WO1998024897A1 - Molecule d'adhesion jonctionnelle (jam), proteine transmembranaire de jonctions serrees - Google Patents

Molecule d'adhesion jonctionnelle (jam), proteine transmembranaire de jonctions serrees Download PDF

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WO1998024897A1
WO1998024897A1 PCT/EP1997/006723 EP9706723W WO9824897A1 WO 1998024897 A1 WO1998024897 A1 WO 1998024897A1 EP 9706723 W EP9706723 W EP 9706723W WO 9824897 A1 WO9824897 A1 WO 9824897A1
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protein
jam
ser
thr
val
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Elisabetta Dejana
Ines Martin Padura
David Simmons
Lisa Williams
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F Hoffmann La Roche AG
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Priority to JP52518498A priority Critical patent/JP2001506847A/ja
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Priority to AU56578/98A priority patent/AU5657898A/en
Priority to CA002273202A priority patent/CA2273202A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • JUNCTIONAL ADHESION MOLECULE JAM
  • the present invention relates to a transmembrane component of tight junction, more precisely a new protein, which is expressed in endothelial cells, epithelial cells, megakaryocytic cells and platelets.
  • This new protein is now denominated junctional adhesion molecule (JAM), and most of the human and the whole of the mouse JAM have been sequenced.
  • JAM junctional adhesion molecule
  • the invention further comprises a cDNA coding for JAM, a structural gene coding for JAM, a recombinant protein or peptide expressed by the structural gene or by a fragment of the gene, an antibody specific for JAM, a modifier of the polymerization of transmembrane JAM, a diagnostic kit comprising the antibody or the modifier, a vaccine adjuvant comprising the modifier, a medicament comprising the modifier, and transgenic animals or cells overexpressing or lacking JAM.
  • An example of the modifier is a monoclonal antibody specifically binding to JAM and preventing the polymerization of JAM at tight junctions, resulting inter alia in the blocking of leukocyte transmigration.
  • Endothelium forms the main barrier to the passage of macromolecules and circulating cells from blood to tissues.
  • Endothelial permeability is in large part regulated by intercellular junctions. These are complex structures formed by transmembrane adhesive molecules linked to a network of cytoplasmic/cytoskeletal proteins. At least four different types of endothelial junctions have been described: tight junctions, gap junctions, adherence junctions and syndesmos (Dejana et al., infra).
  • Intercellular tight junctions are responsible for the control of endothelial and epithelial cell layer permeability [Anderson et al; Curr. Opi ⁇ . Cell. Biol. 5, 772-778 (1993)]. These organelles also regulate leukocyte transmigration, cell polarity and growth. The molecules which constitute the tight junctions are therefore good targets for developing drugs which affect inflammatory reaction, angiogenesis and cell proliferation in general [Dejana et al., FASEB J. 9, 910-918 (1995)]. Leukocyte transmigration through the endothelium or the epithelium in inflammation is associated with edema and tissue damage. To transmigrate leukocytes have to open the tight junctions and go through cell-cell contacts [Carlos and Harlan, Blood 84, 2068-2101 (1994)]. Tools which are able to specifically limit this process have not been disclosed in the prior art.
  • Tumor cells seem to use similar mechanisms to transmigrate through the endothelium and infiltrate tissues. Agents which could prevent tight junction opening would be therefore useful in limiting tumor metastasis.
  • the tight junctions are poorly expressed in epithelial cell derived tumors, and some tight junction components have oncosuppressor activity, i.e., their presence reduces the capacity of the tumor to proliferate and to metastasize [Tsukita et al., J. Cell Biol. 123, 1049-1053 (1993)]. Transfection of the tight junction molecule genes could be seen as a way to limit tumor progression. In addition, since tight junctions are needed for a correct organization of new vessels, inhibition of their organization might prevent angiogenesis and thus inhibit the development of proliferative diseases such as cancer.
  • the present invention is based on the finding of a new transmembrane protein located at tight junction.
  • This new protein is called junctional adhesion molecule and is abbreviated JAM.
  • This JAM protein located at tight junctions, promotes cell to-cell homotypic adhesion.
  • the extracellular parts of the proteins adhere to each other on the same and adjacent cells by protein dimerization, oligomerization or polymerization in a zipper-like fashion and causes a strong reduction in paracellular permeability.
  • the JAM used in the experimental part of this specification is of mouse origin, whereas the JAM of human origin is the one which will be predominantly used as a model for the development of diagnostics and medicaments for use in, e.g., tumor therapy, angiogenesis control, control of Blood Brain Barrier, control of inflammatory response, control of transmigration of leukocytes, control of transmigration of other natural or engineered cells such as gene therapy in the brain.
  • the first aspect of the invention is directed to a protein in glycosylated or unglycosylated form comprising an amino-acid sequence selected from the sequence SEQ ID NO:1
  • homologous sequences having at least 72 % homology to the sequence SEQ ID NO: 1.
  • the percentage of homology may for instance be 75%, 80% or as high as 85 %, or even higher, such as 90 % or 95 %, especially if the homologous sequence originates from a transmembrane protein of the same or closely related species.
  • proteins which have at least 72 % homology to this N-terminal sequence SEQ ID NO: 1 will share both diagnostic and medical properties to such a high degree that they can be used for the various applications of the present invention.
  • Such proteins may be included both naturally occurring analogues and variants of the same or different species as well as synthetic or recombinant equivalents.
  • An example of such a protein having at least 75 % homology to the SEQ ID: 1 is the mouse JAM protein having the amino- acid sequence SEQ ID NO: 2 (mouse)
  • the synthetically or recombinantly produced proteins of the invention will function as competitors at tight junctions.
  • the second aspect of the invention is directed to a DNA sequence coding for a protein of the present invention, specifically a cDNA sequence coding for a protein of the present invention.
  • Specific embodiments of this aspect of the invention are the cDNA sequence SEQ ID NO: 3 (part of human)
  • TCCTCTGCCA CCATTGGGAA CCGGGCAGTG CTGACATGCT CAGAACAAGA TGGTTCCCCA 540
  • GATCCCCTGT CAGCCTCTGA TACTGGAGAA TACAGCTGTG AGGCACGGAA TGGGTATGGG 720
  • AACCCAAACC AATACCTGGC TGTAAAGGCC TCTGAATAAG GACTTTAAGC CTAGCTCCCT 1320
  • cDNA molecules will find their application in gene therapy, and they can be used as an oncosuppressor by transfection in carcinoma cells lacking this molecule.
  • the third aspect of the invention is directed to a gene coding for a protein of the present invention or a peptide derived from the protein.
  • the gene will be used in the production of a protein or peptide of the invention.
  • the flanking regions such as promoter or leader sequences, are preferably chosen with regard to the expression system to be used to promote good production.
  • the codons used in the gene may be selected with regard to the codons most frequently used by the selected expression host, in order to optimize the expression yield. For instance, if yeast is selected as the expression host, the codons may be optimized for yeast.
  • the specific examples of genes of the invention are the protein coding regions of the exemplified cDNAs of the invention, namely the gene having the partial nucleotide sequence SEQ ID NO: 5 (part of human SEQ ID NO: 3)
  • ACATGCTCAG AACAAGATGG TTCCCCACCT TCTGAATACA CCTGGTTCAA AGATGGGATA 540
  • AATCCCACAA CAGGAGAGCT GGTCTTTGAT CCCCTGTCAG CCTCTGATAC TGGAGAATAC 660 AGCTGTGAGG CACGGAATGG GTATGGGACA CCCATGACTT CAAATCGTGT CGCGATGGAA 720
  • the proteins of the present invention can be chemically synthesized using standard methods known in the art, preferably solid state methods, such as the methods of Merrifield (J. Am. Chem. Soc. 85, 2149-2154 [1963]).
  • the proteins of the present invention can be produced using methods of DNA recombinant technology (Sambrook et al. in "Molecular Cloning - A Laboratory Manual", 2nd. ed., Cold Spring Harbor Laboratory [1989]).
  • the fourth aspect of the invention is directed to a recombinant protein or peptide expressed by a structural gene or a fragment of the gene according to the present invention.
  • DNA coding for a protein of the present invention is isolated through expression cloning.
  • a cDNA expression library is constructed from a murine brain EC line (bEnd.3) as previously described (Fawcett et al., Nature 360, 481 [1992]).
  • COS cells are transiently transfected with the cDNA library, stained in suspension with anti JAM antibody and then panned on plastic dishes coated with the appropriate second antibody.
  • a DNA sequence coding for a protein of the present invention is incorporated into a suitable expression vector which produces the requisite expression signals.
  • prokaryotic expression vectors suitable for use in prokaryotic host cells are mentioned, for example, in the aforementioned textbook of Maniatis et al.
  • Such prokaryotic expression vectors which contain the DNA sequences coding for the proteins of the present invention operatively linked with an expression control sequence can be incorporated using conventional methods into any suitable prokaryotic cell.
  • the selection of a suitable prokaryotic cell is determined by different factors which are well-known in the art. Thus, for example, compatibility with the chosen vector, toxicity of the expression product, expression characteristics, necessary biological safety precautions and costs play a role and a compromise between all of these factors must be found.
  • Suitable prokaryotic organisms include gram-negative and gram-positive bacteria, for example, E. coli and B. subtilis strains.
  • Examples of prokaryotic organisms are E. coli strain M15, described as strain OZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-474 (1974) and E. coli W3110 (ATCC No. 27325).
  • E. coli strains In addition to the aforementioned E. coli strains, however, other generally accessible E. coli strains such as E. coli 294 (ATCC No. 31446) and E. coli RR1 (ATCC No. 31343) can also be used.
  • Expression vectors suitable for use in mammalian cells include but are not limited to pBC12MI [ATCC 67109], pSV2dhfr [ATCC 37146], pSVL [Pharmacia, Uppsala, Sweden], pRSVcat [ATCC 37152] and pMSG [Pharmacia, Uppsala].
  • a preferred vector for the expression of the proteins of the present invention is pECE.
  • Mammalian host cells that could be used include, e.g., human Hela, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, CV1 African green monkey kidney cells, quail QC1 -3 cells, Chinese hamster ovary (CHO) cells, mouse L cells and the COS cell lines.
  • the CHO cell line (ATCC CCL 61 ) is preferred.
  • the manner in which the expression of the proteins of the present invention is carried out depends on the chosen expression vector/host cell system.
  • the prokaryotic host organisms which contain a desired expression vector are grown under conditions which are optimal for the growth of the prokaryotic host organisms.
  • the expression of the desired protein is induced.
  • the induction can be carried out by adding an inducer or a derepressor to the growth medium or by altering a physical parameter.
  • the mammalian host cells which contain a desired expression vector are grown under conditions which are optimal for the growth of the mammalian host cells.
  • a typical expression vector contains the promoter element, which mediates the transcription of mRNA, the protein coding sequence, and the signals required for efficient termination and polyadenylation of the transcript. Additional elements may include enhancers and intervening sequences bounded by spliced donor and acceptor sites.
  • Most of the vectors used for the transient expression of a given coding sequence carry the SV40 origin of replication, which allows them to replicate to high copy numbers in cells (e.g. COS cells) that constitutively express the T antigen required to initiate viral DNA synthesis.
  • Transient expression is not limited to COS cells. Any mammalian cell line that can be transfected can be utilized for this purpose.
  • Elements that control a high efficient transcription include the early or the late promoters from SV40 and the the long terminal repeats (LTRs) from retroviruses, e.g. RSV, HIV, HTLVI.
  • LTRs long terminal repeats
  • retroviruses e.g. RSV, HIV, HTLVI.
  • cellular signals can be used (e.g. human- ⁇ - actin-promoter).
  • stable cell lines carrying a gene of interest integrated into the chromosome can be selected upon co-transfection with a selectable marker such as gpt, dhfr, neomycin or hygromycin.
  • the transfected gene can be amplified to express large quantities of a foreign protein.
  • the dihydrofolate reductase (DHFR) is a useful marker to develop lines of cells carrying more than 1000 copies of the gene of interest.
  • the mammalian cells are grown in increasing amounts of methotrexate. Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome.
  • the baculovirus-insect cell vector system can also be used for the production of the proteins of the present invention (for review see Luclow and Summers, Bio/Techno- logy 6, 47-55 [1988]).
  • the proteins produced in insect cells infected with recombinant baculovirus can undergo post-translational processing including N- glycosylation (Smith et al., Proc. Nat. Acad. Sci. USA 82, 8404-8408) and O- glycosylation (Thomsen et al., 12. International Herpesvirus Workshop, University of Philadelphia, Pennsylvania).
  • the proteins of the present invention can be purified from the cell mass or the culture supernatants according to methods of protein chemistry which are known in the art such as, for example, precipitation, e.g., with ammonium sulfate, dialysis, ultrafiltration, gelfiltration, ion-exchange chromatography, SDS-PAGE, isoelectric focusing, affinity chromatography like immunoaffinity chromatography, HPLC on normal or reverse systems or the like.
  • the fifth aspect of the invention is directed to an antibody binding specifically to a protein according to the present invention or a part of the protein.
  • the antibody may be polyclonal or monoclonal.
  • the preparation of monoclonal antibodies of the invention is disclosed.
  • One of the monoclonal antibodies of the invention mAb BV 12 binds specifically to JAM but does not inhibit transmigration of leukocytes through tight junctions, whereas another monoclonal antibody of the invention mAb BV 1 1 not only binds specifically to JAM but also inhibits the transmigration of leukocytes through tight junctions.
  • Both types of antibodies binding specifically to JAM may be used in diagnostics, and in diagnostic kits, e.g., for screening or detection of cell damage, particularly by detection of circulating JAM as a marker of early endothelial cell damage.
  • the sixth aspect of the invention is directed to a modifier of the polymerization of a transmembrane protein according to the present invention.
  • modifier is to be interpreted broadly and to comprise in the present specification and appended claims, both inhibitors and activators of the polymerization of the JAM protein of the invention.
  • the modifiers of the invention will either prevent or promote polymerization of JAM molecules at tight junctions, i.e., the dimerization, oligomerization or polymerization of JAM, or dedimerization, deoligomerization or depolymerization of JAM, respectively, at tight junctions.
  • the modifier of the invention may be any ligand to the protein of the invention which binds to the protein and has the ability to prevent or promote the polymerization of the protein (JAM).
  • the modifier of the invention may have a structure which is complementary to the protein of the invention or a part of the protein.
  • the modifier is selected from the group consisting of polyclonal and monoclonal antibodies specifically binding to the protein according to the invention and inhibiting or inducing the polymerization of said protein, and polymerization-inhibiting or -inducing proteins, peptides, peptidomimetics and organic molecule-ligands derived from the amino-acid sequence of the protein according to the invention.
  • the polyclonal and monoclonal antibodies of the invention can be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein in Nature 256, 495-497 (1975) and Campbell in "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" in Burdon et al., Eds., Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985) as well as by the recombinant DNA method described by Huse et al. in Science 246, 1275-1281 (1989).
  • the antibodies may be prepared in any mammal, including mice, rats, rabbits, goats and humans.
  • the antibody may be a member of one of the following immunoglobulin classes; IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof, and preferably is an IgG antibody.
  • the seventh aspect of the invention is directed to a diagnostic kit comprising as a diagnostic reagent an antibody according to the invention or a modifier according to the invention.
  • the actual diagnostic method which is going to be used will determine possible additional components in the kit, and the kit will preferably be accompanied by instructions for use.
  • An example of a widely used immunological diagnostic method is enzyme linked immunosorbent assay (ELISA), and this has been used in the experimental part of this specification.
  • the eight aspect of the invention is directed to a useful application of the modifier of the invention, namely a vaccine adjuvant comprising a modifier according to the present invention.
  • the ninth aspect of the invention is directed to another useful and desirable application of the modifier of the invention, namely a medicament comprising as an active ingredient a modifier according to the present invention.
  • the tenth aspect of the invention is directed to transgenic animals or cells overexpressing or lacking a protein according to the present invention.
  • Transgenic animals carrying null mutation of JAM created by standard techniques [Hogan et. al., Manipulating the mouse embryo: A laboratory manual. Cold Spring Harbor Laboratory Press N.Y. (1994)] will be used as in vivo models for screening replacing, activating molecules for JAM and for providing the therapeutic potential of JAM in genetherapy in medicine.
  • JAM-overexpressing animals e.g. using promoters selected from NSE, Thy 1 , PDGFB, VE cadherin, Willebrand factor, and transomodulin
  • NSE NSE
  • Thy 1 PDGFB
  • VE cadherin VE cadherin
  • Willebrand factor VE cadherin
  • transomodulin transomodulin
  • Transgenic cells are used for in vitro testing purposes.
  • Hybridomas were produced by fusion of immunized rat splenocytes with Sp2/0 cell line from ATCC (Maryland, USA). Hybridoma supematants were screened by standard enzyme-linked immunoassay (ELISA) for binding to H5V. Positive hybridoma were then characterized by their ability to stain endothelial cell-cell contacts by immunofluorescence microscopy which technique is disclosed below.
  • mAbs were screened on transmigration of leukocytes through the endothelial monolayers (see below).
  • Mabs BV1 1 and BV12 were selected after the first screening, and the corresponding hybridomas were serially cloned twice by the method of limiting dilutions.
  • MAbs isotypes were determined using a rat isotyping kit (Sigma). Ascites were produced by a standard technique [Martin-Padura et al., supra]. Briefly, Nu/Nu (CD1 ) BR mice were primed with intraperitoneal injections of 0.5 ml of pristane 6 days before intraperitoneal injection of 10X10 6 hybridoma cells. Ascites were collected after 2-3 weeks.
  • MAb BV11 was purified from ascites by binding to immobilized protein G (Pharmacia), as described by Martin -Padura et al., supra.
  • Ceils were seeded on glass coverslips and grown to confluence in Medium 199 containing 20 % newborn calf serum before immunofluorescence staining.
  • glass coverslips were coated with human plasma fibronectin (7 ⁇ g /ml).
  • Cells were fixed with MeOH for 4 min and processed for indirect immunofluorescence microscopy as previously described in detail by Lampugnani et al., J. Cell. Biol. 1 18, 151 1 -1522 (1992).
  • JAM distributes selectively at cell-cell contacts in endothelial and epithelial cells. Distribution corresponds to other molecules located at tight junctions such as ZO-1 or cingulin. At confocal electron microscopy JAM localizes at tight junction while it is not found in other regions of cell-cell contacts such as adherence junctions.
  • JAM inhibits leukocyte transmigration
  • monocytes (approximately 92% pure) were obtained from Ficoll-Hypaque separated mononuclear cells by centrifugation on a discontinuous (46%) gradient of isosmotic (285 mOsmol) Percoll (Pharmacia). Polymorphonuclear cells were isolated by dextran sedimentation followed by Lymphoprep gradient and hypotonic lysis of erythrocytes, as previously described by Del Maschio et al., Br. J. Haematol. 72, 329-335 (1989). Cells to be used in suspension were resuspended at 10 7 cells/ml in complete medium and labeled by incubation with 100 mCi 51 Cr for 1 h at room temperature.
  • mAb BV11 and mAb BV12 are both antibodies of the invention.
  • mAb to CD31 is disclosed by Vecchi, A. et. al., Eur. J. Cell Biol. 63, 247-254 (1994) Antibodies were used as hybridoma supernatants at 1 :2 dilution added to the upper chamber. Values are means ⁇ SEM of four experiments./ ** p ⁇ 0.01 by analysis of various and Duncan's test.
  • mAb BV11 monoclonal antibody binding to and neutralizing JAM
  • mAb BV12 monoclonal antibody binding to but not neutralizing JAM.
  • MCP-1 at 100 ng/ml was added to the lower compartment 5 min before monocyte seeding.
  • Antibodies were used as hybridoma supernatants at 1 :2 dilution added to the upper chamber. Values are means ⁇ SEM of two experiments. ** p ⁇ 0.01 by analysis of various and Duncan's test.
  • mAb BV1 1 monoclonal antibody neutralizing JAM .
  • Chemotaxis was induced by addition of fMLP (500 nM) to the lower compartment of the Transwell unit. Antibodies were used as hybridoma supernatants at 1 :2 dilution added to the upper chamber. Values are means ⁇ SEM of two experiments. ** p ⁇ 0.01 by analysis of various and Duncan's test. mAb BV1 1 : monoclonal antibody neutralizing JAM.
  • mice were anesthetized with ether and 5 ml of sterile air were injected under the skin in the back (day 0). After three days pouches were reinjected with 3 ml of sterile air. On day 4, animals received intravenous injection of 200 ⁇ g of monoclonal antibody BV1 1 binding specifically to JAM or the same dose of nonimmune rat IgG (Sigma). On day 6; 1 ml of 1 % carrageenan in saline was injected into the pouch. At different times after carrageenan the animals were anesthetized and the pouches were washed with 1 ml of saline. The lavage fluid was immediately cooled on ice and the volume was recorded. Then 50 ⁇ l were used for cell count after staining with erythrosin.
  • Treatment cell number (x 10 6 )
  • Leukocyte recruitment was induced by injection of carrageenan in sterile saline (1 ml) into six day-old pouches. 200 ⁇ g of purified mAb BV1 1 (mAb binding to JAM) or rat non-immune-lgG (control) were injected intravenously in 200 ⁇ l 12 hours before carrageenan treatment. Animals were killed 48 hours after the treatment. Data are mean ⁇ SD of at least seven animals in two experiments. * p ⁇ 0.002 according to Student ' s t test. In conclusion, as reported in Table 4, the number of polymorphonuclear cells found in the air pouch after carrageenan injection was significantly reduced in the mice treated with the monoclonal antibody BV11 specifically binding to JAM in respect to mice IgG.
  • Plasma exudation was induced by carrageenan in sterile saline (1 ml) in six day-old pouches. 200 ⁇ g of purified mAb BV1 1 (mAb binding to JAM) or rat non-immune-lgG (control) were injected intravenously in 200 ⁇ l 12 hours before carrageenan treatment. Animals were killed 48 hours after the treatment. Data are mean + SD of at least 8 animals in two experiments. * p ⁇ 0.049 according to Student ' s t test.
  • JAM cDNA was isolated through expression cloning.
  • the cDNA expression library was constructed from a murine brain EC line (bEnd.3) as previously described (Fawcett et al., Nature 360, 481 [1992]; Seed, Nature 329, 840 [1987]; Seed and Aruffo, Proc. Natl. Acad. Sci. U.S.A. 84, 3365 [1987]; Simmons et al. Nature 331 , 624 [1988]).
  • the cDNA library was oligo-dT primed bEnd.3 polyA + RNA cloned into pCDM ⁇ (Nature 329, 840-842 [1987]). Plasmid pCDM8 was cut with Hindlll and Notl enzymes and the insert was blunted and subcloned into the Smal restriction site of pECE eucaryotic expression vector (Rutter et al., Cell 45, 721 -732 [1986]) to give the pECE-JAM construct. The construct was then checked for correct orientation by sequence analysis using the dideoxynucleotide chain termination method ("Molecular Cloning", Second Edition, Cold Spring Harbor Laboratory Press (1987) and Ausubel et al.
  • CHO cells were plated at 3-4x10 6 cells per 100 mm petri dish in DMEM with 10% FCS. 24 h after seeding cells were transfected by calcium phosphate precipitation method with 20 ⁇ g of pECE-JAM and 2 ⁇ g of plasmid pSV2 neo (Rutter et al., supra). After 24 h, the DNA-containing medium was replaced by fresh DMEM with 10% FCS and maintained for further 48 h.
  • Transfectants were seeded on Transwell filters, dextran was added to the upper compartment and its passage to the lower compartment was evaluated at 2 hours. Permeability in JAM transfectants was increased by addition of EGTA and cytocalasin D indicating that the activity is Ca ++ dependent and requires an intact actin cytosceleton.
  • JAM transfection significantly reduced the passage of dextran through intercellular junctions.
  • Table 6 reports the effect of permeability increasing agents such as EGTA and cytocalasin D. This also shows that JAM needs Ca++ and an intact actin cytoskeleton to exert its effect.
  • Control 150 ⁇ 10 136 ⁇ 21 166 ⁇ 14 mAb BV 11 920 ⁇ 7 ** 142 ⁇ 16 169 + 19 non-immune serum 167 ⁇ 5 148 ⁇ 9 179 ⁇ 9
  • the mAb BV11 was able to detect JAM protein in solubilized endothelial cells and JAM transfectant cells while it gave negative values using the extracts of cells which do not express JAM such as hemopoietic precursor cell lines and 3T3 fibroblasts.
  • Inhibitors of the JAM self aggregation can be identified by physical methods (light scattering, ultracentrifugation, gelpermeation chromatography, BiaCore etc.) or, as described below, by a two-sided sandwich type immunoassay using the monoclonal antibodies of this invention (mab BV1 1 and mab BV12). Briefly, Immunoplates (Nunc Maxisorb) are coated overnight with 100 ⁇ l/well of a solution of mab BV12 (10 ⁇ g/ml) in 0.1 M sodium bicarbonate buffer.
  • the wells are blocked by addition of blocking buffer (1 % bovine serum albumin in Tris-buffered saline, 0.05% Tween 20 pH 7.5; 100 ⁇ l/well). After three hours the wells are washed and the sample of soluble JAM is added together with the aggregation inhibitor at suitable dilution. After incubation overnight in the cold the sample is removed, the wells are washed and an antibody- enzyme conjugate is added at suitable concentration diluted with blocking buffer.
  • the antibody-enzyme conjugate may be prepared by coupling mab BV12 to activated horseradish peroxidase according to Nakane and Kawaoi (J. Histochem. Cytochem. 22, 1084-1091 [1975]).
  • the plate is washed and incubated with a colorimetric enzyme substrate, e.g., tetramethyl benzidine and hydrogenperoxide.
  • a colorimetric enzyme substrate e.g., tetramethyl benzidine and hydrogenperoxide.
  • Inhibitors of JAM self aggregation are recognized by reduction of the bound peroxidase activity.
  • a similar assay using mab BV11 or BV12 for coating the plate can be used for measuring soluble JAM as diagnostic marker of murine endothelial cell damage / proliferation.
  • AACCCAAACC AATACCTGGC TGTAAAGGCC TCTGAATAAG GACTTTAAGC CTAGCTCCCT 1320
  • ACATGCTCAG AACAAGATGG TTCCCCACCT TCTGAATACA CCTGGTTCAA AGATGGGATA 540
  • AATCCCACAA CAGGAGAGCT GGTCTTTGAT CCCCTGTCAG CCTCTGATAC TGGAGAATAC 660 AGCTGTG AGG C ACGG AATGG GT ATGGG AC A CCC ATG ACTT C AAATCGTGT CGCG ATGG AA 720
  • GCCATGAGGT CAGAGGCTGC ACACATGGAT GCTGTGGAGC TGAATGTGGG GGGCATCGTG 720 GCAGCTGTCC TGGTAACACT GATTCTCCTT GGACTCTTGA TTTTTGGCGT CTGGTTTGCC 780

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  • Photometry And Measurement Of Optical Pulse Characteristics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne une protéine transmembranaire localisée dans des jonctions serrées. La protéine est exprimée dans des cellules endothéliales, épithéliales, mégacaryocytaires ou dans des plaquettes. La protéine est appelée molécule d'adhésion jonctionnelle (JAM). On décrit la séquence amino-acide de la JAM humaine ou murine, ainsi que les séquences d'ADN, les gènes et les protéines recombinées ou peptides exprimés par les gènes ou des fragments de ces gènes. On décrit en outre des anticorps se liant plus particulièrement à la JAM ou partie de la JAM, des modificateurs (inhibiteurs et inducteurs, par exemple) de la polymérisation de la JAM transmembranaire, les anticorps et le modificateur utilisés comme réactifs présentés dans des trousses de diagnostic, ainsi que des emplois du modificateur, en tant qu'adjuvant des vaccins et principe actif, dans des médicaments. Enfin, l'invention concerne aussi des animaux ou des cellules transgéniques qui surexpriment la JAM ou chez qui la JAM est absente.
PCT/EP1997/006723 1996-12-04 1997-12-01 Molecule d'adhesion jonctionnelle (jam), proteine transmembranaire de jonctions serrees Ceased WO1998024897A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP52518498A JP2001506847A (ja) 1996-12-04 1997-12-01 結合接着分子（ｊａｍ）、密着結合の膜貫通蛋白質
EP97952843A EP0948621A1 (fr) 1996-12-04 1997-12-01 Molecule d'adhesion jonctionnelle (jam), proteine transmembranaire de jonctions serrees
AU56578/98A AU5657898A (en) 1996-12-04 1997-12-01 Junctional adhesion molecule (jam), a transmembrane protein of tight junctions
CA002273202A CA2273202A1 (fr) 1996-12-04 1997-12-01 Molecule d'adhesion jonctionnelle (jam), proteine transmembranaire de jonctions serrees

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9604470-6 1996-12-04
SE9604470A SE9604470D0 (sv) 1996-12-04 1996-12-04 Transmembrane component of tight junction

Publications (1)

Publication Number Publication Date
WO1998024897A1 true WO1998024897A1 (fr) 1998-06-11

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PCT/EP1997/006723 Ceased WO1998024897A1 (fr) 1996-12-04 1997-12-01 Molecule d'adhesion jonctionnelle (jam), proteine transmembranaire de jonctions serrees

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EP (1) EP0948621A1 (fr)
JP (1) JP2001506847A (fr)
AU (1) AU5657898A (fr)
CA (1) CA2273202A1 (fr)
SE (1) SE9604470D0 (fr)
WO (1) WO1998024897A1 (fr)
ZA (1) ZA9710794B (fr)

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WO1999027098A3 (fr) * 1997-11-21 1999-11-18 Genentech Inc Antigenes apparentes a a33 et leurs utilisations pharmacologiques
WO2000053749A3 (fr) * 1999-03-11 2000-12-14 Rmf Dictagene Sa Molecules d'adhesion vasculaire et modulation de leur fonction
US6391855B1 (en) 1999-06-02 2002-05-21 Adherex Technologies, Inc. Compounds and methods for modulating junctional adhesion molecule-mediated functions
US6410708B1 (en) 1997-11-21 2002-06-25 Genentech, Inc. Nucleic acids encoding A-33 related antigen polypeptides
EP1513867A4 (fr) * 2001-07-16 2006-02-08 Lilly Co Eli Molecules d'adhesion jonctionnelle extracellulaires
WO2006008076A3 (fr) * 2004-07-16 2006-07-27 Univ Degli Studi Milano Procedes et agents stimulant la reponse immunitaire
US7198917B2 (en) 1997-11-21 2007-04-03 Genentech, Inc. Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US7282565B2 (en) 1998-03-20 2007-10-16 Genentech, Inc. PRO362 polypeptides
US8007798B2 (en) 1997-11-21 2011-08-30 Genentech, Inc. Treatment of complement-associated disorders
US8007797B2 (en) 2006-09-28 2011-08-30 Merck Serono S.A. Junctional adhesion molecule-C (JAM-C) binding compounds and methods of their use
US8088386B2 (en) 1998-03-20 2012-01-03 Genentech, Inc. Treatment of complement-associated disorders
CN101535344B (zh) * 2006-11-24 2013-10-16 皮埃尔法布雷医药公司 新型抗增殖抗体
US9234024B2 (en) 2008-05-06 2016-01-12 Genentech, Inc. Affinity matured CRIg variants

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Cited By (24)

* Cited by examiner, † Cited by third party
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US7273726B2 (en) 1997-11-21 2007-09-25 Genentech, Inc. Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US6838554B2 (en) 1997-11-21 2005-01-04 Genetech, Inc. Nucleic acids encoding proteins that stimulate the proliferation of t-lymphocytes
AU735081B2 (en) * 1997-11-21 2001-06-28 Genentech Inc. A-33 related antigens and their pharmacological uses
WO1999027098A3 (fr) * 1997-11-21 1999-11-18 Genentech Inc Antigenes apparentes a a33 et leurs utilisations pharmacologiques
US6410708B1 (en) 1997-11-21 2002-06-25 Genentech, Inc. Nucleic acids encoding A-33 related antigen polypeptides
US8007798B2 (en) 1997-11-21 2011-08-30 Genentech, Inc. Treatment of complement-associated disorders
US7211400B2 (en) 1997-11-21 2007-05-01 Genentech, Inc. Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US7198917B2 (en) 1997-11-21 2007-04-03 Genentech, Inc. Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US7115713B2 (en) 1997-11-21 2006-10-03 Genentech, Inc. Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
US7282565B2 (en) 1998-03-20 2007-10-16 Genentech, Inc. PRO362 polypeptides
US8088386B2 (en) 1998-03-20 2012-01-03 Genentech, Inc. Treatment of complement-associated disorders
JP4836329B2 (ja) * 1999-03-11 2011-12-14 メルク セローノ エス．エイ． 血管接着分子およびその機能の調節
JP2002537837A (ja) * 1999-03-11 2002-11-12 エールエムエフ・ディクタジェーヌ・ソシエテ・アノニム 血管接着分子およびその機能の調節
US7670826B2 (en) 1999-03-11 2010-03-02 Merck Serono Sa Confluence regulated adhesion molecules useful in modulating vascular permeability
US7393651B2 (en) 1999-03-11 2008-07-01 Laboratoires Serono S.A. Confluence regulated adhesion molecules useful in modulating vascular permeability
WO2000053749A3 (fr) * 1999-03-11 2000-12-14 Rmf Dictagene Sa Molecules d'adhesion vasculaire et modulation de leur fonction
US8143056B2 (en) 1999-03-11 2012-03-27 Merck Serono Sa Vascular adhesion molecules and modulation of their function
CZ303128B6 (cs) * 1999-03-11 2012-04-18 Laboratoires Serono Sa Adhezní molekula 1 regulovaná konfluencí CRAM-1, její kódující nukleová kyselina, protilátky a použití
US6391855B1 (en) 1999-06-02 2002-05-21 Adherex Technologies, Inc. Compounds and methods for modulating junctional adhesion molecule-mediated functions
EP1513867A4 (fr) * 2001-07-16 2006-02-08 Lilly Co Eli Molecules d'adhesion jonctionnelle extracellulaires
WO2006008076A3 (fr) * 2004-07-16 2006-07-27 Univ Degli Studi Milano Procedes et agents stimulant la reponse immunitaire
US8007797B2 (en) 2006-09-28 2011-08-30 Merck Serono S.A. Junctional adhesion molecule-C (JAM-C) binding compounds and methods of their use
CN101535344B (zh) * 2006-11-24 2013-10-16 皮埃尔法布雷医药公司 新型抗增殖抗体
US9234024B2 (en) 2008-05-06 2016-01-12 Genentech, Inc. Affinity matured CRIg variants

Also Published As

Publication number Publication date
JP2001506847A (ja) 2001-05-29
CA2273202A1 (fr) 1998-06-11
SE9604470D0 (sv) 1996-12-04
ZA9710794B (en) 1998-06-04
EP0948621A1 (fr) 1999-10-13
AU5657898A (en) 1998-06-29

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