WO1994013312A1 - Adressine vasculaire de muqueuses, adn codant ladite adressine et expression de ladite adressine - Google Patents

Adressine vasculaire de muqueuses, adn codant ladite adressine et expression de ladite adressine Download PDF

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Publication number
WO1994013312A1
WO1994013312A1 PCT/US1993/011404 US9311404W WO9413312A1 WO 1994013312 A1 WO1994013312 A1 WO 1994013312A1 US 9311404 W US9311404 W US 9311404W WO 9413312 A1 WO9413312 A1 WO 9413312A1
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madcam
binding
fragment
leu
leukocytes
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PCT/US1993/011404
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English (en)
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Eugene Butcher
Michael Briskin
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The Board Of Trustees Of The Leland Stanford Junior University
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Priority to AU56757/94A priority Critical patent/AU5675794A/en
Publication of WO1994013312A1 publication Critical patent/WO1994013312A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the field of this invention is the encoding DNA and expression of mucosal vascular addressin.
  • Blood is the highway of leukocytes, where the leukocytes can move from various tissues, particularly lymphoid tissues, and be transferred to other sites. Since many of the leukocytes monitor for foreign matter and diseased cells, it is necessary that they circulate in a continuous manner to provide protection from disease. Many leukocytes are also involved in providing factors for growth of other cells and aiding in regenerative processes. It is found, however, that certain subsets of leukocytes appear to be programmed to go to different sites. For example, cells will differentially home to mucosal tissue, peripheral lymph nodes, synovial tissue or skin, depending upon the particular proteins which are present on the cell surface. Thus, there are subsets of lymphocytes and monocytes which will preferentially be directed to particularly types of tissue.
  • the process of a cell being directed to a particular site is referred to as "homing. " What this means is that there is a process which allows the cell programmed for a particular site to be directed to that site in the event of injury or disease, or in some instances under normal physiologic circumstances. Part of this process involves endothelial cells, which are part of blood vessels, including high endothelial venules. These cells, present on venules, respond to signals from diseased or injured tissue by upregulating certain surface membrane proteins.
  • the surface membrane proteins referred to as addressins, are capable of specifically binding to particular surface membrane proteins of certain lymphocytes or monocytes, providing for the specificity of the circulating cells. After binding to the endothelial cells, the circulating cells may then be directed to the site of injury or disease by a process referred to as "extravasation.”
  • Nucleotide sequences are provided encoding mammalian mucosal addressin, where the sequences can be used for expressing the addressin or fragments thereof.
  • the addressin or fragments thereof may be used to produce antibodies which may find therapeutic purposes.
  • the addressin or fragments thereof by themselves or joined to other molecules, may find use in tagging leukocytes, inhibiting leukocyte binding to endothelial cells, in isolating leukocytes, in transporting various agents to leukocytes, and the like.
  • EC endothelial cells
  • Methods are provided for isolating or synthesizing DNA which encodes at least a portion of the mucosal addressin, for introducing the DNA into host cells, and for expression of the addressin or fragments thereof.
  • the addressin can serve as a source of the carbohydrate side chains which serve as binding entities to the leukocytes.
  • the Figure depicts the cDNA sequence of murine MAdCAM-1 (Clone 7) and the encoded amino acid sequence.
  • the signal peptide, transmembrane region and single amino acid change (R to L) are bold underlined.
  • the base change is T to G (nucleotide 182).
  • nucleic acid and protein compositions are provided, where the protein compositions are mammalian mucosal addressins or fragments thereof and the nucleic acids are sequences encoding the mucosal addressin or fragments thereof.
  • the mucosal addressin protein in the mouse is characterized by binding to antibodies which have been shown to bind to mucosal addressins, MECA-89 and -367.
  • the mucosal addressin has a structure having three regions, the extracellular region, the transmembrane region, and the cytoplasmic region and when initially expressed, the signal sequence.
  • the mature protein has from about 350-425 amino acids, usually from about 375-400 amino acids. At least about 80% of the mature protein will be the extracytoplasmic portion.
  • the overall structure of the protein is a Type I transmembrane protein.
  • the most end-terminal domain shows high sequence similarity to domain 1 of the cell adhesion molecule ICAM-1.
  • the second immunoglobulin domain (domain 2) shows sequence similarity to the fifth domain of mouse VCAM-1.
  • Domains 1 and 2 are separated by at least 20 amino acids and not more than about 50 amino acids, usually about 35-45 amino acids. This is a typical spacing for separation of two adjacent immunoglobulin-like domains.
  • domain 2 Just proximal to domain 2 is a long stretch rich in prolines, serines and threonines, indicating a site for heavy O-glycosylation.
  • This domain will be at least about 60 amino acids and not more than about 100 amino acids, generally being from about 65-80 amino acids. Of this domain, the majority of serines and threonines are found in about a 35-40 amino acid stretch. In this region, from about 30-50% of the amino acids will be serine or threonine, while from about 5-20% of the amino acids will be proline.
  • Proximal to this region is a third immunoglobulin domain, domain 3. This domain has significant homology with a constant region immunoglobulin domain C2 of the IgAl heavy chain. It should be noted that IgAl is associated with mucosal tissue.
  • the mucosal addressin may come from any species, particularly primate, more particularly human, rodentiae, particularly murine, namely mouse and rat, lagomorpha, ovine, bovine, equine, porcine, feline, canine, guinea pig, etc.
  • the mouse mucosal addressin is characterized by having a signal sequence of 21 amino acids, an extracytoplasmic region of 342 amino acids, a transmembrane sequence of 22 amino acids and a cytoplasmic tail of 20 amino acids.
  • the extracellular region has three potential N-linked glycosylation sites, which do not appear to be used.
  • the mucosal addressins have substantial homology to other adhesion molecules which have been previously reported, namely VCAM-1 and ICAM-1. This homology is particularly found at the N-terminal domain.
  • the subject mucosal addressin will hereinafter be referred to as
  • the subject protein is selectively expressed on HEV of mucosal lymphoid organ HEV and lamina intestinal venules.
  • the subject protein binds to the previously-reported monoclonal antibodies MECA-89 and -367 and specifically binds to the mucosa-selective T-cell lymphoma TKl.
  • MAdCAM-1 is a member of the immunoglobulin superfamily.
  • the gene encoding MAdCAM-1 will generally be joined to other than wild- type sequences. Usually the wild-type DNA will be not more than about 1 cM, generally not more than about 10 kb.
  • the genomic gene will usually be separated from the intact chromosome and may be manipulated as part of a YAC, where fragments will be from about 100-1000 kbp.
  • the cDNA which encodes the subject protein in the mouse has between 1400-1500 nucleotides (nt) with a short, 5'-noncoding region, generally from about 20-40 nt and a 3'-noncoding region of from about 150-250 nt.
  • the mouse cDNA is 1436 nt, with a 5'-noncoding region of 30 nt, and a 3'-noncoding region of 191 nt. Included in the 3' region is a polyadenylation site at position 1400.
  • the initiation codon, ATG is at position 31 of the cDNA.
  • the molecule encoded has 21 amino acids for the signal peptide leaving a mature polypeptide of 384 amino acids.
  • variations of the mucosal addressin may be achieved.
  • removal of the transmembrane sequence either with or without removing the cytoplasmic portion, whereby a soluble secreted form of the mucosal addressin will be obtained.
  • Excising the transmembrane region may be achieved by in vitro mutagenesis, polymerase chain reaction, or other known technique.
  • TNF- or lipopolysaccharide expression of the mucosal addressin in endothelial cells, particularly transformed endothelial cells, can be enhanced by using TNF- or lipopolysaccharide, with relatively high levels of expression occurring within a few hours, generally within about 4-6 hr.
  • glycosylated mature product which may be isolated and purified in accordance with conventional ways.
  • the protein can be obtained in a pure state of at least about 50 wt. % , preferably at least about 75 % and more preferably substantially pure or pure, in that the composition is free of any physiologically active contaminants.
  • compositions provided by the subject invention may be used in a variety of ways.
  • the intact protein, fragments of the protein indicating polypeptide fragments, polysaccharides, and combinations thereof may find application.
  • the DNA encoding the mucosal addressin, the entire reading frame, with or without introns, non-coding regulation elements of the gene, or fragments of the encoding DNA, particularly fragments encoding all or a portion of a functional region or domain can find use.
  • the sequences may be mutated for a variety of reasons, where the mutations may include insertions, deletions and substitutions. Substitutions may be conservative or non-conservative, there will usually be fewer than the greater of 2 substitutions or substitutions equal to 2% of the number of nucleotides. For the protein, the same numbers will apply for the amino acids.
  • Polypeptides of each of the domains may be used to prepare a specific antisera or monoclonal antibodies for a specific site. In this way, binding to each of the domains may be individually inhibited by using antibodies which specifically bind to that domain. These antibodies may then be used to determine the manner in which binding occurs between the addressin and various homing receptors or selectins.
  • the glycosylated domain may be used in a variety of ways, such as providing a source of the sugars which may bind to lectin regions of the selectins, for producing antibodies to the protein and/or saccharide portions of the domain, to serve as inhibitors to binding, to direct compositions to activated leukocytes, and the like.
  • the protein or fragments thereof usually fragments of at least about 12 amino acids, more usually at least about 16 amino acids and preferably at least about 20 amino acids, generally fewer than the entire protein, preferably fewer than 60 amino acids may be employed as agents to block binding of the leukocytes to the mucosal addressin.
  • These peptides may find use in vitro and in vivo, where the peptides may inhibit entry of leukocytes to a site where their presence may contribute to a disease. Thus, one may wish to exclude leukocytes from injured sites, where the leukocytes may enhance inflammation, induce cellular proliferation, destroy native tissue, or the like.
  • Diseases which may be treated include especially intestinal inflammatory disorders including Crohn's disease, ulcerative colitis, regional enteritis, and Celiac disease; but also in inflammatory and autoimmune disorder in other sites where the MAdCAM-1 may be induced abnormally or in pathologic conditions. Also in malignancies, especially of intestinal carcinomas and their metastases, in which MAdCAM-1 may be induced in tumor-associated vessels.
  • the protein or fragments thereof may be modified in a variety of ways for a variety of purposes.
  • the peptides may be joined to immunogens to enhance immunogenicity for the formation of antibodies.
  • the antibodies may be from any convenient source but will normally be produced by laboratory animals, such as mice, rats and rabbits, or in domestic animals, such as cows, sheep, pigs, etc. The manner of producing antisera and monoclonal antibodies is sufficiently well- established as to not require description here.
  • the mouse or other species monoclonal antibodies may be humanized by replacing the constant region, by itself or in combination with one or more conserved framework regions, with regions from human antibodies, so that human IgA, -G, or -M or the like regions may replace the native antibody regions to reduce antigenicity in humans and to provide effector functions.
  • the protein or peptides may be joined to polyethylene glycol, constant regions of immunoglobulins, e.g. IgG, to lipids or the like, to enhance the lifetime of the peptide or protein when administered in vivo.
  • the peptide or protein may be joined to various effector agents, particularly agents which produce a physiological reaction, such as antibodies, toxins, drugs, carbohydrates or cytokines.
  • the peptide or protein may be joined to these various agents in a variety of ways. Where proteins are involved, the sequence encoding the subject peptide or protein may be fused to the other protein to provide for a fusion protein.
  • lipid is to be joined, it may be feasible to provide for the coding sequence which encodes for the linking of the peptide to a lipid through a phosphate linkage, based on known, natural processes of the host cell.
  • various chemical linking agents are known which allow for linking through carboxyl groups, amino groups, thiol groups, or hydroxyl groups, as well as linking through an activated aromatic group, such as is present in tyrosine and tryptophan.
  • a description of these agents may be found in Antibodies, A Laboratory Manual, Harlow, E., Lane, D. ed., Cold Spring Harbor Laboratory, 1988, pp. 319-358.
  • Various synthetic procedures and conditions are known, which need not be described here.
  • the peptides or proteins may be labeled with a wide variety of labels for a variety of purposes.
  • the peptides or proteins may be used in assays for detecting the presence of cells binding to the subject mucosal addressin or fragment thereof.
  • fluorescent labels, enzyme labels, radioactive labels, or the like may be employed for use of the subject peptides or proteins in the assays.
  • Numerous assays are known, such as RIA, ELISA, fluorescence polarization assay, fluorescence-activated cell sorters (FACS), and the like.
  • the peptides or proteins may be bound to a solid support, such as particles, microtiter plate wells, solid surfaces, where the peptides may serve to bind to cells having the appropriate complementary protein.
  • a solid support such as particles, microtiter plate wells, solid surfaces
  • the peptides may serve to bind to cells having the appropriate complementary protein.
  • cells may be selectively removed ex vivo for isolation and characterization, to reduce their population in a host, or the like.
  • the particles may be magnetic particles, pore glass beads, latex particles, agarose particles, sepharose particles, or the like.
  • the various particles may be activated in a wide variety of ways, activated particles being commercially available for covalently linking the peptide or protein to the particle.
  • other surfaces may also be activated by known ways for covalent binding. Alternatively, depending upon the purpose of the binding, with some surfaces, e.g.
  • non-covalent binding may suffice.
  • an innocuous protein will be used to react with any remaining unreacted functional groups and to coat any hot spots which might result in non-specific binding in a subsequent application.
  • the DNA encoding the subject addressin or fragment thereof may be used for a diversity of purposes.
  • the cDNA or fragment thereof may be used to identify the genomic gene which encodes the subject addressin.
  • a cDNA or fragment thereof from one species may be used to probe cDNA or genomic libraries of other species to identify and isolate the gene from the other species.
  • one may use the sequence encoding the mucosal addressin to define antisense sequences, which may then be used to inhibit expression of the gene. In this way, one can inhibit the expression of the mucosal addressin in mucosal endothelial cells, thus inhibiting the homing of lymphocytes via these endothelial cells.
  • the subject peptides may be used in assays to identify drugs, including peptides, saccharides and small synthetic organic molecules which bind to the peptides and have biological activity. These agents may then be used in assays to determine their effectiveness in vivo.
  • the initial assays may take various forms involving direct binding or competitive binding.
  • the subject peptides may be bound to a surface and the radiolabeled agent added.
  • the affinity of the agent may be determined.
  • the proteins or fragments thereof, capable of binding to the complementary binding protein may also be used as antagonists for complex formation and to modulate the various interactions.
  • the protein may serve to home to the complementary binding member and inhibit the binding of the addressin associated with the target cells.
  • the fragment will usually be at least 8 amino acids, more usually at least about 10 amino acids, frequently at least about 16 amino acids, usually not exceeding about 60 amino acids, preferably not exceeding about 36 amino acids.
  • the proteins, fragments thereof, or anti-idiotope antibodies may serve to direct a wide variety of molecules to the target site.
  • neoplastic tissue by administering one of the subject compounds or compositions bound to a therapeutic drug, one can direct the binding of a therapeutic drug to the desired site for retention, and concentration at the desired site to which the MAdCAM-1 expressing endothelial cells bind.
  • a therapeutic drug to the desired site for retention, and concentration at the desired site to which the MAdCAM-1 expressing endothelial cells bind.
  • cytotoxic drugs to the fragments to kill target lymphocytes.
  • empirical determinations will be involved to determine the level of therapeutic agent.
  • the level of therapeutic agent which is administered will depend to a substantial degree on the stability of the protein, its size, the manner of administration, the site of administration, the purpose of the therapy, and the like. Therefore, no simple range may be given which would indicate what levels should be applied for any particular therapy.
  • the proteins will be administered in an appropriate physiologically-acceptable medium, e.g. water, saline, phosphate- buffered saline, mineral oil, Ringer's solution, or the like. Administration will normally be parenteral, particular intravascularly, but may be oral. For the reasons given above, the course of treatment will also vary.
  • cells there may be situations where there is an interest in using cells as therapeutics.
  • the subject peptides By employing the subject peptides, one may isolate particular subsets of cells, which may then be expanded in culture in an appropriate nutrient medium or in co-culture, and may then be used for a therapeutic purpose.
  • the cells may be autologous or allogeneic.
  • the antibodies may also find wide use for detecting the presence of venules in vitro or in vivo, where the subject mucosal addressin is upregulated. By using antibodies, binding of leukocytes to these sites may be inhibited, so as to prevent extravasation to the particular site.
  • the antibodies may also be used to make anti-id antibodies, which may be selected as mimics for the subject peptides or proteins. In this way, the antibodies which will have a substantially different composition from the mucosal addressin, may fulfill the same function by binding to the endothelial cell and block binding of the leukocyte to the venule. Also, the antibodies may be used in the variety of ways indicated above in place of the subject peptides or proteins.
  • the cDNA or genomic DNA encoding MAdCAM-1 can be used in preparing expression constructs.
  • the DNA can be used in any convenient host, both prokaryotic and eukaryotic for the cDNA and invertebrate and vertebrate for splicing capability for the genomic gene. As previously indicated, for glycosylation, eukaryotic, particularly mammalian hosts will be required.
  • the gene encoding MAdCAM-1 may be inserted into a wide variety of generally- available expression cassettes having linkers between a 5' transcriptional and translational initiation region, which may include an enhancing sequence, and may provide for constitutive or inducible transcription, and a 3' transcriptional and translational termination region, which will normally include a polyadenylation signal.
  • an expression cassette by combining the appropriate functional sequences in the appropriate order as indicated above. Numerous transcriptional initiation regions are commercially available or described in the literature.
  • the expression cassette may then be introduced into a vector for replication and expansion.
  • the resulting vector will usually include a marker for selection of hosts containing the construct.
  • the vector may be introduced into the expression host by any convenient means, precipitated DNA, transfection, fusion, electroporation, etc., where the vector may provide for extrachromosomal maintenance or integration into the genome of the host.
  • vectors comprising sequences of a virus for transfection and integration into mammalian cellular hosts. The expression host may then be grown.
  • the product may be secreted and isolated from the supernatant or in the event it is retained in the cytoplasm, the cells may be harvested and the lysate extracted.
  • the product may be purified using electrophoresis, gel chromatography, affinity chromatography, extraction and the like. The following examples are offered by way illustration and not by way limitation.
  • EXPERIMENTAL A transformed mouse endothelioma cell line, Bend3 cells was employed. It was shown to be inducible for the MECA-89 and -367 epitopes with bacterial lipopolysaccharide (LPS) and tumor necrosis factor (TNF- ⁇ ). Serial fluorescence activated cell sorting was used to generate a variant line termed Hi-MAd 4 that expresses much higher levels of the antigen upon TNF- ⁇ stimulation. Hi-MAd 4 cells, stimulated with recombinant TNF- ⁇ for either 6 or 8 hours were pooled and used as the source of RNA for cDNA libraries.
  • LPS bacterial lipopolysaccharide
  • TNF- ⁇ tumor necrosis factor
  • Endothelial cell cDNA libraries were synthesized with a BRL system which generates directionally-oriented cDNA into the ⁇ gtll derivative, ⁇ gt22A.
  • Phage clones (5 X 10 5 ) were screened with a mixture of anti-mucosal addressin antibodies consisting of a rat polyclonal against affinity-purified MAd (1:2000) and the monoclonal antibodies MECA-89 and -367 (5 g/ml) diluted TBS with 1 % BSA and 0.02% sodium azide. Positive clones were detected with an anti-rat IgG conjugated to alkaline phosphatase (Promega) and BioRad BCIP/NBT color detection reagents.
  • the entire sequence of the mucosal addressin cDNA is 1436 nt in length, consisting of a 30 nt 5' untranslated region and a 191 nt 3' untranslated region.
  • a canonical polyadenylation signal AATAAA is located at position 1400.
  • the longest open reading frame of 405 amino acids begins with the first ATG at position 31 and ends with the termination codon TGA at position 1245.
  • Hydrophobicity analysis and rules governing signal peptide cleavage patterns predict a signal peptide of 21 amino acids, leaving a mature polypeptide of 384 amino acids. This sequence predicts a typical transmembrane protein consisting of a 342 amino acid extracellular domain, a 22 amino acid transmembrane region and a short, charged 20 amino acid cytoplasmic tail.
  • the extracellular domain contains 3 potential N-linked glycosylation sites with the consensus sequence NXT/S at position 718, 787 and 111. Studies indicate that these sites are not utilized, since the mucosal addressin is insensitive to peptide N-glycosidase.
  • the largest cDNA inserts from ⁇ clones 7 and 15 ( ⁇ MAd-7 and ⁇ MAd-15) were subcloned into Bluescript vectors (Stratagene) to construct the plasmids pMAd-7 and pMAd-15. Plasmids for sequencing were initially generated by making nested deletions with exonuclease III using Erase A Base (Promega) system.
  • the DNA sequence was determined by the dideoxy method using fluorescent-labeled universal primers and the Applied Biosystems DNA sequence analyzer. Gaps in the sequence were filled in by subcloning small restriction fragments into appropriately-digested Bluescript vectors. Two independent clones were sequenced 100% on both strands and are identical with the exception of a single base substitution which would generate a leucine instead of the arginine shown in pMAd-15 at amino acid 61.
  • the putative hydrophobic signal peptide and transmembrane sequences have a bold underline. Potential N-linked glycosylation sites are present at positions 718, 787 and 111. Cysteines likely to form disulfide bonds in Ig domains are boxed while other cysteines are underlined. The 37 amino acid "mucin domain" between amino acid residues 221 and 257 is outlined with a bold line. The polyadenylation signal ATAAA at position 1400 is indicated in bold.
  • BALB/c mouse liver, kidney, Peyer's patches, brain, peripheral lymph nodes, lung, mesenteric lymph nodes and spleen were hybridized to the 1.4 kb MAd cDNA insert.
  • Northern blots used 1 ⁇ g of poly A+ RNA from cells lines and 2 ⁇ g from tissues which was denatured and electrophoresed through a 1 % agarose formaldehyde gel and transferred to a PVDF (Immobilon, Millipore) membrane by standard capillary blot procedures. Equal amounts (by OD 260 ) of each RNA were electrophoresed and stained with ethidium bromide to ensure equivalent loading of each sample. Hybridization was performed at 65°C using standard conditions. cDNAs were labeled with ⁇ 32 P dCTP by priming with random hexamers.
  • RNA was highly expressed in mesenteric lymph nodes (MLN) and Peyer's patches (PP) and detected at lower levels in spleen and peripheral lymph nodes (PLN). The species were not detected with the pre-B lymphoma Ll-2 or in liver, brain, kidney or thymus.
  • Sall-Notl insert of ⁇ MAd-15 into XhoI-NotI digested pCDM8 was transfected onto COS cell monolayers (along with no DNA as a mock transfected control) using DEAE dextran. After 48 hrs., cells were replated into 3.5 cm dishes to ensure equal plating of all transfectants and 24 hrs. later transfectants were analyzed by immunofiuorescence. Both addressin and mock transfectants were stained with anti-addressin monoclonals MECA-89 or -367 alone or the isotype matched control MEL-14. Staining was detected both visually and by FACS analysis using a phycoerythrin-conjugated goat F(ab') 2 anti rat IgG (Caltag). Transfection efficiencies were approximately 25% for each experiment.
  • COS cell transfectants were plated into 3.5 cm plates as described above.
  • B and T lymphomas were pelleted and resuspended at a density of 2 X 10 6 per ml in RPMI without sodium carbonate, with 2% serum and buffered with 10 mM HEPES, pH 6.7.
  • Cell suspensions were layered (1 ml per plate) onto addressin and mock transfectants and gently rotated on a gyrotary shaker (New Brunswick Scientific Model G-2) at 60 rpm for 30 min. at 25 °C, and then washed 4 times in the same medium by aspirating and gently filling each plate with 1 ml per wash.
  • Assays were fixed in Dulbecco's PBS containing 1 % glutaraldehyde and sodium azide.
  • transfectants were pre-treated with 20 ⁇ g/ml of anti-addressin antibody MECA-367 or isotype matched control 9B5 (anti-CD44) at 1 ml per plate for 20 min. at 25 °C. Solutions were then aspirated and adhesion assays were performed as described above. The average number of cells bound for each experiment was determined by counting cells in 4 random fields.
  • Binding is specific to cells expressing MAd as shown by parallel immunofiuorescence with a non-blocking MAd, MECA-89. Adhesion is inhibited by approximately 95 % by pre-treatment with saturating levels of anti-addressin MAd MECA-367, while pre-treatment with control MAd had no effect. Furthermore, binding was specific for a mucosal HEV binding cell line, as Ll-2 and NS8 cells (lines previously shown not to adhere to HEV) failed to adhere to the COS cell transfectants.
  • the amino acid sequence of the mucosal addressin was analyzed for homology to other proteins in the NBRF database using the program FASTP and the highest degree of homology was to murine ICAM-1. Consistent with the designation of ICAM-1 as a member of the immunoglobulin supergene family, the extracellular domain of MAd can also be folded into a structure containing 3 immunoglobulin-like domains. Each of the 3 domains contains an invariant pair of cysteine residues known to stabilize the Ig loop, with distances of 44, 66 and 47 amino acids between cysteines, respectively.
  • N-terminal domains of ICAM-1 and VCAM-1 have been shown to contain binding sites for their respective lymphocyte counter-receptors LFA-l/a r jSj and VLA-4/ ⁇ 4 j ⁇ 1 .
  • the ⁇ 4 j ⁇ 7 integrin is known to be involved in lymphocyte binding to Peyer's patch-HEV in in vitro assays.
  • the second immunoglobulin domain is most closely related to the fifth domain of mouse VCAM-1.
  • the third immunoglobulin-like domain of the MAd is unique in that the most significant homology is to the CH2 region of human and gorilla IgAl (33% identity).
  • This homology relates two molecules which are highly expressed in mucosal tissues and which both interact with T-cells involved in mucosal immunity.
  • Their binding to mucosal addressin could be strengthened by interaction of the IgA F c receptor or other T-cell surface receptors, which are capable of binding the IgA-1 homologous domain.
  • a proline/serine-threonine-rich region in the amino-terminal portion of this domain which aligns with the hinge region encoded with the IgAl CH2 exon may add flexibility to the addressin and its interaction with ligands on the surface of lymphocytes.
  • GlyCAM-1 mucin-like protein GlyCAM-1
  • the protein core of GlyCAM-1 is also serine/threonine rich and is proposed to serve as a backbone molecule capable of presenting a carbohydrate ligand(s) characterizing the peripheral lymph node addressin.
  • carbohydrate structures are recognized by the L-selectin lymph node homing receptor and by anti-PLN addressin MAb MECA-79.
  • this mucin-like domain in the mucosal addressin can serve as a site for addition of selectin-binding carbohydrates important for recruitment of lymphocyte subsets to the mesenteric lymph node and also, at a lower level, to the Peyer's patches.
  • Proteoglycans can also bind and present heparin-binding cytokines at the cell surface.
  • the mucin-like region may also be available to bind adhesion-promoting cytokine-activating factors required for triggering activation-dependent lymphocyte sticking to the HEV.
  • compositions can be used for diagnostic and therapeutic purposes, for isolation of subsets of lymphocytes or other leukocytes, and the like.
  • ATC CAC CAG GAC CTG GAG GCA GGC TGG GAG CTA CTC TGT GAA GCA TCC 91 lie His Gin Asp Leu Glu Ala Gly Trp Glu Leu Leu Cys Glu Ala Ser
  • Trp Glu Leu Leu Cys Glu Ala Ser Cys Gly Pro Gly Val Thr Val Arg 290 295 300
  • Trp lie Gly Ser Leu Val Leu Gly Leu Leu Ala Leu Val Phe Leu Ala 370 375 380

Abstract

Acides nucléiques et protéines purifiés de mammifères codant des adressines de muqueuses. Les protéines, acides nucléiques et fragments desdits éléments servent une grande diversité d'objectifs pour moduler le guidage des leucocytes vers des muqueuses. De plus, les fragments protéiques peuvent servir d'antigènes pour produire des anticorps spécifiques de domaines particuliers de l'adressine de muqueuses, tandis que les acides nucléiques peuvent être utilisés comme sondes, pour la préparation anti-sens, pour l'introduction dans des hôtes qui peuvent être allogéniques ou xénogéniques à la source d'ADN en vue de l'expression de la protéine, et autres.
PCT/US1993/011404 1992-12-15 1993-11-23 Adressine vasculaire de muqueuses, adn codant ladite adressine et expression de ladite adressine WO1994013312A1 (fr)

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AU56757/94A AU5675794A (en) 1992-12-15 1993-11-23 Mucosal vascular addressin, dna and expression

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US07/990,866 1992-12-15

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WO1996024673A1 (fr) * 1995-02-10 1996-08-15 Leukosite, Inc. Adressines vasculaires de muqueuses et leurs utilisations
EP0948597A1 (fr) * 1996-11-01 1999-10-13 Human Genome Sciences, Inc. MOLECULE 1 D'ADHERENCE CELLULAIRE D'ADRESSINE DE MUQUEUSE HUMAINE (MAdCAM-1) ET VARIANTS D'EPISSURE DE CETTE DERNIERE
US5989552A (en) * 1993-12-24 1999-11-23 Austin Research Institute Antigen carbohydrate compounds and their use in immunotherapy
WO2002096949A1 (fr) * 2001-05-25 2002-12-05 The Council Of The Queensland Institute Of Medical Research Ciblage d'antigenes
US6548643B1 (en) 1994-11-16 2003-04-15 Austin Research Institute Antigen carbohydrate compounds and their use in immunotherapy
US6551593B1 (en) 1995-02-10 2003-04-22 Millennium Pharmaceuticals, Inc. Treatment of Inflammatory bowel disease by inhibiting binding and/or signalling through α 4 β 7 and its ligands and madcam
US7147851B1 (en) 1996-08-15 2006-12-12 Millennium Pharmaceuticals, Inc. Humanized immunoglobulin reactive with α4β7 integrin
WO2007007152A2 (fr) * 2005-07-11 2007-01-18 Pfizer Limited Anticorps anti-madcam utilises pour le traitement de cancers metastatiques et du chlorome
US7750137B2 (en) 1995-09-01 2010-07-06 Millennium Pharmaceuticals, Inc. Mucosal vascular addressins
US7803904B2 (en) 1995-09-01 2010-09-28 Millennium Pharmaceuticals, Inc. Mucosal vascular addressing and uses thereof
US7932372B2 (en) 2004-01-09 2011-04-26 Amgen Fremont Inc. Antibodies to MAdCAM
US8021667B2 (en) 1994-11-16 2011-09-20 Macfarlane Burnet Institute For Medical Research And Public Health Ltd Compositions for immunotherapy and uses thereof
US20120094937A1 (en) * 1996-08-16 2012-04-19 Schering Corporation Mammalian cell surface antigens; related reagents
US8349575B2 (en) 2004-12-07 2013-01-08 The Council Of The Queensland Institute Of Medical Research (Qimr) Relay vaccine
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US9663579B2 (en) 2011-05-02 2017-05-30 Millennium Pharmaceuticals, Inc. Formulation for anti-α4β7 antibody
US10040855B2 (en) 2011-05-02 2018-08-07 Millennium Pharmaceuticals, Inc. Formulation for anti-α4β7 antibody
US11802156B2 (en) 2017-07-14 2023-10-31 Pfizer Inc. Antibodies to MAdCAM

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US5989552A (en) * 1993-12-24 1999-11-23 Austin Research Institute Antigen carbohydrate compounds and their use in immunotherapy
US8021667B2 (en) 1994-11-16 2011-09-20 Macfarlane Burnet Institute For Medical Research And Public Health Ltd Compositions for immunotherapy and uses thereof
US6548643B1 (en) 1994-11-16 2003-04-15 Austin Research Institute Antigen carbohydrate compounds and their use in immunotherapy
US6551593B1 (en) 1995-02-10 2003-04-22 Millennium Pharmaceuticals, Inc. Treatment of Inflammatory bowel disease by inhibiting binding and/or signalling through α 4 β 7 and its ligands and madcam
WO1996024673A1 (fr) * 1995-02-10 1996-08-15 Leukosite, Inc. Adressines vasculaires de muqueuses et leurs utilisations
US7750137B2 (en) 1995-09-01 2010-07-06 Millennium Pharmaceuticals, Inc. Mucosal vascular addressins
US8277808B2 (en) 1995-09-01 2012-10-02 Millennium Pharmaceuticals, Inc. Mucosal vascular addressins and uses thereof
US7803904B2 (en) 1995-09-01 2010-09-28 Millennium Pharmaceuticals, Inc. Mucosal vascular addressing and uses thereof
US7147851B1 (en) 1996-08-15 2006-12-12 Millennium Pharmaceuticals, Inc. Humanized immunoglobulin reactive with α4β7 integrin
US7402410B2 (en) 1996-08-15 2008-07-22 Millennium Pharmaceuticals, Inc. Humanized immunoglobulin reactive with α4β7 integrin
US20120094937A1 (en) * 1996-08-16 2012-04-19 Schering Corporation Mammalian cell surface antigens; related reagents
EP0948597A4 (fr) * 1996-11-01 2002-07-10 Human Genome Sciences Inc MOLECULE 1 D'ADHERENCE CELLULAIRE D'ADRESSINE DE MUQUEUSE HUMAINE (MAdCAM-1) ET VARIANTS D'EPISSURE DE CETTE DERNIERE
EP0948597A1 (fr) * 1996-11-01 1999-10-13 Human Genome Sciences, Inc. MOLECULE 1 D'ADHERENCE CELLULAIRE D'ADRESSINE DE MUQUEUSE HUMAINE (MAdCAM-1) ET VARIANTS D'EPISSURE DE CETTE DERNIERE
US8771701B2 (en) 1997-09-29 2014-07-08 Macfarlane Burnet Institute For Medical Research And Public Health Ltd Compositions for immunotherapy and uses thereof
US8097430B2 (en) 2001-05-25 2012-01-17 The Council Of Queensland Institute Of Medical Research Antigen targeting
WO2002096949A1 (fr) * 2001-05-25 2002-12-05 The Council Of The Queensland Institute Of Medical Research Ciblage d'antigenes
US10259872B2 (en) 2004-01-09 2019-04-16 Pfizer, Inc. Antibodies to MAdCAM
USRE45847E1 (en) 2004-01-09 2016-01-19 Pfizer Inc. Antibodies to MAdCAM
US9328169B2 (en) 2004-01-09 2016-05-03 Pfizer Inc. Human antibodies that bind human MAdCAM
US7932372B2 (en) 2004-01-09 2011-04-26 Amgen Fremont Inc. Antibodies to MAdCAM
US8349575B2 (en) 2004-12-07 2013-01-08 The Council Of The Queensland Institute Of Medical Research (Qimr) Relay vaccine
WO2007007152A2 (fr) * 2005-07-11 2007-01-18 Pfizer Limited Anticorps anti-madcam utilises pour le traitement de cancers metastatiques et du chlorome
WO2007007152A3 (fr) * 2005-07-11 2007-04-19 Pfizer Ltd Anticorps anti-madcam utilises pour le traitement de cancers metastatiques et du chlorome
US9663579B2 (en) 2011-05-02 2017-05-30 Millennium Pharmaceuticals, Inc. Formulation for anti-α4β7 antibody
US10004808B2 (en) 2011-05-02 2018-06-26 Millennium Pharmaceuticals, Inc. Methods of treating ulcerative colitis
US10040855B2 (en) 2011-05-02 2018-08-07 Millennium Pharmaceuticals, Inc. Formulation for anti-α4β7 antibody
US10143752B2 (en) 2011-05-02 2018-12-04 Millennium Pharmaceuticals, Inc. Methods of treating ulcerative colitis
US9764033B2 (en) 2011-05-02 2017-09-19 Millennium Pharmaceuticals, Inc. Formulation for anti-α4β7 antibody
US11560434B2 (en) 2011-05-02 2023-01-24 Millennium Pharmaceuticals, Inc. Formulation for anti-α4β7 antibody
US11802156B2 (en) 2017-07-14 2023-10-31 Pfizer Inc. Antibodies to MAdCAM

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