AU5657898A - Junctional adhesion molecule (jam), a transmembrane protein of tight junctions - Google Patents

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 junctional 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 antibody specifically binding to JAM and preventing the polymerization of JAM at tight junctions, resulting inter alia in the blocking 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 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. Opiπ. 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.

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, 174-178 (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, 1777-1788 (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. 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 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.

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 SEQ ID NO:1

(human)

Met Gly Thr Lys Ala Gin Val Glu Arg Lys Leu Leu Cys Leu Phe lie 1 5 10 15 Leu Ala He Leu Leu Cys Ser Leu Ala Leu Gly Ser Val Thr Val His

20 25 30

Ser Ser Glu Pro Glu Val Arg lie Pro Glu Asn Asn Pro Val Lys Leu 35 40 45

Ser Cys Ala Tyr Ser Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phe 50 55 60

Asp Gin Gly Asp Thr Thr Arg Leu Val Cys Tyr Asn Asn Lys He Thr 65 70 75 80

Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu Pro Thr Gly lie Thr Phe 85 90 95 Lys Ser Val Thr Arg Glu Asp Thr Gly Thr Tyr Thr Cys Met Val Ser

100 105 110 Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys Val Lys Leu He Val 115 120 125

Leu Val Pro Pro Ser Lys Pro Thr Val Asn lie Pro Pro Ser Lys Pro 130 135 140

Thr Val Asn He Pro Ser Ser Ala Thr Thr He Gly Asn Arg Ala Val 145 150 155 160 Leu Thr Cys Ser Glu Gin Asp Gly Ser Pro Pro Ser Glu Tyr Thr Trp

165 170 175

Phe Lys Asp Gly lie Val Met Pro Thr Asn Pro Lys Ser Thr Arg Cys 180 185 190

Leu Gin Gin Leu Phe Leu Ser Ser Leu Asn Pro Thr Thr Gly Glu Leu 195 200 205

Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly Glu Tyr Ser Cys Glu 210 215 220

Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser Asn Arg Val Ala Met 225 230 235 240 Glu Ala Val Asp Gly Asn Val Gly Val He Val Ala Ala Val Leu Val

245 250 255

Thr Leu He Leu Leu Gly He Leu Val Phe Gly lie Trp Phe Pro Tyr 260 265 270

Ser Arg Gly His Phe Asp Arg Thr Lys Lys Gly Thr Ser Ser Lys Lys 275 280 285

Val Val Tyr Ser Gin Pro Ser Ala Arg Ser 290 295

and 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. 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 applications 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 NO: 2 (mouse)

Met Gly Thr Glu Gly Lys Ala Gly Arg Lys Leu Leu Phe Leu Phe Thr 1 5 10 15

Ser Met He Leu Gly Ser Leu Val Gin Gly Lys Gly Ser Val Tyr Thr 20 25 30

Ala Gin Ser Asp Val Gin Val Pro Glu Asn Glu Ser He Lys Leu Thr 35 40 45 Cys Thr Tyr Ser Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phe Val

50 55 60

Gin Gly Ser Thr Thr Ala Leu Val Cys Tyr Asn Ser Gin He Thr Ala 65 70 75 80

Pro Tyr Ala Asp Arg Val Thr Phe Ser Ser Ser Gly He Thr Phe Ser 85 90 95

Ser Val Thr Arg Lys Asp Asn Gly Glu Tyr Thr Cys Met Val Ser Glu 100 105 110

Glu Gly Gly Gin Asn Tyr Gly Glu Val Ser He His Leu Thr Val Leu 115 120 125 Val Pro Pro Ser Lys Pro Thr He Ser Val Pro Ser Ser Val Thr He

130 135 140

Gly Asn Arg Ala Val Leu Thr Cys Ser Glu His Asp Gly Ser Pro Pro 145 150 155 160

Ser Glu Tyr Ser Trp Phe Lys Asp Gly He Ser Met Leu Thr Ala Asp 165 170 175

Ala Lys Lys Thr Arg Ala Phe Met Asn Ser Ser Phe Thr He Asp Pro 180 185 190

Lys Ser Gly Asp Leu He Phe Asp Pro Val Thr Ala Phe Asp Ser Gly 195 200 205 Glu Tyr Tyr Cys Gin Ala Gin Asn Gly Tyr Gly Thr Ala Met Arg Ser

210 215 220 Glu Ala Ala His Met Asp Ala Val Glu Leu Asn Val Gly Gly He Val 225 230 235 240

Ala Ala Val Leu Val Thr Leu He Leu Leu Gly Leu Leu He Phe Gly 245 250 255

Val Trp Phe Ala Tyr Ser Arg Gly Tyr Phe Glu Thr Thr Lys Lys Gly 260 265 270 Thr Ala Pro Gly Lys Lys Val He Tyr Ser Gin Pro Ser Thr Arg Ser

275 280 285

Glu Gly Glu Phe Lys Gin 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)

TCCATTGTGC TCTAAAGCGG GACGCTGATC GCGATGGGGA CAAAGGCGCA AGTCGAGAGG 60

AAACTGTTGT GCCTCTTCAT ATTGGCGATC CTGTTGTGCT CCCTGGCATT GGGCAGTGTT 120

ACAGTGCACT CTTCTGAACC TGAAGTCAGA ATTCCTGAGA ATAATCCTGT GAAGTTGTCC 180

TGTGCCTACT CGGGCTTTTC TTCTCCCCGT GTGGAGTGGA AGTTTGACCA AGGAGACACC 240

ACCAGACTCG TTTGCTATAA TAACAAGATC ACAGCTTCCT ATGAGGACCG GGTGACCTTC 300 TTGCCAACTG GTATCACCTT CAAGTCCGTG ACACGGGAAG ACACTGGGAC ATACACTTGT 360

ATGGTCTCTG AGGAAGGCGG CAACAGCTAT GGGGAGGTCA AGGTCAAGCT CATCGTGCTT 420

GTGCCTCCAT CCAAGCCTAC AGTTAACATC CCTCCATCCA AGCCTACAGT TAACATCCCC 480

TCCTCTGCCA CCATTGGGAA CCGGGCAGTG CTGACATGCT CAGAACAAGA TGGTTCCCCA 540

CCTTCTGAAT ACACCTGGTT CAAAGATGGG ATAGTGATGC CTACGAATCC CAAAAGCACC 600 ∞TTGCCTTCAGCMCTCTT CCTATCTAGT CTGAATCCCA CAACAGGAGA GCTGGTCTTT 660

GATCCCCTGT CAGCCTCTGA TACTGGAGAA TACAGCTGTG AGGCACGGAA TGGGTATGGG 720

ACACCCATGA CTTCAAATCG TGTCGCGATG GAAGCTGTGG ACGGGAATGT GGGGGTCATC 780

GTGGCAGCCG TCCTTGTAAC CCTGATTCTC CTGGGAATCT TGGTTTTTGG CATCTGGTTT 840 CCGTATAGCC GAGGCCACTT TGACAGAACA AAGAAAGGGA CTTCGAGTAA GAAGGTAGTT 900

TACAGCCAGC CTAGTGCCCG AAGT 924 coding for the protein having the amino-acid sequence SEQ ID NO:1

and SEQ ID NO:4 (mouse)

ATACCATTGT GCTGGAAAGG TTGCTGTGCC CGTCGCGTCG GGATTGTAAC TGTAATGGGC 60

ACCGAGGGGA AAGCCGGGAG GAAACTGTTG TTTCTCTTCA CGTCTATGAT CCTGGGCTCT 120

TTGGTACAAG GCAAGGGTTC GGTGTACACT GCTCAATCTG ACGTCCAGGT TCCCGAGAAC 180 GAGTCCATCAAATTGACCTG CACCTACTCT GGCTTCTCCTCTCCCCGAGTGGAGTGGAAG 240

TTCGTCCAAG GCAGCACAAC TGCACTTGTG TGTTATAACA GCCAGATCAC AGCTCCCTAT 300

GCGGACCGAG TCACCTTCTC ATCCAGTGGC ATCACGTTCA GTTCTGTGAC CCGGAAGGAC 360

AATGGAGAGT ATACTTGCAT GGTCTCCGAG GAAGGTGGCC AGAACTACGG GGAGGTCAGC 420

ATCCACCTCA CTGTGCTTGT ACCTCCATCC AAGCCGACGA TCAGTGTCCC CTCCTCTGTC 480 ACCATTGGGA ACAGGGCAGT GCTGACCTGC TCAGAGCATG ATGGTTCCCC ACCCTCTGAA 540

TATTCCTGGT TCAAGGACGG GATATCCATG CTTACAGCAG ATGCCAAGAA AACCCGGGCC 600

TTCATGAATT CTTCATTCAC CATT^pGATCCA AAGTCGGGGG ATCTGATCTT TGACCCCGTG 660

ACAGCCTTTG ATAGTGGTGA ATACTACTGC CAGGCCCAGA ATGGATATGG GACAGCCATG 720

AGGTCAGAGG CTGCACACAT GGATGCTGTG GAGCTGAATG TGGGGGGCAT CGTGGCAGC 780 GTCCTGGTAA CACTGATTCT CCTTGGACTC TTGATTTTTG GCGTCTGGTT TGCCTATAGC 840

CGTGGATACT TTGAAACAAC AAAGAAAGGG ACTGCACCGG GTAAGAAGGT CATTTACAGC 900

CAGCCCAGTA CTCGAAGTGA GGGGGAATTC AAACAGACCT CGTCGTTCCT GGTGTGACCT 960

GCTGCGGCTC CTCCGTTGTC CATTTGCCTT ACTCAGGTGC TACAGGTTCC AGCCCCTGCT 1020

GCTGTAGCTG CACAGGATGC CTTCAATGTC TTCTAGGTCC CACAGGACCC CTTGCTTTTA 1080 TTCTAGCTAG GATATAAATT TAAAAACATC ATCTACTTCC CCCTCCTCTT TCCCACCCTC 1140

CCTCCTTTCC TTACCACCAT TGGGTGGCCC GAGACTAATT ACAAAGTTTT CGTTCCCCAT 1200

TCCTATGTGG GATTGGGCAA GAGTCCTAGA CTAGACAGTA ATAGTGGCTG GGCTGACAGG 1260

AACCCAAACC AATACCTGGC TGTAAAGGCC TCTGAATAAG GACTTTAAGC CTAGCTCCCT 1320

GCTTTCTCCT CCCCGGATGG GGTGCCAGCT ACTCTAGAAG GGGAGCTGCA TAAA 1374

coding for the protein having the amino-acid sequence SEQ ID NO: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 ID NO: 3)

ATGGGGACAA AGGCGCAAGT CGAGAGGAAA CTGTTGTGCC TCTTCATATT GGCGATCCTG 60 TTGTGCTCCC TGGCATTGGG CAGTGTTACA GTGCACTCTT CTGAACCTGA AGTCAGAATT 120

CCTGAGAATA ATCCTGTGAA GTTGTCCTGT GCCTACTCGG GCTTTTCTTC TCCCCGTGTG 180

GAGTGGAAGT TTGACCAAGG AGACACCACC AGACTCGTTT GCTATAATAA CAAGATCACA 240

GCTTCCTATG AGGACCGGGT GACCTTCTTG CCAACTGGTA TCACCTTCAA GTCCGTGACA 300

CGGGAAGACA CTGGGACATA CACTTGTATG GTCTCTGAGG AAGGCGGCAA CAGCTATGGG 360 GAGGTCAAGG TCAAGCTCATCGTGCTTGTG CCTCCATCCA AGCCTACAGTTAACATCCCT 420

CCATCCAAGC CTACAGTTAA CATCCCCTCC TCTGCCACCA TTGGGAACCG GGCAGTGCTG 480

ACATGCTCAG AACAAGATGG TTCCCCACCT TCTGAATACA CCTGGTTCAA AGATGGGATA 540

GTGATGCCTA CGAATCCCAA AAGCACCCGT TGCCTTCAGC AACTCTTCCT ATCTAGTCTG 600

AATCCCACAA CAGGAGAGCT GGTCTTTGAT CCCCTGTCAG CCTCTGATAC TGGAGAATAC 660 AGCTGTGAGG CACGGAATGG GTATGGGACA CCCATGACTT CAAATCGTGT CGCGATGGAA 720

GCTGTGGACG GGAATGTGGG GGTCATCGTG GCAGCCGTCC TTGTAACCCT GATTCTCCTG 780

GGAATCTTGG I 1 1 I IGGCAT CTGGTTTCCG TATAGCCGAG GCCACTTTGA CAGAACAAAG 840

AAAGGGACTT CGAGTAAGAA GGTAGTTTAC AGCCAGCCTA GTGCCCGAAG T 891

coding for the protein having the amino-acid sequence SEQ ID NO:1 and SEQ ID NO:6 (part of mouse SEQ ID NO:4)

ATGGGCACCG AGGGGAAAGC CGGGAGGAAA CTGTTGTTTC TCTTCACGTC TATGATCCTG 60 GGCTCTTTGG TACAAGGCAA GGGTTCGGTG TACACTGCTC AATCTGACGT CCAGGTTCCC 120

GAGAACGAGT CCATCAAATT GACCTGCACC TACTCTGGCT TCTCCTCTCC CCGAGTGGAG 180

TGGAAGTTCG TCCAAGGCAG CACAACTGCA CTTGTGTGTT ATAACAGCCA GATCACAGCT 240 CCCTATGCGG ACCGAGTCAC CTTCTCATCC AGTGGCATCA CGTTCAGTTC TGTGACCCGG 300

AAGGACAATG GAGAGTATAC TTGCATGGTC TCCGAGGAAG GTGGCCAGAA CTACGGGGAG 360

GTCAGCATCC ACCTCACTGT GCTTGTACCT CCATCCAAGC CGACGATCAG TGTCCCCTCC 420

TCTGTCACCA TTGGGAACAG GGCAGTGCTG ACCTGCTCAG AGCATGATGG TTCCCCACCC 480

TCTGAATATT CCTGGTTCAA GGACGGGATA TCCATGCTTA CAGCAGATGC CAAGAAAACC 540 CX3GG∞TTCATGMTTCTTC ATTCACCATTGAT(XAMGTCGGGGGATCTGATCTTTGAC 600

CCCGTGACAG CCTTTGATAG TGGTGAATAC TACTGCCAGG CCCAGAATGG ATATGGGACA 660

GCCATGAGGT CAGAGGCTGC ACACATGGAT GCTGTGGAGC TGAATGTGGG GGGCATCGTG 720

GCAGCTGTCC TGGTAACACT GATTCTCCTT GGACTCTTGA TTTTTGGCGT CTGGTTTGCC 780

TATAGCCGTG GATACTTTGA AACAACAAAG AAAGGGACTG CACCGGGTAA GAAGGTCATT 840 TACAGCCAGC CCAGTACTCG AAGTGAGGGG GAATTCAAAC AGACCTCGTC GTTCCTGGTG 900

coding for the protein having the amino-acid sequence SEQ ID NO: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., Cold 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 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.

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 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 late 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-β- 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 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. 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 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.

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, 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). 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 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.

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, atherosclerosis, 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.

Example 1

Generation of mAbs with JAM-neutralizinα 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 (1994). 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 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.

In order to select neutralizing mAbs, 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 10X10⁶ 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.

Example 2

Localization of JAM at tight junctions

Immunofluorescence microscopy

Ceils 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 coverslips were coated with human plasma fibronectin (7μg /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). Briefly, incubation with the primary antibody (mAb BV1 1 or mAb BV 12) was followed by rhodamine-conjugated secondary antibody (Dakopatts) in the presence of fluorescein-labelled phalloidin (1μg/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 laser 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 μm 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 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.

Example 3

JAM inhibits leukocyte transmigration

A. in vitro assay in transwell units

2x10⁵ endothelial cells were seeded on human plasma fibronectin (7μg/ml, Sigma) precoated polycarbonate membrane of Transwell units (24 mm diameter; 8.0 μm 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 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 10⁷ cells/ml in complete medium and labeled by incubation with 100 mCi ⁵¹ Cr for 1 h at room temperature. Cells were then washed extensively and resuspended at 1.8 x 10⁶ cells/ml in complete medium. An aliquot (1.5 ml) of radiolabelied 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 BV1 1 190346 ± 23926^**

Irrelevant lgG2b 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) 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 BV1 1 on MCP-1 -induced monocyte transmigration through an endothelial monolayer.

Treatment none MCP-1

Control 323000 ± 38400 825767 ± 14351 mAb BV1 1 172833 ± 38933^** 488967 ± 13926^*

Irrelevant lgG2b 366100 ± 38400 852250 ± 16214

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 .

Table 3. Effect of mAb BV1 1 on polymorphonuclear cell (PMN) migration through an endothelial monolayer.

Treatment none fMLP (10 nM)

Control 25566 ± 3486 69400 ± 2275 mAb BV11 24852 ± 3854 24852 ± 1916^** Irrelevant lgG2b 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< 0.01 by analysis of various and Duncan's test. mAb BV1 1 : monoclonal antibody neutralizing JAM.

In conclusion, as reported in Tables 1 , 2 and 3, addition of monoclonal antibody BV1 1 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 vivo 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 μg 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 μl were used for cell count after staining with erythrosin.

The results are presented in Table 4 below.

Table 4. Effect of mAb BV1 1 on neutrophil recruitment in vivo

Treatment cell number (x 10⁶)

Control 5.769 ± 0.932 mAb BV1 1 3.629 ± 0.217^*

Leukocyte recruitment was induced by injection of carrageenan in sterile saline (1 ml) into six day-old pouches. 200 μg of purified mAb BV1 1 (mAb binding to JAM) or rat non-immune-lgG (control) were injected intravenously in 200 μl 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.

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 exudate volume. Plasma exudation was induced by carrageenan in sterile saline (1 ml) in six day-old pouches. 200 μg of purified mAb BV1 1 (mAb binding to JAM) or rat non-immune-lgG (control) were injected intravenously in 200 μl 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 paracellular permeability bv JAM

A. Constructs and Transfection

Constructs preparation and transfection procedures were performed according to Breviario et al., Arterioscler. Throm. Vase. Biol. 15, 1229-1239 (1995). 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 pCDMδ (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. (Eds) "Current Protocols in Molecular Biology, "Green Publishing Associates / W ley- 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 μg of pECE-JAM and 2 μg 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 detached and plated at 1x10⁶ per 100 mm petri dish and cultured in selective medium with 600 μg/ml G418 (Geneticin, GIBCO). Resistant colonies were isolated and tested for BV1 1 antigen expression by immunofluorescence staining and immunoprecipitation analysis. Positive cells were cloned by limiting dilution and expanded for further studies.

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 endothelial cells were seeded at 1.5x10⁴ per 6. ,5 mm in Transwell units (polycarbonate filter, 0.4 mm pore, Costar) and cultured to confluency for 5 days. Then, culture medium was replaced with serum-free medium and fluorescein isothiocyanate- dextran (1 mg/ml, Sigma) was added to the upper chamber. At different times, 100 μl from the lower 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.

Table 6. Effect of JAM transfection on paracellular permeability.

Transfectant cells % Permeability

Control 100.0 ± 2.5

JAM 46.5 ± 2.0

JAM + EGTA 5mM 110.0 ± 4.4

JAM + Cyt D 91.0 ± 6.5 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.

Example 5

Use of mAb BV11 to detect JAM in ELISA assay

ELISA sandwich

An ELISA standard protocol was followed [Peri et al., J. Immunol. Meth. 174, 249- 257, (1994)]. Briefly, 96 well ELISA plates (Falcon) were coated with 50 μl/well 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 1 :2000, Sigma) was incubated for 1 h at RT and then 100 μl 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 endothelial cells 3T3 fibroblasts Mel(hemopoietic line)

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

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.

Example 6

Expression of soluble JAM

An extracellular JAM fragment was cloned by PCR [Saiki et al., Science 239, 487 (1988)] 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 Hindlll and the resulting DNA was ligated into the cloningsite of the baculovirus expression vector pFASTBACI (Gibco BRL). The vector was transformed into DHIOBac 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 transfected 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 BV1 1 and mab BV12). Briefly, Immunoplates (Nunc Maxisorb) are coated overnight with 100 μl/well of a solution of mab BV12 (10 μg/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 μl/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.

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 / 965542 hlr ch

(ϋ) TTTLE OF INVENTION: Transmembrane component of tight junction

(iii) NUMBEROF SEQUENCES: 6

(iv) COMPUTER READABLEFORM:

(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 9604470-6

(B) FILING DATE: 04-DEC-1996

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISΗCS: (A) LENGTH: 298 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: both (ϋ) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

Met Gly Thr Lys Ala Gin Val Glu Arg Lys Leu Leu Cys Leu Phe lie 1 5 10 15 Leu Ala lie Leu Leu Cys Ser Leu Ala Leu Gly Ser Val Thr Val His

20 25 30

Ser Ser Glu Pro Glu Val Arg He Pro Glu Asn Asn Pro Val Lys Leu 35 40 45 Ser Cys Ala Tyr Ser Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phe 50 55 60

Asp Gin Gly Asp Thr Thr Arg Leu Val Cys Tyr Asn Asn Lys lie Thr 65 70 75 80

Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu Pro Thr Gly He Thr Phe 85 90 95

Lys Ser Val Thr Arg Glu Asp Thr Gly Thr Tyr Thr Cys Met Val Ser 100 105 110

Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys Val Lys Leu lie Val 115 120 125

Leu Val Pro Pro Ser Lys Pro Thr Val Asn He Pro Pro Ser Lys Pro 130 135 140 Thr Val Asn lie Pro Ser Ser Ala Thr Thr He Gly Asn Arg Ala Val

145 150 155 160

Leu Thr Cys Ser Glu Gin Asp Gly Ser Pro Pro Ser Glu Tyr Thr Trp 165 170 175

Phe Lys Asp Gly lie Val Met Pro Thr Asn Pro Lys Ser Thr Arg Cys 180 185 190

Leu Gin Gin Leu Phe Leu Ser Ser Leu Asn Pro Thr Thr Gly Glu Leu 195 200 205

Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly Glu Tyr Ser Cys Glu 210 215 220 Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser Asn Arg Val Ala Met

225 230 235 240

Glu Ala Val Asp Gly Asn Val Gly Val He Val Ala Ala Val Leu Val 245 250 255

Thr Leu He Leu Leu Gly lie Leu Val Phe Gly lie Tip Phe Pro Tyr 260 265 270

Ser Arg Gly His Phe Asp Arg Thr Lys Lys Gly Thr Ser Ser Lys Lys 275 280 285

Val Val Tyr Ser Gin Pro Ser Ala Arg Ser 290 295

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 300 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: both

(ϋ) MOLECULE TYPE: protein

(iϋ) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Disulfide-bond (B) LOCATION:49..108 (ix) FEATURE:

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: 152..212

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Gly Thr Glu Gly Lys Ala Gly Arg Lys Leu Leu Phe Leu Phe Thr 1 5 10 15 Ser Met He Leu Gly Ser Leu Val Gin Gly Lys Gly Ser Val Tyr Thr

20 25 30

Ala Gin Ser Asp Val Gin Val Pro Glu Asn Glu Ser lie Lys Leu Thr 35 40 45

Cys Thr Tyr Ser Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phe Val 50 55 60

Gin Gly Ser Thr Thr Ala Leu Val Cys Tyr Asn Ser Gin lie Thr Ala 65 70 75 80

Pro Tyr Ala Asp Arg Val Thr Phe Ser Ser Ser Gly He Thr Phe Ser 85 90 95 Ser Val Thr Arg Lys Asp Asn Gly Glu Tyr Thr Cys Met Val Ser Glu

100 105 110

Glu Gly Gly Gin Asn Tyr Gly Glu Val Ser lie His Leu Thr Val Leu 115 120 125

Val Pro Pro Ser Lys Pro Thr He Ser Val Pro Ser Ser Val Thr He 130 135 140

Gly Asn Arg Ala Val Leu Thr Cys Ser Glu His Asp Gly Ser Pro Pro 145 150 155 160

Ser Glu Tyr Ser Trp Phe Lys Asp Gly He Ser Met Leu Thr Ala Asp 165 170 175 Ala Lys Lys Thr Arg Ala Phe Met Asn Ser Ser Phe Thr He Asp Pro 180 185 190

Lys Ser Gly Asp Leu He Phe Asp Pro Val Thr Ala Phe Asp Ser Gly 195 200 205

Glu Tyr Tyr Cys Gin Ala Gin Asn Gly Tyr Gly Thr Ala Met Arg Ser 210 215 220 Glu Ala Ala His Met Asp Ala Val Glu Leu Asn Val Gly Gly He Val

225 230 235 240

Ala Ala Val Leu Val Thr Leu He Leu Leu Gly Leu Leu He Phe Gly 245 250 255

Val Trp Phe Ala Tyr Ser Arg Gly Tyr Phe Glu Thr Thr Lys Lys Gly 260 265 270

Thr Ala Pro Gly Lys Lys Val He Tyr Ser Gin Pro Ser Thr Arg Ser 275 280 285

Glu Gly Glu Phe Lys Gin Thr Ser Ser Phe Leu Val 290 295 300

(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 (ϋ) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHEΗCAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

TCCATTGTGC TCTAAAGCGG GACGCTGATC GCGATGGGGA CAAAGGCGCA AGTCGAGAGG 60

AAACTGTTGT GCCTCTTCAT ATTGGCGATC CTGTTGTGCT CCCTGGCATT GGGCAGTGTT 120

ACAGTGCACT CTTCTGAACC TGAAGTCAGA ATTCCTGAGA ATAATCCTGT GAAGTTGTCC 180 TGTGCCTACT CGGGCTTTTC TTCTCCCCGT GTGGAGTGGA AGTTTGACCA AGGAGACACC 240

ACCAGACTCG TTTGCTATAA TAACAAGATC ACAGCTTCCT ATGAGGACCG GGTGACCTTC 300

TTGCCAACTG GTATCACCTT CAAGTCCGTG ACACGGGAAG ACACTGGGAC ATACACTTGT 360 ATGGTCTCTG AGGAAGGCGG CAACAGCTAT GGGGAGGTCA AGGTCAAGCT CATCGTGCTT 420 GTGCCTCCAT CCAAGCCTAC AGTTAACATC CCTCCATCCA AGCCTACAGT TAACATCCCC 480 TCCTCTGCCA CCATTGGGAA CCGGGCAGTG CTGACATGCT CAGAACAAGA TGGTTCCCCA 540 CCTTCTGAAT ACACCTGGTT CAAAGATGGG ATAGTGATGC CTACGAATCC CAAAAGCACC 600 CGTTGCCTTC AGCAACTCTT CCTATCTAGT CTGAATCCCA CAACAGGAGA GCTGGTCTTT 660 GATCCCCTGT CAGCCTCTGA TACTGGAGAA TACAGCTGTG AGGCACGGAA TGGGTATGGG 720 ACACCCATGA CTTCAAATCG TGTCGCGATG GAAGCTGTGG ACGGGAATGT GGGGGTCATC 780 GTGGCAGCCG TCCTTGTAAC CCTGATTCTC CTGGGAATCT TGGTTTTTGG CATCTGGTTT 840 CCGTATAGCC GAGGCCACTT TGACAGAACA AAGAAAGGGA CTTCGAGTAA GAAGGTAGTT 9ω TACAGCCAGC CTAGTGCCCG AAGT 924

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1374 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: cDNA to mRNA

(hi) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

ATACCATTGT GCTGGAAAGG TTGCTGTGCC CGTCGCGTCG GGATTGTAAC TGTAATGGGC 60

ACCGAGGGGA AAGCCGGGAG GAAACTGTTG TTTCTCTTCA CGTCTATGAT CCTGGGCTCT 120

TTGGTACAAG GCAAGGGTTC GGTGTACACT GCTCAATCTG ACGTCCAGGT TCCCGAGAAC 180 GAGTCCATCA AATTGACCTG CACCTACTCT GGCTTCTCCT CTCCCCGAGT GGAGTGGAAG 240

TTCGTCCAAG GCAGCACAAC TGCACTTGTG TGTTATAACA GCCAGATCAC AGCTCCCTAT 300

GCGGACCGAG TCACCTTCTC ATCCAGTGGC ATCACGTTCA GTTCTGTGAC CCGGAAGGAC 360

AATGGAGAGT ATACTTGCAT GGTCTCCGAG GAAGGTGGCC AGAACTACGG GGAGGTCAGC 420

ATCCACCTCA CTGTGCTTGT ACCTCCATCC AAGCCGACGA TCAGTGTCCC CTCCTCTGTC 480 ACCATTGGGA ACAGGGCAGT GCTGACCTGC TCAGAGCATG ATGGTTCCCC ACCCTCTGAA 540

TATTCCTGGT TCAAGGACGG GATATCCATG CTTACAGCAG ATGCCAAGAA AACCCGGGCC 600 TTCATGAATT CTTCATTCAC CATTGATCCA AAGTCGGGGG ATCTGATCTT TGACCCCGTG 660

ACAGCCTTTG ATAGTGGTGA ATACTACTGC CAGGCCCAGA ATGGATATGG GACAGCCATG 720

AGGTCAGAGG CTGCACACAT GGATGCTGTG GAGCTGAATG TGGGGGGCAT CGTGGCAGCT 780

GTCCTGGTAA CACTGATTCT CCTTGGACTC TTGATTTTTG GCGTCTGGTT TGCCTATAGC MO CGTGGATACT TTGAAACAAC AAAGAAAGGG ACTGCACCGG GTAAGAAGGT CATTTACAGC 900

CAGCCCAGTA CTCGAAGTGA GGGGGAATTC AAACAGACCT CGTCGTTCCT GGTGTGACCT 960

GCTGCGGCTC CTCCGTTGTC CATTTGCCTT ACTCAGGTGC TACAGGTTCC AGCCCCTGCT 1020

GCTGTAGCTG CACAGGATGC CTTCAATGTC TTCTAGGTCC CACAGGACCC CTTGCTTTTA 1080

TTCTAGCTAG GATATAAATT TAAAAACATC ATCTACTTCC CCCTCCTCTT TCCCACCCTC 1140 CCTCCTTTCC TTACCACCAT TGGGTGGCCC GAGACTAATT ACAAAGTTTT CGTTCCCCAT 1200

TCCTATGTGG GATTGGGCAA GAGTCCTAGA CTAGACAGTA ATAGTGGCTG GGCTGACAGG 1260

AACCCAAACC AATACCTGGC TGTAAAGGCC TCTGAATAAG GACTTTAAGC CTAGCTCCCT 1320

GCTTTCTCCT CCCCGGATGG GGTGCCAGCT ACTCTAGAAG GGGAGCTGCA TAAA 1374

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 891 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ϋ) MOLECULE TYPE: cDNA to mRNA

(hi) HYPOTHEΗCAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ATGGGGACAA AGGCGCAAGT CGAGAGGAAA CTGTTGTGCC TCTTCATATT GGCGATCCTG 60

TTGTGCTCCC TGGCATTGGG CAGTGTTACA GTGCACTCTT CTGAACCTGA AGTCAGAATT 120

CCTG AG AATA ATCCTGTG AA GTTGTCCTGT GCCT ACTCGG GCTTTTCTTC TCCCCGTGTG 180

GAGTGGAAGT TTGACCAAGG AGACACCACC AGACTCGTTT GCTATAATAA CAAGATCACA 240

GCTTCCTATG AGGACCGGGT GACCTTCTTG CCAACTGGTA TCACCTTCAA GTCCGTGACA 300 CGGGAAGACA CTGGGACATA CACTTGTATG GTCTCTGAGG AAGGCGGCAA CAGCTATGGG 360

GAGGTCAAGG TCAAGCTCAT CGTGCTTGTG CCTCCATCCA AGCCTACAGT TAACATCCCT 420 CCATCCAAGC CTACAGTTAA CATCCCCTCC TCTGCCACCA TTGGGAACCG GGCAGTGCTG 480

ACATGCTCAG AACAAGATGG TTCCCCACCT TCTGAATACA CCTGGTTCAA AGATGGGATA 540

GTGATGCCTA CGAATCCCAA AAGCACCCGT TGCCTTCAGC AACTCTTCCT ATCTAGTCTG 600

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

GCTGTGGACG GGAATGTGGG GGTCATCGTG GCAGCCGTCC TTGTAACCCT GATTCTCCTG 780

GGAATCTTGG TTTTTGGCAT CTGGTTTCCG TATAGCCGAG GCCACTTTGA CAGAACAAAG 840

AAAGGGACTT CGAGTAAGAA GGTAGTTTAC AGCCAGCCTA GTGCCCGAAG T 891

(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

(ϋ) MOLECULE TYPE: cDNA to mRNA

(hi) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

ATGGGCACCG AGGGGAAAGC CGGGAGGAAA CTGTTGTTTC TCTTCACGTC TATGATCCTG 60

GGCTCTTTGG TACAAGGCAA GGGTTCGGTG TACACTGCTC AATCTGACGT CCAGGTTCCC 120

GAGAACGAGT CCATCAAATT GACCTGCACC TACTCTGGCT TCTCCTCTCC CCGAGTGGAG 180 TGGAAGTTCG TCCAAGGCAG CACAACTGCA CTTGTGTGTT ATAACAGCCA GATCACAGCT 240

CCCTATGCGG ACCGAGTCAC CTTCTCATCC AGTGGCATCA CGTTCAGTTC TGTGACCCGG 300

AAGGACAATG GAGAGTATAC TTGCATGGTC TCCGAGGAAG GTGGCCAGAA CTACGGGGAG 360

GTCAGCATCC ACCTCACTGT GCTTGTACCT CCATCCAAGC CGACGATCAG TGTCCCCTCC 420

TCTGTCACCA TTGGGAACAG GGCAGTGCTG ACCTGCTCAG AGCATGATGG TTCCCCACCC 480 TCTGAATATT CCTGGTTCAA GGACGGGATA TCCATGCTTA CAGCAGATGC CAAGAAAACC 540

CGGGCCTTCA TGAATTCTTC ATTCACCATT GATCCAAAGT CGGGGGATCT GATCTTTGAC 600

CCCGTGACAG CCTTTGATAG TGGTGAATAC TACTGCCAGG CCCAGAATGG ATATGGGACA 660

GCCATGAGGT CAGAGGCTGC ACACATGGAT GCTGTGGAGC TGAATGTGGG GGGCATCGTG 720 GCAGCTGTCC TGGTAACACT GATTCTCCTT GGACTCTTGA TTTTTGGCGT CTGGTTTGCC 780

TATAGCCGTG GATACTTTGA AACAACAAAG AAAGGGACTG CACCGGGTAA GAAGGTCATT 840 TACAGCCAGC CCAGTACTCG AAGTGAGGGG GAATTCAAAC AGACCTCGTC GTTCCTGGTG 900

Claims (21)

CLAI MS

1. Protein in glycosylated or unglycosylated form comprising an amino-acid sequence selected from the sequence SEQ ID NO:1 and homologous sequences having at least 72% homology thereto.

2. Protein according to claim 1 , wherein said sequence is SEQ ID NO:2

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

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

5. Gene coding for a protein as claimed in claim 1 or a peptide derived therefrom.

6. Gene according to claim 5 selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6.

7. Vector comprising a DNA sequence as claimed in claim 4 or a gene as claimed in claim 5 or 6.

8. Organisms transformed with a vector as claimed in claim 7.

9. Recombinant protein or peptide expressed by a gene or a fragment of the gene according to claim 5 or 6.

10. Antibody binding specifically to a protein according to claim 1 or a part of the protein.

11. Modifier of the polymerization of a transmembrane protein defined in claim 1.

12. Modifier according to claim 1 1 selected from the group consisting of antibodies specifically binding to the protein defined in claim 1 and inhibiting or inducing the polymerization of the protein, and polymerization-inhibiting or -inducing proteins, peptides, peptidomimetics and organic molecule-ligands derived from the amino- acid sequence of the protein defined in claim 1.

13. Diagnostic kit comprising as a diagnostic reagent an antibody according to claim 10 or a modifier according to claim 1 1 or 12.

14. Vaccine adjuvant comprising a modifier according to claim 1 1 or 12.

15. Medicament comprising as an active ingredient a modifier according to claim 11 or 12.

16. The use of a protein according to claim 1 for identifying and isolating modifiers of its polymerization.

17. The use of a modifier according to claim 1 1 or 12 for the preparation of medicaments.

18. A method for producing a protein as claimed in claim 1 or 2, comprising cultivating an organism as claimed in claim 8 in a suitable medium and optionally isolating said protein.

19. Proteins as claimed in claim 1 or 2 prepared by the method of claim 18.

20. A modifier according to claim 11 or 12 as active ingredient in medicaments.

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