.
  • Pemphigus antibodies identify a cell surface glycoprotein synthesized by human and mouse keratinocytes. J. Clin. Invest. 70 , 281-288.
  • Cadherins a molecular family important in selective cell-cell adhesion.
  • N-cadherin gene maps to human chromosome 18 and is not linked to the E-cadherin gene. J. Neurochem. 55 , 805-812.
  • glycoprotein DGI a component of intercellular desmosome junctions, is related to the cadherin family of cell adhesion molecules. Proc. Natl. Acad. Sci. USA 88 , 4796-4800.

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Abstract

Le pemphigus vulgaris (PV) est une maladie épidermique mettant en danger la vie du malade, dans laquelle des auto-anticorps dirigés contre une glycoprotéine de 130-kD de la surface des cellules kératinocytes, c'est-à-dire l'antigène de PV (PVA), provoquent la perte de l'adhésion intercellulaire, ce qui occasionne des cloques épidermiques. L'invention se rapporte à des séquences d'ADN codant la totalité de la séquence d'aminoacides de PVA. L'invention se rapporte également à des structures recombinées contenant la séquence d'ADN de PVA, ainsi qu'à des cellules hôtes transformées au moyen desdites structures. De plus, l'invention se rapporte à des procédés de diagnostic et de traitement de personnes souffrant de la maladie de phemphigus vulgaris.
PCT/US1992/009933 1991-11-27 1992-11-24 Sequences de nucleotides et d'acides amines de l'antigene de pemphigus vulgaris et procede d'utilisation WO1993010813A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0759771A1 (fr) * 1995-03-07 1997-03-05 President And Fellows Of Harvard College Identification des auto-antigenes et des non auto-antigenes intervenant dans les affections auto-immunes
US7084247B2 (en) 2004-03-11 2006-08-01 Peptimmune, Inc. Identification of self and non-self antigens implicated in autoimmune diseases
US9255131B2 (en) 2004-05-11 2016-02-09 Ganymed Pharmaceuticals Ag Identification of surface-associated antigens for tumor diagnosis and therapy

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
D.M. GLOVER, "DNA Cloning Volume II A Practical Approach", published February 1986, by IRL PRESS (OXFORD, ENGLAND), pages 191-211, and 213-239. *
G.J. TORTORA et al., "Microbiology an Introduction", published 1989, by BENJAMIN/CUMMINGS (CA), pages 446-447. *
JOURNAL OF CLINICAL INVESTIGATIONS, Volume 74, issued August 1984, J.R. STANLEY et al., "Distinction Between Epidermal Antigens Binding Pemphigus Vulgaris and Pemphigus Foliaceus Autoantibodies", pages 313-320. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 80, issued March 1983, R.A YOUNG et al., "Efficient Isolation of Genes by Using Antibody Probes", pages 1194-1198. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 83, issued October 1986, J.C.R. JONES et al., "A Cell Surface Desmosome-Associated Component: Identification of a Tissue-Specific Cell Adhesion Molecule", pages 7282-7286. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0759771A1 (fr) * 1995-03-07 1997-03-05 President And Fellows Of Harvard College Identification des auto-antigenes et des non auto-antigenes intervenant dans les affections auto-immunes
EP0759771A4 (fr) * 1995-03-07 1999-12-29 Harvard College Identification des auto-antigenes et des non auto-antigenes intervenant dans les affections auto-immunes
US7255861B1 (en) 1995-03-07 2007-08-14 President And Fellows Of Harvard College Preparations for inducing immunotolerance and uses therefor
US7084247B2 (en) 2004-03-11 2006-08-01 Peptimmune, Inc. Identification of self and non-self antigens implicated in autoimmune diseases
US9255131B2 (en) 2004-05-11 2016-02-09 Ganymed Pharmaceuticals Ag Identification of surface-associated antigens for tumor diagnosis and therapy
US9533043B2 (en) 2004-05-11 2017-01-03 Biontech Ag Identification of surface-associated antigens for tumor diagnosis and therapy
US10724103B2 (en) 2004-05-11 2020-07-28 Biontech Ag Identification of surface associated antigen FLJ31461 for tumor diagnosis and therapy

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