CA2273202A1 - Junctional adhesion molecule (jam), a transmembrane protein of tight junctions - Google Patents

Abstract

A transmembrane protein located at tight junctions is disclosed. The protein is expressed in endothelial cells, epithelial cells, megakaryocytic cells and platelets. This protein is called junctional adhesion molecule (JAM). The amino-acid sequence of human JAM and of murine JAM are presented. In addition, the DNA sequences, the genes and recombinant proteins or peptides expressed by the genes or fragments of the genes are disclosed. Furthermore, antibodies binding specifically to JAM or a part of JAM and modifiers, i.e. inhibitors and inducers, of the polymerization of the transmembrane JAM are disclosed, together with the antibodies and the modifier as reagents in diagnostic kits, as well as applications of the modifier as vaccine adjuvant and as active ingredient in medicaments. Finally, transgenic animals or cells overexpressing or lacking JAM are comprised by the disclosure.

Description

JUNCTIONAL ADHESION MOLECULE (JAM), A TRANSMEMBRANE PROTEIN OF TIGHT JUNCTIONS
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 functional adhesion molecule (JAM), and most of the human and the whole of the mouse JAM
have been sequenced. 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 IS antibody specifically binding to JAM and preventing the polymerization of JAM at tight junctions, resulting inter alia in the blacking of leukocyte transmigration.
Background The 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 cytoplasmicicytoskeletal 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. Opin. Cell. Biol. 5, (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)].

WO 98!24897 PCT/EP97/06723 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 nat 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.
in addition, tight junctions are particularly important in the brain microvasculature where they are responsible for a tight control of permeability between the blood and the central nervous system [Risau and Wolburg, Trends Neurosci. 13, 174-(1990)). This endothelial barrier constitutes a strong obstacle to the access of useful drugs to the brain as for instance the penetration of chemotherapeutics for the cure of cerebral tumors. Methods to open or to repair the blood brain barrier without induction of endothelial damage have not yet been described in the literature.
Finally, 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 molecular organization of tight junctions is so far only partially characterized .
Only one transmembrane protein which is specific for tight junctions has been identified and denominated occludin [Furuse et al., J. Cell Biol. 123, 1777-(1993)]. This protein connects specific cytoskeletal proteins inside the cells. The adhesive properties of occludin and its capacity to promote homotypic cell-to-cell interaction are not conclusively proven yet.

WO 98/24897 PC'F/EP97106723 Description of the invention The present invention is based on the finding of a new transmembrane protein located at tight junction. This new protein is called functional adhesion molecule S 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 1S response) control of transmigration of leukocytes, control of transmigration of other natural or engineered cells such as gene therapy in the brain.
Thus, 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 SECT ID N0:1 (human) Met Gfy Thr Lys Ala Gln Val Glu Arg Lys Leu Leu Cys Leu Phe Ile 2S Leu Ala Ile Leu Leu Cys Ser Leu Ala Leu Gly Ser Val Thr Val His Ser Ser Glu Pro Glu Val Arg Ile Pro Glu Asn Asn Pro Val Lys Leu _ Ser Cys Ala Tyr Ser Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phe Asp Gln Gly Asp Thr Thr Arg Leu VaI Cys Tyr Asn Asn Lys Ile Thr Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu Pro Thr Gly Ile Thr Phe 85 ~ 90 95 Lys Ser Val Thr Arg Glu Asp Thr Gly Thr Tyr Thr Cys Met Val Ser Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys Val Lys Leu Ile Val Leu Val Pro Pro Ser Lys Pro Thr Val Asn Ile Pro Pro Ser Lys Pro Thr Val Asn Ile Pro Ser Ser Ala Thr Thr ile Gly Asn Arg Ala Vai 145 150 . 155 160 Leu Thr Cys Ser Glu Gln Asp Gly Ser Pro P-ro Ser Glu Tyr Thr Trp Phe Lys Asp GIy Ile Val Met Pro Thr Asn Pro Lys Ser Thr Arg Cys Leu Gln Gln Leu Phe Leu Ser Ser Leu Asn Pro Thr Thr Gly Glu Leu Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly Glu Tyr Ser Cys Glu Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser Asn Arg Val Ala Met Glu Ala Val Asp Gly Asn Val Gly Val Ile Val Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gly Ile Leu Val Phe Gly Ile Trp Phe Pro Tyr Ser Arg Gly His Phe Asp Arg Thr Lys Lys Gly Thr Ser Ser Lys Lys Val Val Tyr Ser Gln Pro Ser Ala Arg Ser and homologous sequences having at least 72 % homology to the sequence SEQ
ID N0: 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. However, it is anticipated that proteins which have at least 72 %
homology to this N-terminal sequence SEQ ID NO: 1 ( some amino-acid residues at the C-terminal are missing from the full length protein) will share both diagnostic and medical properties to such a high degree that they can be used for the various appiications of the present invention. Among 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 N0:,2 (mouse) Met Gly Thr Glu ~1y Lys Ala Gly Arg Lys Leu Leu Phe Leu Phe Thr Ser Met Ile Leu Gly Ser Leu Val Gln Gly Lys Gly Ser Val Tyr Thr Ala Gln Ser Asp Val Gln Val Pro Glu Asn Glu Ser Ile Lys Leu Thr Cys Thr Tyr Ser Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phe Val Gln Gly Ser Thr Thr Ala Leu Val Cys Tyr Asn Ser Gln Ile Thr Ala 65 70 75 gp Pro Tyr Ala Asp Arg Val Thr Phe Ser Ser Ser Gly Ile Thr Phe Ser Ser Val Thr Arg Lys Asp Asn Gly Glu Tyr Thr Cys Met Val Ser Glu Glu Gly Gly Gln Asn Tyr Gly Glu Val Ser Ile His Leu Thr Val Leu Val Pro Pro Ser Lys Pro Thr !le Ser Val Pro Ser Ser Val Thr Ile Gly Asn Arg Ala Val Leu Thr Cys Ser Glu His Asp Gly Ser Pro Pro Ser Glu Tyr Ser Trp Phe Lys Asp Giy Ile Ser Met Leu Thr Ala Asp Ala Lys Lys Thr Arg Ala Phe Met Asn Ser Ser Phe Thr Ile Asp Pro 180 185 1g0 Lys Ser Gly Asp Leu Ile Phe Asp Pro Vat Thr Ala Phe Asp Ser Gly Glu Tyr Tyr Cys Gln Ala Gln Asn Gly Tyr Gly Thr Ala Met Arg Ser 210 215 220 __ WO 98/24897 PCTlEP97/06723 Glu Ala Ala His Met Asp Ala Val Glu Leu Asn Val Giy Gfy Ile Val Ala Ala Vai Leu Val Thr Leu Ile Leu Leu Gly Leu Leu Ile Phe Gly Val Trp Phe Ala Tyr Ser Arg Gly Tyr Phe Glu Thr Thr Lys Lys Gly Thr Ala Pro Gly Lys Lys Val 11e Tyr Ser Gln Pro Ser Thr Arg Ser Glu Gly Glu Phe Lys Gln Thr Ser Ser Phe Leu Val 290 295 300 .
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) ATACACTTGT

CATCGTGCTT

TAACATCCCC

TGGTTCCCGA

CCTTCTGAAT ACACCTGGTT CAAAGATGG~ ATAGTGATGC CTACGAATCC 600 CAAAAGCACC

GCTGGTCTTT

TGGGTATGGG

GGGGGTCATC

CATCTGGTTT

_ _ coding for the protein having the amino-acid sequence SEQ ID N0:1 and SEQ ID N0:4-(mouse) ACCGAGGGGA AAGCCGGGAG GAAACTGTTG T'iTCTCTTCA CGTCTATGAT CCTGGGCTCT120 BO

GGAGTGGAAG

ACCCTCTGAA

TGACCCCGTG

GACAGCCATG

CGTGGCAGC

GTCCTGGTAA CACTGATTCT CCTTGGACTC TTGATTT'TTG GCGTCTGGTT 840 TGCCTATAGC

CATTTACAGC

GGTGTGACCT

GCTGCGGCTC CTCCGTfGTC CATTTGCCTT ACTCAGGTGC TACAGGTTCC 7 CTTGCTTTfA 080 TCCCACCCTC

CCTCCTTfCC TTACCACCAT TGGGTGGCCC GAGACTAATT ACAAAGTTTT 1200 CGTTCCCCAT

GGCTGACAGG

CTAGCTCCCT

GCTfTCTCCT CCCCGGATGG GGTGCCAGCT ACTCTAGAAG GGGAGCTGCA 1374 TAAA

coding for the protein having the amino-acid sequence SEQ ID N0:2.

The 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. Further, 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 1D NO: 3) CCTGAGAATA ATCCTGTGAA GTTGTCCTGT GCCTACTCGG GCT'TTTCTTC TCCCCGTGTG 180 GCTTCCTATG AGGACCGGGT GACC'TTCTTG CCAACTGGTA TCACCTTCAA GTCCGTGACA 300 coding for the protein having the amino-acid sequence SEQ ID N0:1 and SEQ ID N0:6 _g_ (part of mouse SEQ ID N0:4) TATGATCCTG

CCGAGTGGAG

GATCACAGCT

O TGTGACCCGG

CTACGGGGAG

TGTCCCCTCC

TTCCCCACCC

CAAGAAAACC

CGGGCCTTCA TGAATTCTfC ATTCACCATT GATCCAAAGT C~GGC;GGATCT 600 GATCTTTGAC

CCCGTGACAG CCTTfGATAG TGGTGAATAC TACTGCCAGG CCCAGAATGG 660 ATATGGGACA

GGGCATCGTG

GCAGCTGTCC TGGTAACACT GATTCTCCTT GGACTCTTGA TT'TTTGGCGT 780 CTGGTTTGCC

GAAGGTCATT

GTTCCTGGTG

coding for the protein having the amino-acid sequence SEQ ID N0:2.
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]). Alternatively, 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., Cofd Spring Harbor Laboratory [1989]). Thus 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.
Preferably, 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.
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). 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 pBCl2Ml [ATCC 67109]) pSV2dhfr [ATCC 37146], pSVL [Pharmacia, Uppsala, SwedenJ, 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, H_9 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.
Usually) the prokaryotic host organisms which contain a desired expression vector are grown-under conditions which are optimal for the growth of the prokaryotic host _ 11 organisms. At the end of the exponential growth, when the increase in cell number per unit time decreases, 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 fate promoters from SV40 and the the long terminal repeats (LTRs) from retroviruses, e.g. RSV, HIV) HTLVI. However, also cellular signals can be used (e.g. human-~i-actin-promoter).
Alternatively) 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. _._ Now, the transfected gene can be amplified to_express large quantities of a foreign protein. The dihydrofolate reductase (DHFR) is a useful marker to develop fines 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 tines 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~echno-_ 12 _ 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. In the experimental part of this specification 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 11 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.
The term "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. Thus, the modifiers of the invention will either prevent or promote polymerization of JAM molecules at tight junctions, i.e., the dimerization, oiigomerization 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). For example, the modifier of the invention may have a structure which is complementary to the protein of the invention or a part of the protein.
However, in a preferred embodiment of this aspect of the invention 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 Ge 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 24fi, 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.

WO 98!24897 PCT/EP97/06723 _ - 14 -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.
_ Examples of various applications of the modifiers of the invention are - leukocyte infiltration - tumor cell metastasis angiogenesis - endothelial permeability - detection of early endothelial cell damage - adjuvants of oral vaccines) and - making gut junctions more permeable to antigens, thus indicating use as a - medicament for the therapeutic or prophylactic treatment of acute and chronic inflammatory diseases, organ transplantation, myocardial ischemia, atheroscierosis, cancer, diabetic retinopathy, psoriasis, reumathoid artritis) intestinal infection.
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) will be used for screening in vivo for the therapeutic use of modifiers of JAM polymerization in medicine.
Transgenic cells are used for in vitro testing purposes.
Having now generally described this invention, the same will become better understood by reference to the specific examples, which are included herein for purpose of illustration only and are not intended to be limiting unless otherwise specified.
Exam~nle 1 Generation of mAbs with JAM-neutralizing activity Antibodies binding specifically to JAM were produced in the laboratory by standard hybridoma techniques as described in Martin-Padura et al., J. Biol. Chem.
269,6124-6132 (19J4). Briefly, Lewis rat was immunized with a murine endothelial cell line (H5V) [Garlanda et al., Proc. Natl. Acad. Sci. USA 91,7291-7295 (1994)].
Hybridomas were produced by fusion of immunized rat splenocytes with Sp2/0 cell line from ATCC (Maryland, USA). Hybridoma supernatants were screened by standard enzyme-linked immunoassay (ELISA) for binding to HSV. Positive hybridoma were then characterized by their ability to stain endothelial cell-cell contacts by immunofluorescence microscopy which technique is disclosed below.
In order to select neutralizing mAbs, mAbs were screened on transmigration of leukocytes through the endothelial monolayers (see below). Mabs BV11 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, NuINu (CD1 ) BR mice were primed with intraperitoneaf injections of 0.5 ml of pristane 6 days before intraperitoneal injection of 1 OX106 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.
Exam lid a 2 Localization of JAM at tiiqh~junc-Immunofluorescence microscopy Cells were seeded on glass coverslips and grown to confluence in Medium 199 containing 20 % newborn calf serum before immunofluorescence staining. For some cell types, glass coversiips were coated with human plasma fibronectin (7p,g lml).

WO 98!24897 PCTlEP97/06723 _ 16 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. 118, 1511-1522 (1992). Briefly, incubation with the primary antibody (mAb BV11 or mAb BV 12) was followed by rhodamine-conjugated secondary antibody (Dakopatts) in the presence of fluorescein-labelled phalloidin (1 ~,g/ml) with several washes with 0.1 % BSA in PBS between the various steps. Coverslips were then mounted in Mowiol 4-88 (Calbiochem). A Zeiss Axiophot microscope was used for observation and image recording on Kodak TMax P3200 films.
Confocal laser-scanning immunofluoresce microscopy was done on a Zeiss LSM
410 UV (Carl Zeiss). For simultaneous double-label fluorescence, an Argon ion laser operating at 488 nm and a Helium-Neon user operating at 543 nm were used together with a band-pass filter combination of 510-525 and 590-610 for visualization of FICT and rhodamine fluorescence) respectively. RGB images were taken in high resolution mode using 1.024 X 1.024 image points (pixels) and 2s scan times.
Regularly noise levels were reduced by several line averagings of the scans.
Projection images were created from 0.8 ~.m optical sections of tissue preparation or cell layers. To verify the distribution of JAM and cingulin, the individual images were separated electronically by a 20 pixel off-set along the abscissa.
JAM distributes selectively at cell-cell contacts in endothelial and epithelial cells.
Distribution corresponds to other molecules located at tight junctions such as 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.
Exam! la a 3 JAM inhibits leukocyrte transmigration A. in vitro assay in transwell units 2x105 endothelial cells were seeded on human plasma fibronectin (7p,g/ml, Sigma) precoated polycarbonate membrane of Transwell units (24 mm diameter; 8.0 p,m pore size) and cultured for 4 to 5 days to confluency. Cultured medium from both upper and lower chambers were replaced with fresh medium and in some experiments mAbs were added to endothelial cell monolayers for 30 min and then allowed to stay during the transmigration assay. Monocytes were obtained from the _ 17 _ peripheral blood of normal healthy donors as described by Colotta et al., J.
Immunol. 132, 936 (1984). Briefly, 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 7 0~ cells/ml in complete medium and labeled by incubation with 100 mCi 5' Cr for 1 h at room temperature. Cells were then washed extensively and resuspended at 1.8 x 106 cellslml in complete medium. An aliquot (1.5 ml) of radiolabelled cells was then added to each well and incubated for 60 min at 37°C. Non-adherent cells were then removed by washing gently with PBS plus 2% FCS from the upper chamber (non-adherent fraction). Transmigrated cells were collected from the lower chamber medium and removed by scraping with cotton - buds on the opposite face of the filter. These two fractions were pooled (migrated fraction). The intact EC monolayer together with the adherent leukocytes were collected by cutting the polycarbonate membrane (adherent fraction).
Radioactivity of the three fractions was measured in a counter (Beckman).
The results are presented in Tables 1, 2 and 3 below.
Table 1. Spontaneous monocyte transmigration in vitro through an endothelial monolayer in the presence of different monoclonal antibodies.
Treatment cell number Control 437713 14351 mAb BV11_ ~ 190346 23926**

Irrelevant IgG2b 403027 16214 mAb BV12 381903 ~ 9213 mAb to CD31 - 415150 ~ 12213 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) _ 18 _ 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.
Table 2. Effect of mAb BV11 on MCP-1-induced monocyte transmigration through an endothelial monolayer.
Treatment none MCP-1 Control 323000 ~ 38400 825767 ~ 14351 mAb BV11 172833 ~ 38933** 488967 ~ 13926**
Irrelevant IgG2b 366100 ~ 38400 852250 ~ 16214 MCP-1 at 100 nglml 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 BV11: monoclonal antibody neutralizing JAM .
Table 3. Effect of mAb BV11 on polymorphonuclear cell (PMN) migration through an endothelial monolayer.
Treatment none fMLP (10 nM) Control 25566 ~ 3486 69400 ~ 2275 mAb BV 11 24852 t 3854 24852 ~ 1916**
Irrelevant IgG2b 26890 ~ 3890 68890 ~ 1934 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<

WO 98!24897 PGT/EP97/06723 _ 19 _ 0.01 by analysis of various and Duncan's test. mAb BV11: monoclonal antibody neutralizing JAM.
. In conclusion, as reported in Tables 1, 2 and 3, addition of monoclonal antibody BV11 specifically binding to JAM resulted in the inhibition of both monocyte and polymorphonuclear cell transmigration. The antibody did not significantly alter the number of cells which remained adherent to the filter.
B, in vfvo assay: measurement of leukocyte recruitment in the air pouch model 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 p.g of monoclonal antibody BV11 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 p.l were used for cell count after staining with erythrosin.
The results are presented in Table 4 below.
Table 4. Effect of mAb BV11 on neutrophii recruitment in vivo Treatment cell number (x 106) Control 5.769 ~ 0.932 mAb BV11 3.629 + 0.217*
Leukocyte recruitment was induced by injection of carrageenan in sterile saline (1 ml) into six day-old pouches. 200 p.g of purified mAb BV11 (mAb binding to JAM) or rat non-immune-IgG (control) were injected intravenously in 200 p.l 12 hours before carrageenan treatment. Animals were killed 48 hours after the treatment. Data are mean ~ SD of at feast seven animals in two experiments. * p< 0.002 according to Students 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.
Table 5. Effect of mAb BV11 on paracellular permeability in vivo Treatment Control mAb BV11 Exudate (ml) 1.49 ~ 0.125 1.28 ~ 0.217*
Measurement of permeability in the air pouch model was evaluated as collected 15- exudate volume. Plasma exudation was induced by carrageenan in sterile saline (1 ml) in six day-old pouches. 200 p.g of purified mAb BV11 (mAb binding to JAM) or rat non-immune-IgG (control) were injected intravenously in 200 p.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.
Example 4 Inhibition of oaracellular permeability byr JAM
A. Constructs and Transfection Constructs preparation and transfection procedures were performed according to Breviario et al., Arterioscler. Throm. Vasc. Biol. 15, 1229-1239 (1995). JAM
cDNA was isolated through expression cloning. The cDNA expression library was constructed from a marine brain EC line (bEnd.3) as previously described (Fawcett et ai., 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 pCDM8 (Nature 329, 840-842 [1987]). Plasmid pCDMB was cut with Hindlll and Notl enzymes and the insert was blunted and subcloned into the Smal restriction site of pECE
eucaryotic expression vector (Ratter et al., Cell 45, 721-732 (1986)) to give the pECE-JAM

_ - 21 -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 ai.
(Eds) "Current Protocols in Molecular Biology) "Green Publishing Associates /
Wiley-Interscience, New York {1990)). CHO cells were plated at 3-4x106cells per 100 mm petri dish in DMEM with 10% FCS. 24 h after seeding cells were transfected by calcium phosphate precipitation method with 20 p.g of pECE-JAM and 2 p.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. Then cells were IO detached and plated at 1 x106 per 100 mm petri dish and cultured in selective medium with 600 ~g/ml 6418 (Geneticin, GIBCO). Resistant colonies were isolated and tested for BV11 antigen expression by immunofluorescence staining and immunoprecipitation analysis. Positive cells were cloned by limiting dilution and expanded for further studies.
IS
B. Measurement of dextran passage in Transwell units Procedure to measuring permeability across the cell monolayer in Transwell units is extensively described in Breviario et al., supra. Briefly) JAM transfectant or endothe-lial cells were seeded at 1.5x104 per 6.,5 mm in Transwell units (pofycarbonate 20 filter, 0.4 mm pore, Costar) and cultured to confluency for 5 days. Then, culture medium was replaced with serum-free medium and fiuorescein isothiocyanate-dextran (1 mg/ml) Sigma) was added to the upper chamber. At different times, ~I from the tower compartment were withdrawn and assayed by fluorimeter (excitation wavelength set at 492 nm and emission at 520 nm).
The results are presented in Table 6.
Tabte 6. Effect of JAM transfection on paracellular permeability.
Transfectant cells % Permeability Control 100.0 t 2.5 JAM 46.5 t 2.0 JAM + EGTA 5mM 110.0 t 4.4 JAM + Cyt D 91.0 ~ 6.5 WO 98124897 PC3'1EP97106723 Values are means ~ SEM of three independent experiments) ** p< 0.01 by analysis of various and Duncan's test.
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. -In conclusion, as reported in Table 6, JAM transfection significantly reduced the passage of dextran through intercellular junctions. As reference the Table also 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.
is Exam I
~Jse of mAb BV11 to detect JAM in ELISA assay ELISA sandwich An ELISA standard protocol was followed [Peri et al., J. Inimunol. Meth. 174, 257, (1994)j. Briefly, 96 well ELISA plates (Falcon) were coated with 50 p.Uwell of rabbit anti-JAM serum diluted 1 /3000 in 15 mM carbonate buffer, pH 9.6 and incubated overnight at 4°C. After incubation, plates were washed three times with PBS + 0.05% Tween 20 (washing buffer). Non-specific binding was blocked with 5%
dry milk in washing buffer for 2h at room temperature . After wash, JAM-containing samples were added for 2h at 37°C. Then, plates were washed and incubated with mAb BV11 for 1 h at 37°C. Peroxidase conjugated anti-rat IgG (diluted-~:2000, Sigma) was incubated for 1 h at RT and then 100 p.l chromogen substrate was added.
Adsorbance values were read at 405 nm.
The results are presented in Table 7.

Table 7. Detection of JAM (O.D.) on different cells in ELISA
Treatment endothelia! cells 3T3 fibroblasts Mel(hemopoietic line) Control 150 10 136 21 166 14 mAbBV 11 9207"* 14216 16919 non-immune serum 167 5 148 9 179 9 Control: only secondary antibody was added. Rabbit anti-JAM serum or non-immune rabbit serum were used at dilution 1 /3000. Adsorbance values are means ~ SEM of four replicates/ '* p< 0.01 by analysis of various and Duncan's test.
In conclusion, in ELISA 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.
Exam) I~ a 6 E~r~ression of soluble JAM
An extracellular JAM fragment was cloned by PCR [Saiki et al., Science 239, (1988)j from full length JAM cDNA introducing a Kpn1 restriction site upstream the start codon and a stop codon at position 775 just in front a Hindlll restriction site. The amplified DNA was cut by the restriction enzymes Kpn1 and Hindtll and the resulting DNA was ligated into the cloningsite of the baculovirus expression vector pFASTBAC1 (Gibco BRL}. The vector was transformed into DHlOBac cells (Gibco BRL), the transformants were plated and stained with x-Gal according to the recommendation of the manufacturer. After selection of a recombinant vector) the DNA was transfeeted into Sf9 cells. Soluble JAM was detected in the cell culture medium seven days after transfection with the vector or three days after infection with the recombinant baculovirus.
Purified soluble JAM aggregates by itself depending on the solvent conditions.
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 BV11 and mab BV12). Briefly, lmmunoplates (Nunc Maxisorb) are coated overnight with 100 p.l/well of a solution of mab BV12 (10 p.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 ~I/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. 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.

WO 98124897 PCTlEP97106723 _ 2g _ SEQUENCE LISTING
( 1 ) GENERAL INFORMATION:

(i) APPLICANT:
-(A) NAME: F.Hoffmann-La Roche AG

(B) STREET: Grenzacherstrasse 124 (C) CITY: Basle (D) STATE: BS

(E) COUNTRY: Switzerland (F) POSTAL CODE (ZIP): CH-4070 (G) TELEPHONE: 061 - 688 42 56 (H) TELEFAX: 061 - 688 13 95 (I) TELEX: 962292 l 965542 hlr ch (ii) TITLE OF INVENTION: Transmembrane component of tight junction (iii) NUMBER OF SEQUENCES: 6 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: System 7.1 (Macintosh) (D) SOFTWARE: Word 5.0 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: SE 960 4470-6 (B) FILING DATE: 04-DEC-1996 (2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 298 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: both (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met Gly Thr Lys Ala Gln Val Glu Arg Lys Leu Leu Cys Leu Phe Ile Leu Ala Ile Leu Leu Cys Ser Leu Ala Leu Gly Ser VaI Thr Val His Ser Ser Glu Pro Glu Val Arg Ile Pro Glu Asn Asn Pro Val Lys Leu Ser SyOs Ala Tyr Ser Gly S~e Ser Ser Pro Arg 6~ Glu Trp Lys Phe Asp Gln Gly Asp Thr Thr Arg Leu Val Cys Tyr Asn Asn Lys Ile Thr Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu Pro Thr Gly lle Thr Phe Lys Ser Val Thr Arg Glu Asp Thr Gly Thr Tyr Thr Cys Met Val Ser Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys Val Lys Leu Ile Val Leu Val Pro Pro Ser Lys Pro Thr~al Asn Ile Pro Pro Ser Lys Pro Thr Val Asn Ile Pro Ser Ser Ala Thr Thr Ile Gly Asn Arg Ala Val Leu Thr Cys Ser Glu Gln Asp Gly Ser Pro Pro Ser Glu Tyr Thr Trp Phe Lys Asp Gly Ile Val Met Pro Thr Asn Pro Lys Ser Thr Arg Cys Leu Gln GIn Leu Phe Leu Ser Ser Leu Asn Pro Thr Thr Gly Glu Leu Val Phe Asp Pro Leu Ser AIa Ser Asp Thr Gly GIu Tyr Ser Cys Glu Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser Asn Arg Val Ala Met Glu Ala Val Asp Gly Asn Val Gly Val Ile Val AIa Ala Val Leu Val Thr Leu lle Leu Leu Gly Ile Leu Val Phe Gly Ile Trp Phe Pro Tyr Ser Arg Gly His Phe Asp Arg Thr Lys Lys Gly Thr Ser Ser Lys Lys Val Val Tyr Ser Gln Pro Ser Ala Arg Ser (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 amino acids WO 98124897 PCT/EP97/Ob723 (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: both (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal (ix) FEA1'LJRE:
(A) NAME/IG;Y: Disulfide-bond (B) LOCATION:49..108 (ix) FEAT~JRE:
(A) NAME/IGrY: Disulfide-bond (B) LOCATION:152..212 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Gly Thr Glu ~ ly Lys Ala Gly Arg Lys Leu Leu Phe Leu Phe Thr Ser Met De Leu Gly Ser Leu Val Gln Gly Lys Gly Ser Val Tyr Thr Ala Gln Ser Asp Val Gln Val Pro Glu Asn Glu Ser Ile Lys Leu Thr Cys Thr Tyr Ser Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phc Val 50 s5 60 Gln Gly Ser Thr Thr Ala Leu Val Cys Tyr Asn Ser Gln Ile Thr Ala 65 70 7s 80 Pro Tyr Ala Asp Arg Val Thr Phe Ser Ser Ser Gly Ile Thr Phe Ser 8s 90 9s Ser Val Thr «g Lys Asp Asn Gly Glu Tyr Thr Cys Met Val Ser Glu ios llo Glu Gly Gly Gln Asn Tyr Gly GIu Val Ser Ile His Leu Thr Val Leu Val Pro Pro Ser Lys Pro Thr Ile Ser Val Pro Ser Ser Val Thr Ile Gly Asn Arg Ala Val Leu Thr Cys Ser Glu His Asp Gly Ser Pro Pro s0 145 150 155 16p Ser Glu Tyr Ser Trp Phe Lys Asp Gly De Ser Met Leu Thr Ala Asp 16s 1~0 l~s Ala Lys Lys Thr Arg Ala Phe Met Asn Ser Ser Phe Thr Ile Asp Pro Lys Ser Gly Asp Leu Ile Phe Asp Pro Val Thr Ala Phe Asp Ser Gly Glu Tyr Tyr Cys Gln Ala Gln Asn Gly Tyr Gly Thr Ala Met Arg Ser Glu Ala Ala His Met Asp Ala Val Glu Leu Asn Val Giy Gly Ile Val Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gly Leu Leu Ile Phe Gly Val Trp Phe Ala Tyr Ser Arg Gly Tyr Phe Glu Thr Thr Lys Lys Gly Thr Ala Pro Gly Lys Lys Val Ile Tyr Ser Gln Pro Ser Thr Arg Ser Glu Gly Glu Phe Lys Gln Thr Ser Ser Phe Leu Val (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 924 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC~JLE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

TGTGCCTACT CGGGCTTTI'C TTCTCCCCGT GTGGAGTGGA AGTTTGACCA AGGAGACACC240 (2) INFORMATION FOR SEQ ID NO: 4:
_. (i) SEQUENCE CI-IARACTERISTICS:
(A) LENGTH: 1374 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

w ACAGCCTTTG ATAGTGGTGA ATACTACTGC CAGGCCCAGA ATGGATATGG GACAGCCATG 720 S

GTCCTGGTAA CACTGATTCT CCTTGGACTC TTGATiTITG GCGTCTGGTT TGCCTATAGC 840 CCTCCTl'TCC TTACCACCAT TGGGTGGCCC GAGACTAATT ACAAAGTTTT CGTTCCCCAT 1200 (2) INFORMATION FOR SEQ 1D NO: S:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTIi: 891 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC'LTLE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO ---(xi) SEQUENCE DESCRIPTTON: SEQ D7 NO: 5:

_ _ AATCCCACAA CAGGAGAGCT GGTCTTTGAT CCCCTGTCAG CCTCTGATAC TGGAGAA,TAC 660 (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 900 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTTON: SEQ 117 NO: 6:

GGCTC~T~GG TACAAGGCAA GGGTTCGGTG TACACTGCTC AATCTGACGT CCAGGTTCCC 120 CCCGTGACAG CC'ITTGATAG TGGTGAATAC TACTGCCAGG CCCAGAATGG ATATGGGACA 660 - _ -. _ 32 -GCAGCTGTCC TGGTAACACT GATTCTCCTT GGACTCTTGA TZTI'TGGCGT CTGGTTTGCC 780

Claims (20)

1. A protein in glycosylated or unglycosylated form which is a junctional adhesion molecule (JAM) and comprises the amino acid of SEQ ID NO:1 or a homologous sequence having at least 72% homology to SEQ ID NO:1,

2. The protein according to claim 1 having the sequence of SEQ ID NO:2.

3. A DNA sequence coding for a protein as claimed in claim 1 or in claim 2.

4. The DNA sequence of claim 3 selected from the group consisting of SEQ ID
NO:3 and SEQ ID NO:4.

5. A gene comprising a DNA sequence as claimed in anyone of claims 3 or 4, preferably a gene selected from the group consisting of SEQ ID NO:5 and SEQ
ID NO:6.

6. The DNA of claim 4, which has oncosuppressor activity by transfection in carcinoma cells.

7. A fragment of the DNA as claimed in claims 3 or 4 encoding an extracellular JAM
fragment.

8. A vector comprising a DNA sequence as claimed in claim 3 or 4 or a gene as claimed in claim 5 or a fragment as claimed in claim 8.

9. A non-human organism transformed with a vector as claimed in claim 8.

10. A process for the preparation of a polypeptide which is a modifier of the dimerization, oligomerization or polymerization of a transmembrane protein of tight junctions, which process comprises:
a) culturing an organism comprising a DNA sequence as claimed in claims 3 or 4 or a gene as claimed in claim 5 or a DNA fragment as claimed in claim 7; and optionally b) isolating the expression product from the organism or from the medium.

11. A polypeptide obtainable by a process as claimed in claim 10.

12. An antibody which specifically binds to the protein as claimed in claims 1 or 2.

13. The antibody of claim 12 which is a modifier ef the dimerization, oligomerization or polymerization of a transmembrane protein of tight junctions.

14, A modifier of the dimerization, oligomerization or polymerization of a transmembrane protein of tight junctions which is a polymerization inhibiting or polymerization inducing protein, a peptide, a peptidomimetic or an organic molecule-ligand which is derived from the amino acid sequence as defined in claims 1 or 2 and which modifier is capable of either inhibiting or inducing the polymerization of the transmembrane protein of tight junctions.

15. A diagnostic kit comprising as a diagnostic reagent an antibody as claimed in claim 13 or a modifier as claimed in claim 14.

16. A vaccine adjuvant comprising an antibody as claimed in claim 13 or a modifier as claimed in claim 14.

17. A medicament comprising as an active ingredient an antibody as claimed in claim 13 or a modifier as claimed in claim 14.

18. The use of a junctional adhesion molecule as claimed in claims 1 or 2 for identifying and isolating modifiers for its dimerization, oligomerization or polymerization.

19. The use of an antibody as claimed in claim 13 or a modifier as claimed in claim 14 for the preparation of a medicament.

20. The invention substantially as hereinbefore described, especially with reference to the examples.