WO1998020110A1 - HUMAN MUCOSAL ADDRESSIN CELL ADHESION MOLECULE-1 (MAdCAM-1) AND SPLICE VARIANTS THEREOF - Google Patents
HUMAN MUCOSAL ADDRESSIN CELL ADHESION MOLECULE-1 (MAdCAM-1) AND SPLICE VARIANTS THEREOF Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- MAdCAM-1 Human Mucosal Addressin Cell Adhesion Molecule- 1 (MAdCAM-1) and Splice Variants Thereof
- the present invention relates to novel cell surface adhesion molecules.
- isolated nucleic acid molecules are provided encoding a human mucosal vascular addressin cell adhesion molecule (MAdCAM-l(a)), as well as 4 splice variants thereof, designated MAdCAM-l(b), -1(c). -1(d), and -1(e).
- MAdCAM-l(a-e) polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same.
- the invention further relates to screening methods for identifying agonists and antagonists of MAdCAM-l(a-e) activity. Also provided are diagnostic methods for detecting cancer or a pathological inflammatory condition, and therapeutic methods for treating an individual in need of a reduction in the activity of any of MAdCAM-l(a-e).
- the invention provides isolated genomic DNA molecules comprising the 5 exons which comprise the genes which encode any of MAdCAM-l(a-e), as well as the 5' flanking region which includes the promoter for these genes.
- the invention relates to a method of screening compounds for the ability to regulate expression of any of MAdCAM- l(a-e) from their promoter.
- the invention also relates to a method of selectively expressing genes on gut endothelia.
- MAdCAM-1 (mucosal vascular addressin cell adhesion molecule- 1) is a mouse endothelial cell-surface adhesion molecule that interacts with the ⁇ 7 integrin LPAM-1 ( ⁇ 4 ⁇ 7), and participates in directing the traffic of leukocytes to mucosal and inflamed vasculature.
- MAdCAM-1 may also play a role in mediating the entry of antigen-nonspecific leukocytes into such sites since it is able to recognize both VLA-4 and LPAM-1 on activated monocytes/ macrophages (Leung et al, Immunol. Cell Biol. (1996); (in press)).
- a recombinant MAdCAM-1-IgFc chimera constructed from cDNA clones supported the adhesion of peripheral blood and spleen cells from a range of animal species, and binding was mediated by ⁇ 4 integrins (Yi et al, Scand. J. Immunol. 42: 235-47 (1995)).
- Transcripts encoding mouse MAdCAM-1 are detectable in various mouse tissues including mesenteric lymph nodes (MLN), Peyer's patches, spleen, and peripheral lymph nodes (PLN), but are absent from a pre-B lymphoma, liver, brain, and kidney (Briskin et al, Nature 363:461-64 (1993)).
- Complementary DNAs for MAdCAM-1 encode an immunoglobulin (Ig)-like molecule that bears strong homology with the addressins VCAM- 1 and ICAM- 1 , which are the endothelial ligands for the leukocyte integrins VLA-4 and LFA-1, respectively (Briskin et al, Nature 363:461-64 (1993)).
- the multidomain MAdCAM-1 structure comprises an N-terminal Ig domain that is similar to the N-terminal domains of ICAM-1 (32%) and VCAM-1 (28%); a second Ig domain that is similar to the fifth domain of VCAM-1 (30%); and a third Ig domain that shares similarity (33%) with the C ⁇ 2 domain of IgAl (Briskin et al, Nature 363:461-64 (1993)).
- the first Ig domain for MAdCAM-1 can support cell binding via LPAM-1 , but the second domain is needed to provide the full binding function of the receptor (Briskin et al, J. Immunol. 156: 719-26 (1996)). Between Ig domains two and three is a serine/threonine/proline-rich mucin domain that is decorated with carbohydrate determinants recognized by L-selectin.
- MAdCAM-1 purified from mesenteric lymph nodes is able to support the rolling of lymphocytes under shear, in a fashion similar to the selectin-dependent rolling of lymphocytes under shear and to the selectin-dependent rolling of neutrophils which precedes leukocyte extravasation (Berg et al, Nature 366: 695- 98 (1993)).
- the selectin-binding carbohydrate determinants are likely to be generated by cell-type specific glycosyltransferases. since neither stimulated bEnd.3 enthothelioma cells (Berg et al, Nature 366: 695-98 (1993)), nor recombinant MAdCAM-1 (Briskin et al, J. Immunol.
- MAdCAM-1 has dual functions in that it engages in both primary contact formation via L-selectin and LPAM-1, and adhesion strengthening via LPAM-1. In certain cell types the interaction of MAdCAM-1 with VLA-4 may play a contributory role.
- Murine MAdCAM-1 is located on chromosome 10 and contains 5 exons, with the mucin-like region and the third Ig domain encoded together in exon 4.
- An alternatively spliced murine MAdCAM-1 mRNA has been identified that lacks the IgA/mucin homologous exon 4-encoded segment.
- the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding any of the MAdC AM- 1 polypeptides, designated MAdCAM-l(a-e), wherein MAdCAM-l(a) has the amino acid sequence shown in FIG. 1 (SEQ ID NO:2); MAdCAM-l(b) has the amino acid sequence shown in FIG. 2 (SEQ ID NO:4); MAdCAM-l(c) has the amino acid sequence shown in FIG. 3 (SEQ ID NO:6); MAdCAM-l(d) has the amino acid sequence shown in FIG. 4 (SEQ ID NO:8); and MAdCAM-l(e) has the amino acid sequence shown in FIG. 5 (SEQ ID NO: 10).
- the invention also relates to isolated genomic DNA molecules comprising the 5 exons which, in various combinations, comprise the coding region of any of the MAdC AM- 1 splice variants (MAdCAM-l(a-e)), as well as sequence located 5' to the start codon of the first exon, which includes the promoter for the MAdCAM-1 splice variants.
- the genomic DNA sequence of the 5 exons encoding the MAdC AM- 1 proteins, as well as the genomic DNA sequence of the sequence located 5' to the start codon of the first exon, is shown in FIG. 6.
- the sequence of the 5' flanking region, which includes the promoter for the genes encoding any of MAdC AM- l(a-e). is given in SEQ ID NO:33.
- the sequences of exons 1-5 are given in SEQ ID NOS:34, 35, 36, 37. and 38, respectively (hereinafter referred to as SEQ ID NOS:34-38).
- the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for the production of any of the MAdCAM-l(a- e) polypeptides or peptides (including peptides corresponding to exons 1-5, described above) by recombinant techniques.
- the present invention also relates to an isolated nucleic acid molecule comprising a polynucleotide encoding any of the MAdC AM- 1 polypeptides encoded by the genomic clone deposited in a bacterial host as ATCC Deposit Number 97758 on October 10, 1996.
- the nucleotide sequence determined by sequencing portions of the deposited genomic DNA, which is shown in FIG. 6, includes the sequence of the 5' flanking region, given in SEQ ID NO:33, as well as the sequences of exons 1-5, given in SEQ ID NOS:34-38, respectively.
- the invention further provides isolated MAdC AM- 1 polypeptides
- MAdCAM-l(a-e) having an amino acid sequence encoded by a polynucleotide described herein.
- the present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by any of the MAdCAM-1 polypeptides (designated MAdCAM-l(a-e)), which involves contacting cells which express the desired MAdCAM-1 polypeptides with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
- a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by any of the MAdCAM-1 polypeptides designated MAdCAM-l(a-e)
- the invention also provides a diagnostic method useful during diagnosis of an inflammatory disorder.
- An additional aspect of the invention is related to a method for treating an individual in need of a decreased level of MAdCAM-l(a-e) activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of MAdCAM-l(a-e)-mediated adhesion.
- Preferred antagonists for use in the present invention are MAdCAM- l(a-e)-specific antibodies, as well as soluble forms of MAdCAM-l(a-e).
- the invention also includes isolated genomic DNA molecules comprising the 5' flanking region of MAdCAM-l(a-e), including the promoter for these genes, yet another aspect of the invention is related to a method for identifying compounds capable of enhancing or inhibiting expression of any of MAdCAM-l(a-e). Because MAdCAM-1 is selectively expressed on HEV and on lamina limba venules, the promoter can also be used to selectively target therapeutic genes to the gut endothelia.
- FIGS. 1 A and IB show the nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ ID NO:2) sequences of MAdCAM-l(a).
- the protein has a leader sequence of about 17 amino acid residues (first underlined region), followed by an extracellular domain.
- the second underlined region corresponds to the transmembrane domain, and is followed by the intracellular domain.
- the predicted amino acid sequence of the mature MAdC AM- 1 (a) protein (which lacks the leader sequence) is also shown in FIG. 1 (SEQ ID NO:2).
- FIGS. 2A and 2B show the nucleotide (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:4) sequences of MAdCAM-l(b).
- the protein has a leader sequence of about 17 amino acid residues (first underlined region), followed by an extracellular domain.
- the second underlined region corresponds to the transmembrane domain, and is followed by the intracellular domain.
- the predicted amino acid sequence of the mature MAdC AM- 1(b) protein (which lacks the leader sequence) is also shown in FIG. 2 (SEQ ID NO:4).
- FIG. 3 shows the nucleotide (SEQ ID NO:5) and deduced amino acid (SEQ ID NO:6) sequences of MAdC AM- 1(c).
- the protein has a leader sequence of about 17 amino acid residues (first underlined region), followed by an extracellular domain.
- the second underlined region corresponds to the transmembrane domain, and is followed by the intracellular domain.
- the predicted amino acid sequence of the mature MAdC AM- 1 (b) protein (which lacks the leader sequence) is also shown in FIG. 3 (SEQ ID NO:6).
- FIGS. 4 A and 4B show the nucleotide (SEQ ID NO: 7) and deduced amino acid (SEQ ID NO:8) sequences of MAdCAM-l(d).
- the protein has a leader sequence of about 17 amino acid residues (first underlined region), followed by an extracellular domain.
- the second underlined region corresponds to the transmembrane domain, and is followed by the intracellular domain.
- the predicted amino acid sequence of the mature MAdC AM- 1(d) protein (which lacks the leader sequence) is also shown in FIG. 4 (SEQ ID NO:8).
- FIGS. 5A and 5B show the nucleotide (SEQ ID NO:9) and deduced amino acid (SEQ ID NO: 10) sequences of MAdC AM- 1(e).
- the protein has a leader sequence of about 17 amino acid residues (first underlined region), followed by an extracellular domain.
- the second underlined region corresponds to the transmembrane domain, and is followed by the intracellular domain.
- the predicted amino acid sequence of the mature MAdC AM- 1 (e) protein (which lacks the leader sequence) is also shown in FIG. 5 (SEQ ID NO: 10).
- FIGS. 6A and 6B show the nucleotide sequence of genomic DNA encoding the region 5' to the gene encoding MAdCAM-1 (SEQ ID NO:33). Also shown are exons 1-5 (SEQ ID NOS:34-38, respectively), which comprise the genes which encode any of MAdCAM-l(a-e). Lower case letters represent intron sequence.
- FIGS. 7A and 7B show the regions of similarity between the predicted amino acid sequences of the human MAdCAM-l(a-e) proteins (SEQ ID NOS:2, 4, 6, 8, 10, respectively), mouse MAdCAM-1 (SEQ ID NO:46), and the predicted amino acid sequence of human MAdCAM-1 from Shyjan et al, J Immunol. 75f5(8):2851-2857 (1996) (SEQ ID NO:47).
- FIG. 8 shows an analysis of the MAdCAM-l(a) amino acid sequence.
- Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
- amino acid residues 52- 80, 164-296 and 228-321 in FIG. 1 correspond to the shown highly antigenic regions of the MAdCAM-l(a) protein.
- FIG. 9A shows the isolation of MAdC AM- 1 (a) cDNA.
- MAdC AM- 1 (a) cDNAs were initially identified as expressed sequence tags (ESTs), clones HEBBC23X and Y, in an EST database created from an early stage human brain cDNA library. The insert of clone HEBBC23Y was subsequently used to isolate clone MAD-C1 from a human cosmid library. Complementary DNA encoding the 5'-end of human MAdCAM-l(a) was obtained by PCR using PCR primers designed from HEBBC23X and MAD-C1 , yielding PCR clone PCR1-5'. The upper FIG.
- FIG. 9B shows nucleotide and deduced amino acid sequence of human
- MAdCAM-l(a) SEQ ID NOS:l and 2).
- the numbers in the right-hand margin show nucleotide and amino acid positions, respectively.
- the initiation methionine has been assigned to position 1 by comparison with the mouse MAdCAM-l(a) sequence.
- the putative signal peptide and transmembrane domains are underlined.
- the major (residues 226 to 273) mucin domain is boxed, and the minor mucin (residues 278 to 31 1) domain is italicized, and cysteines expected to form disulphide bonds in the two immunoglobulin domains are circled.
- a potential polyadenylation signal site is overlined.
- FIGS. 10A and 10B show a comparison of the major mucin domain of human MAdCAM-l(a) with the imperfect repeats of the mucin domain of the intestinal mucin MUC-2.
- FIG. 10A the six octomer repeats comprising the major mucin domain of MAdCAM-l(a) have been aligned (SEQ ID NOS:49, 50,
- FIGS. 11 A and 1 IB show an identification of MAdC AM- 1 splice variants
- FIG. 11 A partial sequences of MAdCAM-1 splice variants encoding the second Ig domain and the major mucin domain or parts thereof have been aligned.
- HEBBC23Y which is missing 3 mucin repeats, was identified as an EST.
- Sequences 3. 5 and 7 are missing a major portion of the second Ig domain and 3 to 6 mucin repeats were isolated as PCR products following amplification from fetal brain RNA.
- FIG. 1 IB sequences of acceptor and donor splice sites in MAdCAM-1 variants are shown.
- FIG. 12 shows proposed structures for MAdCAM-1 splice variants.
- the Ig domains are shown as ovals, and the mucin domains are represented as decorated rods, where the minor mucin domain is less decorated.
- FIG. 13 shows the DNA sequence of the 5'-fianking region of the human MAdC AM- 1 gene (SEQ IDNO:33) and comparison with the mouse MAdC AM- 1 promoter (SEQ ID NO:48). Numbers refer to nucleotide positions and are relative to the translational start codon, which is underlined. Potential transcriptional factor binding sites identified in the human and mouse 5 '-flanking regions are underlined. Identical nucleotides shared by the human and mouse sequences are denoted by vertical lines.
- FIGS. 14 A, 14B and 14C show that the 5 '-flanking region of the human MAdC AM- 1 gene has promoter activity in the human dermal endothelial cell line HMEC.
- Figure 14A is a schematic representation of the basic luciferase vector pGL-2/B, and the expression vectors pGL-2/B-718+ and pGL-2/B-718- derived from it, which contain a 700 bp 5'-flanking region (-718 to +20 relative to the translational start) in sense and antisense orientations, respectively.
- Figure 14B and 14C show the relative luciferase activity directed by the expression vectors in the human dermal endothelial cell line HMEC. The results are from two separate experiments where promoter activity is expressed as the relative photon count above the background control of cells transfected with no DNA.
- the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding any one of the MAdCAM-l(a-e) polypeptides having the amino acid sequences shown in FIGS. 1-5 (SEQ ID NOs:2, 4, 6, 8, 10), respectively, which was determined by sequencing a cloned cDNA.
- the MAdC AM- 1 (a-e) proteins of the present invention share sequence homology with mouse MAdCAM-1 (FIG. 7A and 7B) (SEQ ID NO:46).
- the nucleotide sequence shown in FIG. 1 (SEQ ID NO:l) was obtained by sequencing the HEBBC23 clone.
- SEQ ID NO:3 was obtained by sequencing the HSKCW36 clone, which encodes MAdCAM-l(b), a splicing variant of the deposited cDNA clone described below.
- the nucleotide sequence shown in FIG. 3(SEQ ID NO:5) was obtained by sequencing the MAdCAM-lc clone, which encodes MAdCAM-l(c), a splicing variant of the deposited cDNA clone described below.
- the nucleotide sequence shown in FIG. 4 (SEQ ID NO:7) was obtained by sequencing the MAdCAM-ld clone, which encodes MAdC AM- 1(d), a splicing variant of the deposited cDNA clone described below.
- the nucleotide sequence shown in FIG. 5(SEQ ID NO:9) was obtained by sequencing the MAdCAM-le clone, which encodes MAdC AM- 1(e), a splicing variant of the deposited cDNA clone described below.
- the invention also relates to isolated genomic DNA molecules comprising the 5 exons (all of which are shown in Fig. 6) which comprise the coding region of any of the MAdCAM- 1 splice variants (MAdCAM- 1 (a-e)), as well as sequence located 5' to the start codon of the first exon, which includes the promoter for the MAdCAM- 1 splice variants.
- a genomic clone comprising this genomic DNA was deposited on October 10, 1996, at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, and given accession number 97758.
- the sequence of the 5' flanking region, which includes the promoter for the genes encoding any of MAdCAM- 1 (a-e) is given in SEQ ID NO:33.
- exons 1-5 are given in SEQ ID NOS:34-38, respectively.
- Example 6 gives further description of how the 5 exons shown in FIG. 6, or portions thereof, can be combined in order to generate the splice variants of MAd-C AM- 1.
- the present invention also relates to isolated nucleic acid molecules comprising a polynucleotide encoding the MAdCAM- 1 (a) polypeptide encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 97759 on October 10, 1996.
- the deposited clone is contained in the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA).
- Nucleic Acid Molecules Nucleic Acid Molecules
- nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about
- the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
- a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
- a nucleic acid molecule of the present invention encoding any of the MAdCAM- 1 (a-e) polypeptides may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
- the nucleic acid molecules described in FIGS. 1 -5 (SEQ ID NOs: 1 , 3, 5, 7, 9) were discovered in a cDNA library derived from human fetal brain cells. The genes were also identified in cDNA libraries from the following tissues: small intestine, colon, spleen, and pancreas. The determined nucleotide sequences of the MAdCAM- 1 (a-e) cDNAs of FIGS.
- MAdCAM- 1 (a-e) has an initiation codon at positions 1-3 of their respective nucleotide sequence in FIGS. 1-5 (SEQ ID NOs: 1, 3, 5, 7, 9), and each has a predicted leader sequence of about 17 amino acid residues.
- the mature MAdCAM- 1 (a-e) polypeptides will of course lack this leader sequence.
- the deduced molecular weights of complete MAdCAM- 1 (a-e) polypeptides are about 40, 38, 27, 32 and 32.4 kDa, respectively.
- the invention in another aspect, relates to isolated genomic DNA molecules comprising the 5 exons which comprise the coding region of any of the MAdCAM-1 splice variants (MAdCAM- 1 (a-e)), as well as sequence located 5' to the start codon of the first exon, which includes the promoter for the MAdCAM- 1 splice variants.
- the sequence of the 5' flanking region, which includes the promoter for the genes encoding any of MAdCAM- 1 (a-e) is given in SEQ ID NO:33.
- the sequences of exons 1-5 are given in SEQ ID NOS:34-38, respectively.
- the invention provides isolated nucleic acid molecules comprising the genomic DNA sequence contained in the clone deposited as ATCC Deposit No. 97758 on October 10, 1996.
- the present invention also relates to isolated nucleic acid molecules comprising a polynucleotide encoding the MAdCAM- 1(a) polypeptide encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 97759 on October 10, 1996.
- the amino acid sequence of the mature MAdCAM- 1(a) protein is shown in FIG. 1, amino acid residues 18-382 (SEQ ID NO:2).
- the present invention also provides the mature form(s) of the MAdCAM- 1 (a-e) proteins of the present invention.
- proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
- Most mammalian cells and even insect cells cleave secreted proteins with the same specificity.
- cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein.
- the present invention provides a nucleotide sequence encoding the mature amino acid sequence of the polypeptide.
- the mature MAdCAM- 1 (a-e) proteins shown in FIGS. 1-5 is meant the mature form(s) of the MAdCAM- 1 proteins produced by expression in a mammalian cell (e.g., COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the cDNA clone contained in the vector in the deposited host.
- the actual mature MAdCAM- 1 (a-e) polypeptides may or may not differ from the predicted "mature" MAdCAM- 1 (a-e) polypeptides shown in FIGS 1-5, depending on the accuracy of the predicted cleavage site based on computer analysis.
- the predicted amino acid sequence of the complete MAdCAM- 1 (a-e) polypeptides of the present invention were analyzed by a computer program ("PSORT") (K. Nakai and M. Kanehisa, Genomics 14:897-911 (1992)), which is an expert system for predicting the cellular location of a protein based on the amino acid sequence.
- PSORT computer program
- McGeoch and von Heinje are incorporated.
- the analysis by the PSORT program predicted the cleavage sites between amino acids
- the predicted leader sequence of the MAdCAM- 1 (a-e) proteins of the present invention are predicted to be about 17 amino acids in length, but may be anywhere in the range of about 14 to about 22 amino acids.
- the predicted polypeptide corresponding to MAdCAM- 1 (a) comprises about 382 amino acids, but may be anywhere in the range of 368-396 amino acids.
- MAdCAM- 1(b) comprises about 366 amino acids, but may be anywhere in the range of 348-382 amino acids.
- the predicted polypeptide corresponding to MAdCAM- 1(c) comprises about 263 amino acids, but may be anywhere in the range of 250-276 amino acids.
- the predicted polypeptide corresponding to MAdCAM- 1(d) comprises about 310 amino acids, but may be anywhere in the range of 294-325 amino acids.
- the predicted polypeptide corresponding to MAdCAM- 1(e) comprises about 289 amino acids, but may be anywhere in the range of 275-304 amino acids.
- nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
- the DNA may be double-stranded or single-stranded.
- Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
- isolated nucleic acid molecule(s) is intended a nucleic acid molecule,
- DNA or RNA which has been removed from its native environment
- recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
- isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
- Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention.
- Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
- Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in FIGS. 1-5 (SEQ ID NOS. 1-5 (SEQ ID NOS. 1-5 (SEQ ID NOS. 1-5 (SEQ ID NOS. 1-5).
- the invention also includes DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode any of the MAdCAM- 1 (a-e) proteins.
- the genetic code is well known in the art.
- the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in FIGS. 1-6 (SEQ ID NOs:l, 3, 5, 7, 9, 33, 34, 35, 36, 37, and 38, respectively), or a nucleic acid molecule having a sequence complementary to one of the above sequences.
- isolated molecules particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the MAdCAM- l(a-e) genes in human tissue, for instance, by northern blot analysis.
- the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
- a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the nucleotide sequences shown in FIGS. 1-6 is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
- fragments 50-1 150 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence shown in FIGS. 1 -6 (SEQ ID NOs:l, 3, 5, 7, 9, 33, 34, 35, 36, 37, and 38, respectively).
- a fragment at least 20 nt in length for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the nucleotide sequences as shown in FIGS. 1-6 (SEQ ID NOs:l, 3, 5, 7, 9, 33, 34, 35, 36, 37, and 38, respectively).
- nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions, or the transmembrane domain, or the extracellular domain, or the intracellular domain, of the MAdCAM- 1 (a-e) proteins.
- nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about 52 to about 80 in FIG. 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 164 to about 196 in FIG. 1 (SEQ ID NO:2); and a polypeptide comprising amino acid residues from about 278 to about 321 in FIG. 1 (SEQ ID NO:2).
- the inventors have determined that the above polypeptide fragments are antigenic regions of the MAdCAM- 1 (a-e) proteins. Methods for determining other such epitope-bearing portions of the MAdCAM- 1 (a-e) proteins are described in detail below).
- nucleic acid fragments include the genomic region 5' to the MAdCAM- 1 gene (nucleotides residue 1 through 718 of SEQ ID NO:33), and fragments which correspond to exon 1 (nucleotide residues 1-52 of SEQ ID NO:34), exon 2 (nucleotide residues 11-295 of SEQ ID NO:35), exon 3 (nucleotide residues 11- 340 of SEQ ID NO:36), exon 4 (nucleotide residues 11-343 of SEQ ID NO:37), and exon 5 (nucleotide residues 11-608 of SEQ ID NO:38) all of which are shown in FIG. 6.
- Example 6 which clearly mark functional domains in the molecule, will be helpful in designing variant forms of MAdCAM- 1 for use in therapy (see below).
- the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above.
- stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50%> formamide, 5x SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10%> dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA. followed by washing the filters in O.lx SSC at about 65 °C.
- a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
- a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly(A) tract of any of the MAdCAM- 1 (a-e) cDNAs shown in FIGS.
- nucleic acid molecules of the present invention which encode any of the MAdCAM- 1 (a-e) polypeptides may include, but are not limited to, those encoding the amino acid sequence of the mature polypeptides, by themselves; the coding sequence for the mature polypeptides and additional sequences, such as those encoding the about 17 amino acid leader or secretory sequence, such as a pre-, or pro- or prepro-protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5 ' and 3 ' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
- the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
- the marker amino acid sequence is a hexa-histidine peptide.
- the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
- hexa-histidine provides for convenient purification of the fusion protein.
- the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37: 767 (1984).
- other such fusion proteins include any of the MAdCAM- 1 (a-e) polypeptides fused to Fc at the N- or C-terminus.
- the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the MAdCAM- 1 (a-e) proteins.
- Variants may occur naturally, such as a natural allelic variant.
- allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
- variants include those produced by nucleotide substitutions, deletions or additions, which may involve one or more nucleotides.
- the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the MAdCAM- 1 (a-e) proteins or portions thereof. Also especially preferred in this regard are conservative substitutions.
- nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90%o identical, and more preferably at least 95%. 96%>, 97%, 98% or 99%> identical to (a) a nucleotide sequences encoding the full-length MAdCAM-l(a-e) polypeptides having the complete amino acid sequence in FIGS. 1-5 (SEQ ID NOs:2, 4, 6, 8, 10, respectively), including the predicted leader sequence; (b) a nucleotide sequence encoding the mature MAdCAM- 1 (a-e) polypeptides
- full-length polypeptide with the leader removed having the amino acid sequences at positions 18-382 in FIG. 1 (SEQ ID NO:2), 18-366 in FIG. 2 (SEQ ID NO:4), 18-263 in FIG. 3 (SEQ ID NO:6), 18-310 in FIG. 4 (SEQ ID NO:8), or 18-290 in FIG.
- MAdCAM- 1 promoter wherein the nucleotide sequence is given in SEQ ID NO:33; (g) a nucleotide sequence encoding exon 1, 2, 3, 4 or 5 of MAdCAM-1, having the sequence given in SEQ ID NOS:34, 35, 36, 37 and 38, respectively; and (h) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f) or (g), above.
- polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding any of the MAdCAM- 1 (a-e) polypeptides is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding any of the MAdC AM - l(a-e) polypeptides.
- a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
- These mutations of the reference sequence may occur at the 5 ' or 3 ' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
- nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequences shown in FIGS. 1-6 or to the nucleotides sequence of the deposited genomic clone, or to the deposited cDNA clone, can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences.
- Bestfit program Wiconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences.
- the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
- the present application is directed to nucleic acid molecules at least 90%, 95%o, 96%), 97%o, 98%o or 99% identical to the nucleic acid sequences shown in FIGS. 1-6 (SEQ ID NOs:l, 3, 5, 7, 9, 33, 34, 35, 36, 37, and 38, respectively), or to the nucleic acid sequence of the deposited genomic DNA, irrespective of whether they encode a polypeptide having the activity of any of MAdCAM- 1 (a-e).
- nucleic acid molecule does not encode a polypeptide having MAdCAM- 1 (a-e) activity
- a-e MAdCAM- 1
- PCR polymerase chain reaction
- nucleic acid molecules of the present invention that do not encode a polypeptide having the activity of any of MAdCAM- 1 (a-e) include, inter alia, (1) isolating the gene encoding MAdCAM- 1 (a-e) or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the gene encoding MAdCAM- 1 (a-e), as described in Verma et al, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and Northern Blot analysis for detecting mRNA expression of any of MAdCAM- 1 (a-e) in specific tissues.
- FISH in situ hybridization
- nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to any of the nucleic acid sequences shown in FIGS. 1-6 (SEQ ID NOs:l, 3, 5, 7, 9, 33, 34, 35, 36, 37, and 38, respectively), or to the nucleic acid sequence of the deposited genomic DNA which does, in fact, encode a polypeptide having the protein activity of any of
- MAdCAM-l(a-e) a polypeptide having the protein activity of any of MAdCAM- 1 (a-e)
- the protein activity of any of MAdCAM- 1 (a-e) can be measured by using a variation of the Stamper- Woodruff in vitro lymphocyte-endothelial cell binding assay (J. Exp. Med.
- the assay involves contacting a cell which expresses ⁇ 4 ⁇ 7 (such as TK1 cells) and thus binds to cells expressing any of MAdCAM- 1 (a-e), with cells expressing any of the MAdCAM- 1 (a-e) molecules of the invention, and measuring the resultant adhesion between the two types of cells.
- a cell expressing the protein activity of any of MAdCAM- 1 (a-e) will bind to the cells expressing ⁇ 4 ⁇ 7
- a cell expressing a protein which does not bind to ⁇ 4 ⁇ 7 will be considered not to have the activity of any of MAdCAM- 1 (a-e).
- nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown in FIGS. 1-5 will encode a polypeptide "having the protein activity of any of MAdCAM- 1 (a-e)."
- degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
- nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having the protein activity of any of MAdCAM- 1 (a-e). This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g.. replacing one aliphatic amino acid with a second aliphatic amino acid).
- the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of any of the
- MAdCAM- 1 (a-e) polypeptides or fragments thereof by recombinant techniques are provided.
- the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
- a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
- the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retro viral LTRs, to name a few.
- an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retro viral LTRs, to name a few.
- the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
- the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
- the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
- bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells
- fungal cells such as yeast cells
- insect cells such as Drosophila S2 and Spodoptera Sf9 cells
- animal cells such as
- CHO, COS and Bowes melanoma cells CHO, COS and Bowes melanoma cells; and plant cells.
- Appropriate culture mediums and conditions for the above-described host cells are known in the art.
- vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors. Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A. available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
- preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
- Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).
- the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
- a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins.
- EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
- the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
- Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations.
- human proteins such as. hIL5- has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471 (1995).
- the MAdCAM- 1 (a-e) proteins can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
- Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
- polypeptides of the present invention may be glycosylated or may be non-glycosylated.
- polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
- the invention further provides isolated MAdCAM- 1 (a-e) polypeptides having the amino acid sequence given in FIG. 1-5 (SEQ ID NO:2, 4, 6, 8, 10, respectively), or a peptide or polypeptide comprising a portion of the above polypeptides, as well as any of the polypeptides encoded by the nucleotide sequence of exons 1-5 of FIG 6 (SEQ ID NOS:34-38).
- the invention further includes variations of the MAdCAM- 1 (a-e) polypeptides which show substantial MAdCAM- 1 (a-e) polypeptide activity or which include regions of any of the MAdCAM- 1 (a-e) proteins such as the protein portions discussed below.
- Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
- further guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247. 1306-1310 (1990).
- the fragment, derivative or analog of the polypeptide shown in FIGS 1-5 may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
- a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
- substituted amino acid residue may or may not
- changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1). TABLE 1. Conservative Amino Acid Substitutions.
- Amino acids in the MAdCAM- 1 (a-e) polypeptides of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vitro proliferative activity. Sites that are critical for protein activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).
- polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
- a recombinantly produced version of any of the MAdCAM- 1 (a-e) polypeptides can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
- polypeptides of the present invention include any of the polypeptides of FIGS. 1-5(SEQ ID NOS:2, 4, 6, 8, 10, respectively) including the leader, any of the mature polypeptides of FIGS. 1-5 (SEQ ID NOS:2, 4, 6, 8, 10, respectively) minus the leader (i.e., the mature protein), any of the polypeptides of FIGS. 1-5(SEQ ID NOS:2, 4, 6, 8, 10, respectively) minus the leader, the extracellular domain of any of the polypeptides of FIGS. 1-5(SEQ ID NOS:2, 4, 6, 8, 10, respectively), the intracellular domain of any of the polypeptides of FIGS.
- polypeptide variants of MAdCAM- 1 can be recombinantly prepared by combining exons, or portions of exons, of the sequences shown in FIG. 6 (SEQ ID NOS:34-38).
- polypeptides are also included in the invention.
- polypeptides which are at least 80%> identical, more preferably at least 90%) or 95% identical, still more preferably at least 96%, 97%, 98%o or 99% identical to the above-mentioned polypeptides, and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
- a reference amino acid sequence of any of the MAdCAM- 1 (a-e) polypeptides is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of any of the MAdCAM- 1 (a-e) polypeptides.
- up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
- alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polypeptide is at least 90%,
- the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5%o of the total number of amino acid residues in the reference sequence are allowed.
- polypeptide of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
- the invention provides a peptide or polypeptide comprising an epitope-bearing portion of the invention described hererin.
- the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
- An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.
- a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
- the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al, Proc. Natl. Acad. Sci. USA 57:3998- 4002 (1983).
- peptides or polypeptides bearing an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
- relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G.,
- Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
- Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al, Cell 37:161-11% (1984) at 777.
- Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
- Non-limiting examples of antigenic polypeptides or peptides that can be used to generate antibodies specific to any of the MAdCAM- 1 (a-e) polypeptides include: a polypeptide comprising amino acid residues from about 52 to about 80 in FIG. 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about 164 to about 196 in FIG.
- polypeptide fragments are antigenic regions of the endokine alpha protein.
- the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 52:5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,21 1 to Houghten et al. (1986).
- SMPS Simultaneous Multiple Peptide Synthesis
- the invention also relates to the diagnosis of a pathological inflammatory condition by identifying the presence of an enhanced level of one or more of the MAdCAM- 1 (a-e) proteins or mRNA encoding these proteins, as compared to a corresponding "standard" mammal, i.e., a mammal of the same species not having the pathological inflammatory condition.
- a pathological inflammatory condition include transplantation rejection, arthritis, rheumatoid arthritis, infection, dermatosis, inflammatory bowel disease, and autommune disease, including chronic relapsing experimental autoimmune encephalitis (EAE).
- tissue in mammals with cancer express significantly enhanced levels of one or more of the MAdCAM- 1 (a-e) proteins and mRNA encoding these proteins when compared to a corresponding "standard" mammal, i.e., a mammal of the same species not having the cancer.
- enhanced levels of any of the MAdCAM- 1 (a-e) proteins can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with cancer when compared to sera from mammals of the same species not having the cancer.
- the invention provides a diagnostic method useful during tumor diagnosis, which involves assaying the expression level of the gene encoding any of the MAdCAM- 1 (a-e) proteins in mammalian cells or body fluid and comparing the gene expression level with a standard expression level for that same gene, whereby an increase in the gene expression level over the standard is indicative of certain tumors.
- the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced expression of any of the MAdCAM- 1 (a-e) genes will experience a worse clinical outcome relative to patients expressing the relevant gene at a lower level.
- test the expression level of the gene encoding one or more of the MAdCAM- 1 (a-e) proteins is intended qualitatively or quantitatively measuring or estimating the level of one or more of the MAdCAM- 1 (a-e) proteins or the level of the mRNA encoding one or more of the MAdCAM- 1 (a-e) proteins in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the protein level or mRNA level of the same MAdCAM- l(a-e)in a second biological sample).
- the level of the MAdCAM- 1 (a-e) protein or mRNA level in the first biological sample is measured or estimated and compared to a standard protein level or mRNA level for the same protein, the standard being taken from a second biological sample obtained from an individual not having the cancer.
- a standard protein level or mRNA level for one or more of MAdCAM- 1 (a-e) is known, it can be used repeatedly as a standard for comparison.
- biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source which contains one or more of the MAdCAM- l(a-e)proteins or the mRNA encoding them.
- Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain a secreted mature protein, and ovarian, prostate, heart, placenta, pancreas liver, spleen, lung, breast and umbilical tissue.
- the present invention is useful for detecting cancer in mammals.
- the invention is useful during diagnosis of the of following types of cancers in mammals: lymphoma, leukemia, and metastatic tumors.
- Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
- Total cellular RNA can be isolated from a biological sample using the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 7(52. 156-159 (1987). Levels of mRNA encoding any of the MAdCAM- 1 (a-e) proteins are then assayed using any appropriate method. These include Northern blot analysis, S 1 nuclease mapping, the polymerase chain reaction (PCR). reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
- PCR polymerase chain reaction
- RT-PCR reverse transcription in combination with the polymerase chain reaction
- RT-LCR reverse transcription in combination with the ligase chain reaction
- Assaying protein levels of any of MAdC AM- 1 (a-e) in a biological sample can occur using antibody-based techniques.
- expression of any of the MAdCAM- 1 (a-e) polypeptides in tissues can be studied with classical immunohistological methods. (Jalkanen, M.. et al, J. Cell. Biol 707.976-985 (1985); Jalkanen, M, et al, J. Cell . Biol. 705.3087-3096 (1987)).
- Suitable antibody-based methods useful for detecting MAdCAM- 1 (a-e) protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
- Suitable labels are known in the art, and include enzyme labels, such as glucose oxidase, and radioisotopes, such as iodine ( 125 1, 121 I), carbon ( l4 C), sulfur ( 35 S), tritium ( H), indium (“ 2 In), and technetium ( 99m Tc). and fluorescent labels, such as fluorescein and rhodamine, and biotin.
- the nucleic acid molecules of the present invention are also valuable for chromosome identification.
- the sequence is specifically targeted to and can hybridize with human chromosome 19pl3.3.
- the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
- the cDNA herein disclosed is used to clone genomic DNA of any of the genes encoding MAdCAM- 1 (a-e) proteins. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA then is used for in situ chromosome mapping using well known techniques for this purpose.
- sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes.
- Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
- This technique can be used with probes from the cDNA as short as 50 or 60 bp.
- Verma et al Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988).
- the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V.
- circulating lymphocytes are believed to express a receptor for one or more of the MAdC AM - 1 proteins (MAdCAM- 1 (a-e)), bind to the MAdCAM- 1 protein on mucosal venules via this receptor, and then migrate through the venules to the epithelium, where acute inflammation results. Therefore, the administration of a therapeutic composition capable of blocking the migration of leukocytes via MAdCAM- 1 polypeptides (MAdCAM- 1 (a-e)) (i.e., an antagonist of the activity of any of MAdCAM- 1 (a-e)) could be an effective therapeutic treatment for minimizing tissue damage in many abnormal inflammatory conditions, especially where the inflammation is chronic or acute. Such conditions include transplantation rejection, arthritis, rheumatoid arthritis, infection, dermatosis, inflammatory bowel disease, and autommune disease, including chronic relapsing experimental autoimmune encephalitis (EAE).
- EAE chronic relapsing experimental autoimmune encephalitis
- the invention also relates to a therapeutic method for treating an individual in need of a reduction in the activity of any of MAdCAM- 1 (a-e) by administering to the individual a therapeutically effective amount of a composition comprising an antagonist of MAdCAM-l(a-e) activity.
- a composition comprising an antagonist of MAdCAM-l(a-e) activity.
- Such compounds include anti -MAdC AM- 1 antibodies or fragments thereof, as well as compounds such as solubilized ⁇ 4 ⁇ 7 .
- Such individuals can include those suffering from abnormal inflammatory conditions, especially where the inflammation is chronic or acute.
- the invention also includes using such compositions as a "preventative" treatment before detection of an inflammatory state, so as to prevent the development of inflammation in a patient at high risk for the same, such as, for example, transplant patients.
- the invention is further directed to antibody-based therapies which involve administering an antibody directed against any of MAdCAM- l(a- e), to a mammalian, preferably human, patient for treating one or more of the above-described disorders.
- antibody-based therapies which involve administering an antibody directed against any of MAdCAM- l(a- e), to a mammalian, preferably human, patient for treating one or more of the above-described disorders.
- Methods for producing such anti-MAdCAM-1 polyclonal and monoclonal antibodies are described in detail above.
- Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
- a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding any of the MAdCAM- 1 (a-e) polypeptides locally or systemically in the body. Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
- the antagonists of MAdCAM- 1 (a-e) activity of the invention may also include soluble forms of any of the MAdCAM- 1 (a-e) polypeptides.
- the administration of soluble forms of any of the MAdCAM- 1 (a-e) polypeptides may block leukocyte adhesion to endothelium at sites of inflammation.
- soluble forms of any of the MAdCAM- 1 (a-e) polypeptides may block leukocyte adhesion to endothelium at sites of inflammation.
- the invention further provides a method of treating an individual in need of a decreased level of MAdCAM- l(a- e)-mediated adhesion comprising administering to such an individual a pharmaceutical composition comprising an effective amount of antagonist of any of the MAdCAM- 1 (a-e) polypeptides of the invention.
- Such antagonists include anti-MAdCAM-1 antibodies or fragments or derivatives thereof, as well as compounds such as solubilized ⁇ 4 ⁇ 7 , or soluble forms of any of MAdCAM- l(a- e), which are effective to decrease the activity level of the desired MAdCAM- l(a-e) protein in such an individual.
- the total pharmaceutically effective amount of one or more of the antagonists, including antibodies, soluble forms of ⁇ 4 ⁇ 7 , and soluble forms of the MAdCAM- 1 (a-e) polypeptides, administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion.
- this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
- the desired antagonist of the MAdCAM- 1 (a-e) polypeptides is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour. either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.
- compositions containing one or more of the antagonists of the MAdCAM- 1 (a-e) polypeptides of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
- pharmaceutically acceptable carrier is meant a non- toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
- parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
- the antagonist to be used is an antibody, fragment thereof, or derivative thereof
- Such antibodies, fragments, or regions will preferably have an affinity for any of human MAdCAM- 1 (a-e), expressed as Ka, of at least 10 8 M " ', more preferably, at least IO 9 M "1 , such as 5 X 10 8 M "1 , 8 X 10 8 M “1 , 2 X IO 9 M "1 , 4 X IO 9 M ', 6 X
- Preferred for human therapeutic use are high affinity murine and murine/human or human/human chimeric antibodies, and fragments, regions and derivatives having potent in vivo MAdCAM- 1 -inhibiting and/or neutralizing activity, according to the present invention, e.g., that block MAdCAM-1- mediated cell adhesion activity, in vivo, in situ, and in vitro.
- yet another aspect of the invention is related to a method for identifying compounds capable of enhancing or inhibiting expression of any of MAdCAM- 1 (a-e).
- reporter plasmids are constructed by linking a portion of the DNA located 5' to the transcription start site of any of MAdCAM- 1 (a-e) in front of a reporter gene.
- Such constructs are then transfected into appropriate cell lines.
- Compounds that are to be tested for their ability to increase or decrease expression from the MAdCAM- 1 promoter are then administered to the cell bearing the reporter construct, and the effect of each compound on reporter gene expression is determined by comparing that level of expression to the expression level in a control cell bearing the reporter construct, where the test compound has not been administered to the control cell.
- nucleic acid molecules of the present invention can be generated as follows.
- the MAdCAM-1 gene promoter region is obtained by amplification using the polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- the amplified fragment is then inserted into an appropriate plamid (such as, for example, pCAT TM (Promega, Madison, WI)).
- Nested deletion plasmids are then generated using the commercially available "Erase-a-Base” System (Promega, Madison, WI) as described in
- the nucleic acid molecules of the present invention can include the MAdCAM- 1 promoter and ds-acting enhancer and/or silencer elements capable of affecting gene transcription.
- these isolated nucleic acid molecules of the present invention are referred to below as " MAdCAM- 1 transcriptional regulatory elements" or “transcriptional elements. "
- nested deletion reporter plasmids can be generated containing a transcriptional element of the present invention linked in front of the chloramphenicol acetyltransferase (CAT) reporter gene.
- CAT chloramphenicol acetyltransferase
- Such recombinant DNA molecules of the present invention actually generated by the inventors include transcriptional elements inserted, in both orientations, into the Xbal site of pBLCAT2 vector (Luckow, B., Sch ⁇ tz, G., Nucleic Acids Res. 75:5490 (1987)).
- a recombinant DNA molecule containing a transcriptional element of the present invention is used to transiently transfect an appropriate cell line such as, for example, human choriocarcinoma cell lines
- the hGH transient expression system can also be used (Selden et al., Mol. Cell Biol. 6:3173-3179 (1986)) or other systems that are based on the expression of ⁇ -galactosidase (An et al, Mol. Cell. Biol. 2: 1628-1632 (1982)) and xanthine-guanine phosphoribosyl transferase (Chu et al, Nucleic Acids Res. 75:2921-2930 (1985)).
- a transcriptional element of the present invention may be inserted into an appropriate vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T. , et al, infra, and are well known in the art. Clones containing a transcriptional element of the present invention may be identified by any means which specifically selects for a MAdCAM- 1 enhancer or silencer region DNA such as, for example by hybridization with an appropriate nucleic acid probe(s) containing a sequence complementary to all or part of the transcriptional element.
- Oligonucleotide probes specific for a transcriptional element of the present invention can be designed simply by reference to SEQ ID No:33. Techniques for nucleic acid hybridization and clone identification are disclosed by Maniatis, T. , et al. , (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)), and by Hames, B.D. , et al. , (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)). To facilitate the detection of the desired clone containing a transcriptional element of the present invention, the above-described nucleic acid probe may be labeled with a detectable group.
- Such detectable groups can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention. Particularly useful are radioactive labels, such as 32 P, 3 H, 14 C, 35 S, 125 I, or the like. Any radioactive label may be employed which provides for an adequate signal and has a sufficient half-life.
- the oligonucleotide may be radioactively labeled, for example, by "nick- translation" by well-known means, as described in, for example, Rigby, P.J.W. , et al , J. Mol. Biol.
- polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group. See, for example, Leary, J.J. , et al , Proc. Natl. Acad. Sci. USA 50:4045 (1983); Renz, M. , et al , Nucl. Acids Res. 72:3435 (1984); and Renz, M. , EMBO J. 6:817 (1983).
- a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
- heterologous protein is intended to refer to a peptide sequence that is heterologous to the transcriptional regulatory elements of the invention.
- teaching herein will also apply to the expression of genetic sequences encoding the MAdCAM- 1 protein, or splice variants thereof, by such transcriptional regulatory elements.
- the reporter genes for use in the screening assay described below can code for either the MAdCAM- 1 protein, or splice variants thereof, or a heterologous protein.
- detection of reporter gene expression can be at the mRNA level, such as, for example, detection of MAdCAM- 1 mRNA.
- an operable linkage is a linkage in which a desired sequence is connected to a transcriptional or translational regulatory sequence (or sequences) in such a way as to place expression (or operation) of the desired sequence under the influence or control of the regulatory sequence.
- Two DNA sequences are said to be operably linked if induction of promoter function results in the transcription of the reporter gene and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation (if reporter protein activity is necessary for detection of reporter gene expression), (2) in- terfere with the ability of the expression regulatory sequences to direct reporter gene expression, or (3) interfere with the ability of reporter gene to be transcribed by the promoter region sequence.
- a promoter would be operably linked to a DNA sequence if the promoter were capable of affecting transcription of that DNA sequence.
- a transcriptional regulatory element of the present invention that enhances or represses gene expression may be operably-linked to such a promoter.
- Exact placement of the element in the nucleotide chain is not critical as long as the element is located at a position from which the desired effects on the operably linked promoter may be revealed.
- a nucleic acid molecule, such as DNA is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are operably linked to the nucleotide sequence which encodes the polypeptide.
- all transcriptional and translational regulatory elements (or signals) that are operably linked to a heterologous gene should be recognizable by the appropriate host. By “recognizable” in a host is meant that such signals are functional in such host.
- the MAdCAM- 1 transcriptional regulatory elements of the present invention may be operably linked to a heterologous gene (such as a reporter gene), preferably in an expression vector, and introduced into a host cell, preferably a eukaryotic cell, to assay reporter gene expression.
- a heterologous gene such as a reporter gene
- Preferred eukaryotic cells include choriocarcinoma cell lines, breast cancer cell lines, prostate carcinoma cell lines and kidney cell lines.
- translation of eukaryotic mRNA is initiated at the codon that encodes the first methionine.
- the linkage between a eukaryotic promoter and a reporter gene does not contain any intervening codons that are capable of encoding a methionine.
- the presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as the DNA encoding the heterologous protein) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the reporter gene).
- a fusion product of a reporter protein may be constructed.
- the sequence coding for the reporter protein may be linked to a signal sequence which will allow secretion of the protein from, or the compartmentalization of the protein in, a particular host.
- signal sequences may be designed with or without specific protease sites such that the signal peptide sequence is amenable to subsequent removal.
- the native signal sequence for this protein may be used.
- the transcriptional regulatory elements of the invention can be selected to allow for repression or activation, so that expression of the operably linked reporter genes can be modulated. Translational signals are not necessary when it is desired to express antisense RNA sequences or to assay reporter gene expression via mRNA detection.
- the non-transcribed and/or non-translated regions 3' to the reporter gene can be obtained by the above-described cloning methods.
- the 3'- non-transcribed region may be retained for its transcriptional termination regulatory sequence elements; the 3 '-non-translated region may be retained for its translational termination regulatory sequence elements, or for those elements that direct polyadenylation in eukaryotic cells. Where the native expression control sequences signals do not function satisfactorily host cell, then sequences functional in the host cell may be substituted.
- reporter gene expression may occur through the transient expression of the introduced sequence.
- Genetically stable transformants may be constructed with vector systems, or transformation systems, whereby the reporter gene is integrated into the host chromosome. Such integration may occur de novo within the cell or, in a most preferred embodiment, be assisted by transformation with a vector that functionally inserts itself into the host chromosome.
- Vectors capable of chromosomal insertion include, for example, retroviral vectors, transposons or other DNA elements which promote integration of DNA sequences in chromosomes, especially DNA sequence homologous to a desired chromosomal insertion site.
- Cells that have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers that allow for selection of host cells which that the desired sequence.
- the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.
- the selectable marker gene can either be directly linked to the reporter gene, or introduced into the same cell by co- transfection.
- the introduced sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose, as outlined below.
- Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
- Preferred eukaryotic plasmids include those derived from the bovine papilloma virus, vaccinia virus, and SV40. Such plasmids are well known in the art and are commonly or commercially available. For example, mammalian expression vector systems in which it is possible to cotransfect with a helper virus to amplify plasmid copy number, and, integrate the plasmid into the chromosomes of host cells have been described (Perkins, A.S. et al , Mol. Cell Biol. 5: 1123 (1983); Clontech, Palo Alto, California).
- vectors derived from pCAT-Basic, pCAT-Enhancer and pCAT-Promoter vectors are particularly preferred.
- the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transfection, electroporation or delivery by liposomes.
- DEAE dextrin, calcium phosphate, and preferably, the transfection reagent DOTAP may be useful in the transfection protocol.
- reporter gene results in the production mRNA and, if desired, reporter protein. According to the invention, this expression can take place in a continuous manner in the transformed cells, or in a controlled manner.
- the reporter protein is isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like. Alternatively, levels of reporter protein expression can be assayed according to conventional protein assays, such as, for example, the CAT expression system.
- the MAdCAM- 1 transcriptional regulatory elements of the present invention are useful for screening drugs, ligands and/or other trans-acting agents to determine which are capable of affecting expression of MAdCAM- 1 or any splice variant thereof.
- trans-acting factors can be identified by their ability to up-regulate or down-regulate MAdCAM- 1 expression.
- MAdCAM- 1 transacting agent a drug, ligand, or other compound capable interacting, either directly or indirectly, with a MAdCAM- 1 transcriptional regulatory element of the present invention to enhance or repress gene expression.
- Such MAdCAM- 1 tr ⁇ ns-acting elements which interact directly with a transcriptional regulatory element of the present invention include those, which, for example, bind directly to the element and either enhance or repress gene expression.
- MAdCAM- 1 tr ⁇ ns-acting agents which interact indirectly with a transcriptional regulatory element of the present invention include those which, for example, bind to and induce activity of a second tr ⁇ ns-acting agent (e.g.
- tram-acting agent a triplex-forming oligonucleotide.
- Administration of a suitable oligonucleotide will result in the formation of a triple helix between the oligonucleotide and the MAdCAM- 1 promoter, which will inhibit transcription from that promoter (Ebbinghaus, S.W. et ⁇ , Gene Therapy 3: 287-297 (1996);
- the invention provides a screening assay for determining whether any given compound is capable of up-regulating or down- regulating expression from the MAdCAM- 1 promoter, leading to an increase or decrease of MAdCAM- 1 production.
- the screening assay involves (1) providing a host cell transfected with a recombinant nucleic acid molecule containing a MAdCAM- 1 transcriptional regulatory element of the present invention and a reporter gene, wherein the transcriptional element is operably linked to the reporter gene; (2) administering a candidate MAdCAM- 1 transacting agent to the transfected host cell; and (3) determining the effect on reporter gene expression.
- the invention provides a screening assay for the identification of substances capable of altering the expression from the
- MAdCAM- 1 promoter comprising:
- step (b) measuring the level of expression of said reporter gene in a control cell, wherein said control cell is transformed with the recombinant DNA molecule of step (a); and (c) comparing the level of expression of said reporter gene in said test cell to the level of said reporter gene in said control cell.
- Suitable and preferred host cells, transfection methods, expression vectors, promoters, and reporter genes, are described above and will be known in the art. Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
- Example 1 Expression and Purification of any of MAdCAM- 1 (a-e) in E. coli
- the DNA sequence encoding any of the mature MAdCAM- 1 (a-e) proteins is amplified using PCR oligonucleotide primers specific to the amino terminal sequences of the desired MAdCAM- 1 (a-e) protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences respectively.
- the plasmid HEBBC23 is used, along with the primers given below.
- the plasmid HSKCW36 is used, along with the primers given below.
- the plasmid MAdCAM- lc is used, along with the primers given below.
- the plasmid MAdCAM-ld is used, along with the primers given below.
- the plasmid is used, along with the primers given below.
- MAdCAM- 1 e is used, along with the primers given below.
- the 5' oligonucleotide primer has the sequence 5'cgc ccatgg gc cag tec etc cag gtg 3' (SEQ ID NO:l 1) containing the underlined Ncol restriction site, which encodes 17 nucleotides of the coding sequence of the gene encoding any of the MAdCAM-l(a-e) proteins shown in FIGS. 1-5 (SEQ ID NOs:l, 3, 5, 7, 9), respectively, beginning immediately after the signal peptide.
- the 3 ' primer has the sequence 5 ' cgc aagctt tea ggg cag ctg gtc ace cgc 3 ' (SEQ ID NO: 12) containing the underlined Hindlll restriction site followed by nucleotides complementary to nucleotides 940-967 of FIG. 1 , which follow immediately after the coding sequence of any of MAdCAM- 1 (a-e).
- restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQE60. which are used for bacterial expression in these examples. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 9131 1). pQE60 encodes ampicillin antibiotic resistance ("Amp r ”) and contains a bacterial origin of replication ("ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a 6-His tag and restriction enzyme sites.
- the amplified DNA encoding any of MAdCAM- 1 (a-e) and the vector pQE60 both are digested with Ncol and Hindlll and the digested DNAs are then ligated together. Insertion of the DNA encoding any of the MAdCAM- 1 (a-e) proteins into the restricted pQE60 vector places the coding region of MAdC AM- l(a-e) downstream of and operably linked to the vector's IPTG-inducible promoter and in-frame with an initiating AUG appropriately positioned for translation of the appropriate MAdCAM- 1 (a-e) protein. The ligation mixture is transformed into competent E coli cells using standard procedures.
- This strain which is only one of many that are suitable for expressing any of the MAdCAM- 1 (a-e) proteins, is available commercially from Qiagen.
- Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis.
- Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
- the O/N culture is used to inoculate a large culture, at a dilution of approximately 1 : 100 to 1 :250.
- the cells are grown to an optical density at 600nm
- IPTG Isopropyl-B-D-thiogalactopyranoside
- the 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2X phosphate-buffered saline ("PBS"), thereby removing the urea, exchanging the buffer and refolding the protein.
- PBS 2X phosphate-buffered saline
- the protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered.
- the sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 ⁇ g/ml.
- Example 2 Cloning and Expression of any of the MAdCAM-1 (a-e) proteins in a Baculovirus Expression System
- the plasmid shuttle vector pA2 is used to insert the cloned DNA encoding the complete protein, including its naturally associated secretary signal (leader) sequence, into a baculovirus to express any of the mature proteins MAdCAM- 1 (a-e), using standard methods as described in Summers et al, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
- This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI and Asp718.
- the polyadenylation site of the simian virus 40 (“SV40") is used for efficient polyadenylation.
- the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene.
- the inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
- baculovirus vectors could be used in place of the vector above, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required.
- Such vectors are described, for instance, in Luckow et al, Virology 770:31-39.
- the cDNA sequence encoding any of the full length MAdCAM- 1 (a-e) proteins is amplified using PCR oligonucleotide primers corresponding to the 5 ' and 3 ' sequences of the gene.
- the plasmid HEBBC23 is used, along with the primers given below.
- the plasmid HSKCW36 is used, along with the primers given below.
- the plasmid plasmid HSKCW36 is used, along with the primers given below.
- MAdCAM- lc is used, along with the primers given below.
- the plasmid MAdCAM-ld is used, along with the primers given below.
- the plasmid MAdCAM- le is used, along with the primers given below.
- the 5 ' primer has the sequence 5 'cgc ggatcc gcc ate atg gat ttc gga ctg gcc 3 ' (SEQ ID NO: 13) containing the underlined BamHI restriction enzyme site followed by 18 bases of the sequence of the relevant MAdCAM- 1 (a-e) protein shown in FIGS. 1-5, respectively.
- the 5 ' end of the amplified fragment encoding the relevant MAdCAM- 1 (a- e) protein provides an efficient signal peptide.
- An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196: 947-950 (1987) is appropriately located in the vector portion of the construct.
- the 3' primer has the sequence 5 'cgc ggtacc tea ctt gaa ggg gtc caa gc 3' (SEQ ID NO: 14) containing the underlined Asp718 restriction site followed by nucleotides complementary to nucleotides 1 183-1199 of FIG. 1 , which follow immediately after the coding sequence of any of MAdCAM- 1 (a-e).
- the cDNA sequence encoding the extracellular soluble domain of any of the MAdCAM- 1 (a-e) proteins is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
- the plasmid HEBBC23 is used, along with the primers given below.
- the plasmid HSKCW36 is used, along with the primers given below.
- the plasmid MAdCAM- lc is used, along with the primers given below.
- the plasmid is used, along with the primers given below.
- MAdCAM-ld is used, along with the primers given below.
- the plasmid MAdCAM- 1 e is used, along with the primers given below.
- the 5 ' primer has the sequence 5 'cgc ggatcc gcc ate atg gat ttc gga ctg gcc 3' (SEQ ID NO: 15), containing the underlined BamHI restriction enzyme site followed by 18 bases of the sequence of the relevant MAdCAM- 1 (a-e) protein shown in FIGS. 1-5, respectively. Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding the relevant MAdCAM-l(a- e) protein provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M.. J. Mol. Biol. 196:
- the 3' primer has the sequence 5 'cgc ggtacc tea ggg cag ctg gtc ace cgc 3' (SEQ ID NO: 16) containing the underlined Asp718 restriction site followed by nucleotides complementary to nucleotides 940-967 of FIG. 1, which follow immediately after the coding sequence of any of MAdCAM- 1 (a-e).
- the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with BamHI and Asp718 and again is purified on a 1% agarose gel. This fragment is designated herein F2.
- the plasmid is digested with the restriction enzymes BamHI and Asp718 and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
- the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated herein "V2".
- Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase.
- E. coli HB101 cells are transformed with ligation mix and spread on culture plates.
- Bacteria are identified that contain the plasmid with the desired human gene encoding MAdCAM- 1 (a-e) by digesting DNA from individual colonies using Xbal and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
- This plasmid is designated herein pBacMAdCAM-l(a-e) (i.e., if MAdCAM- 1(a) is cloned, the plasmid is pBacMAdCAM-l(a), while if
- MAdCAM- 1(b) is cloned, the plasmid is pBACMAdCAM-l(b), etc.).
- l ⁇ g of BaculoGoldTM virus DNA and 5 ⁇ g of the plasmid pBacMAdCAM-l(a-e) are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ⁇ l Lipofectin plus 90 ⁇ l Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution.
- Sf9 insect cells ATCC CRL 1711
- the plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10%o fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27°C for four days. After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, cited above. An agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay" of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).
- the virus is added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4°C. A clone containing any of the properly inserted genes encoding
- MAdCAM- 1 (a-e) is identified by DNA analysis including restriction mapping and sequencing. This is designated herein as V-MAdCAM-l(a-e), i.e., V- MAdCAM-l(a), or V-MAdCAM-l(b), etc., depending on which MAdCAM-1 variant is cloned.
- Sf9 cells are grown in Grace's medium supplemented with 10%> heat- inactivated FBS. The cells are infected with the recombinant baculovirus V- MAdCAM-l(a-e) at a multiplicity of infection ("MOI") of about 2 (about 1 to about 3).
- MOI multiplicity of infection
- the cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteins are visualized by SDS-PAGE and autoradiography.
- SV40 origin of replication This allows the replication of the vector to high copy numbers in cells (e.g. COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this purpose.
- a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g.
- LTRs long terminal repeats
- Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
- Mammalian host cells that could be used include, human Hela, 283, H9 and Jurkart cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells.
- the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
- the co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
- the transfected gene can also be amplified to express large amounts of the encoded protein.
- the DHFR dihydrofolate reductase
- GS glutamine synthase
- the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used for the production of proteins.
- the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al. , Molecular and Cellular Biology, 438-4470 (March, 1985)) plus a fragment of the CMV -enhancer (Boshart et al, Cell 47:521-530 (1985)). Multiple cloning sites, e.g. with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest.
- the vectors contain in addition the 3 ' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
- the expression plasmid, pMAdCAM-l(a-e) HA is made by cloning a cDNA encoding one of MAdCAM- 1 (a-e) into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
- the expression vector pcDNAI/amp contains: (1) an E.coli origin of replication effective for propagation in E.
- coli and other prokaryotic cells (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
- a DNA fragment encoding the relevant MAdCAM- 1 (a-e) protein is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
- the plasmid construction strategy is as follows. The cDNA encoding the relevant MAdCAM- 1 (a-e) is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of the desired MAdCAM- 1 (a-e) in E. coli.
- Suitable primers include the following, which are used in this example.
- the DNA sequence encoding the full length protein of any of MAdC AM- l(a-e) is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
- the 5' primer has the sequence 5' cgc ggatcc gcc ate atg gat ttc gga ctg gcc
- the 3' primer has the sequence 5' cgc ggtacc tea ctt gaa ggg gtc caa gc 3' (SEQ ID NO: 18) containing the underlined Asp718 restriction followed by nucleotides complementary to nucleotides 1 183-1 199 of the MAdCAM-l(a) coding sequence given in FIG. 1.
- the 5' primer containing the underlined BamHI site, an AUG start codon and 18 codons of the 5' coding region has the following sequence: 5' cgc ggatcc gcc ate atg gat ttc gga ctg gcc 3' (SEQ ID NO:
- the 3' primer containing an Xbal site, a stop codon, and 3 ' coding sequence for the extracellular domain, has the following sequence:
- the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with Hindlll and Xhol and then ligated.
- the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 1 1099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of a fragment encoding the relevant MAdCAM- 1 (a-e).
- COS cells are transfected with an expression vector, as described above, using DEAE-
- MAdC AM- 1 (a-e)HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; Cold
- the cells are labeled by incubation in media containing 33 S-cysteine for 8 hours.
- the cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.
- Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
- Plasmid pCl is used for the expression of any of the MAdCAM- 1 (a-e) proteins.
- Plasmid pCl is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
- a selective medium alpha minus MEM, Life Technologies
- MTX methotrexate
- DHFR target enzyme
- a second gene is linked to the DHFR gene it is usually co-amplified and over-expressed. It is state of the art to develop cell lines carrying more than 1,000 copies of the genes. Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome(s).
- Plasmid pCl contains for the expression of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molecular and Cellular biology, March 1985, 438-4470) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al.. Cell 41 :521 -530, 1985). Downstream of the promoter is a BamHI restriction enzyme cleavage site that allows the integration of the genes. Behind this cloning site the plasmid contains translational stop codons in all three reading frames followed by the 3' intron and the polyadenylation site of the rat preproinsulin gene.
- LTR long terminal repeat
- CMV cytomegalovirus
- high efficient promoters can also be used for the expression, e.g., the human ⁇ -actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
- the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well.
- Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g. G418 plus methotrexate.
- the plasmid pCl is digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.
- DNA sequence encoding the full length protein of any of MAdCAM- 1 (a-e) is amplified using PCR oligonucleotide primers corresponding to the 5' and
- the 5' primer has the sequence 5' cgc ggatcc gcc ate atg gat ttc gga ctg gcc 3' (SEQ ID NO:17) containing the underlined BamHI restriction enzyme site followed by 18 bases of the sequence of the relevant MAdCAM- 1 (a-e) gene shown in FIGS. 1-5 (SEQ ID NOs:l , 3, 5, 7, 9), respectively.
- Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding any of human MAdCAM-l(a-e) provides an efficient signal peptide.
- An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987) is appropriately located in the vector portion of the construct.
- the 3' primer has the sequence 5' cgc ggtacc tea ctt gaa ggg gtc caa gc 3' (SEQ ID NO: 18) containing the underlined Asp718 restriction followed by nucleotides complementary to nucleotides 1 183-1199 of the MAdCAM-l(a) coding sequence given in FIG. 1.
- MAdCAM- 1 (a-e) proteins is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
- the 5' primer has the sequence 5' cgc ggatcc gcc ate atg gat ttc gga ctg gcc
- the 3' primer has the sequence 5' cgc ggtacc tea ggg cag ctg gtc ace cgc
- amplified fragments are isolated from a 1 % agarose gel as described above and then digested with the endonuclease BamHI and then purified again on a 1%) agarose gel.
- the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
- E.coli HB101 cells are then transformed and bacteria identified that contained the plasmid pCl inserted in the correct orientation using the restriction enzyme BamHI. The sequence of the inserted gene is confirmed by DNA sequencing.
- Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection.
- 5 ⁇ g of the expression plasmid CI are cotransfected with 0.5 ⁇ g of the plasmid pSVneo using the lipofecting method (Feigner et al., supra).
- the plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
- the cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
- the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M). The same procedure is repeated until clones grow at a concentration of 100 ⁇ M.
- the expression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE.
- the probe was purified using a CHROMA SPIN- 100TM column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe was then used to examine various human tissues for mRNA corresponding to any of MAdCAM- 1(a).
- MTN Multiple Tissue Northern
- H human tissues
- IM human immune system tissues
- Human MAdCAM- 1 cDNA was initially identified as an expressed sequence tag (EST) following screens for homology in an EST cDNA database (Adams, M.D., et al. Nature 577:3-17 (1995); Adams, M.D. et al. Science
- a MAdCAM- 1 genomic clone was subsequently isolated by screening a cosmid library constructed in the cosmid vector pCV007 (Choo, K. H., et al, Gene 46: 277 (1986)). The library was replica plated onto Gene-Screen Plus filters (DuPont, Boston, MA), and screened as described previously (Leung, E., et al. Int. Immunol. 5: 551-558 (1993)) with the insert of the MAdCAM- 1 EST clone labeled by random hexanucleotide priming (see Example 6). DNA sequencing
- DNA sequences were determined by cycle sequencing using Applied Biosystems automated DNA sequenators (The Centre for Gene Technology, School of Biological Sciences, University of Auckland, Auckland, New Zealand; and at Human Genome Sciences Inc., Rockville, Maryland).
- the complete composite MAdCAM- 1 sequencer obtained from genomic and cDNA clones was determined on both strands using a combination of universal Ml 3 primers, and primers specific for human MAdCAM- 1 sequences.
- a MAdCAM- 1 genomic clone was subsequently isolated by screening a cosmid library constructed in the cosmid vector pCV007 (Choo, K. H., et al, Gene 46: 277 (1986)).
- the library was replica plated onto Gene-Screen Plus filters (DuPont, Boston, MA), and screened as described previously (Leung, E., et al. Int. Immunol. 5: 551-558 (1993)) with the insert of the MAdCAM- 1 EST clone labeled by random hexanucleotide priming.
- RNA from human fetal brain (Clontech) in reverse transcriptase (RT) buffer (BRL, Gaithersburg, MD) was heated to 70 °C for 3 min and then cooled on ice. All four dNTPs were added to a final concentration of 0.5 mM, together with 500 ng of random hexamer primers, and 400 U of Superscript RT (BRL,
- the LI 03 primer corresponds to the sequence 347-405 of the human MAdCAM-1.
- An aliquot of 2.5 ⁇ l of the PCR reaction was reamplified for 25 cycles using the L203 primer, and the nested primer L50- (SEQ ID NO:27) (5 '-GCTGGT CCGGGAAGGCGTACACAA GGAGCTGC-3 ') corresponding to nucleotides 321-352, with the same annealing temperature.
- the PCR product was ethanol precipitated and ligated into an EcoRV digested, Taq polymerase 3 ' dTTP-tailed pBluescript vector, and sequenced.
- MTN (Clontech) filters were screened with the insert of the MAdCAM-1 EST clone labeled by random hexanucleotide priming.
- the conditions of hybridization were 1% SDS, 2 x SSC, 10% (w/v) dextran sulphate, 100 ⁇ g/ml denatured salmon sperm DNA, and 50% (v/v) deionized formamide at 50 °C. Filters were washed twice in 0.1 x SSC, 0.1% SDS at 50 °C for 30 min. and autoradiographed using XAR-5 film and Cronex Lightning Plus screens.
- MAdCAM-1 by using the BLAST algorithm (Altschul, S. F., et al. J. Mol. Biol. 275:403-410 (1990)). Partial overlapping MAdCAM- 1 cDNA clones HEBBC23X and HEBBC23Y were initially identified from an early stage human brain cDNA library ( Figure 8A). They were sequenced on both strands and together encoded the MAdCAM- 1 sequence from a position corresponding to amino acid residue 89 of the mouse MAdCAM- 1 cDNA clone pMAd-7, to the end of the 3 '-untranslated region.
- the early stage brain library was rescreened, as well as five other brain, pancreatic, and adult and fetal spleen cDNA libraries, but no clones that extended the sequence were obtained.
- fetal brain mRNA was subjected to rapid amplification of cDNA ends (RACE), but despite exhaustive attempts the MAdCAM- 1 5 '-sequence remained elusive.
- RACE rapid amplification of cDNA ends
- MAdCAM- 1 HEBBC23X cDNA clone the genomic clone MAD-C1, and the 5'- PCR product are given in Fig. 8.
- the nucleotide sequence of 1546 bp ends with the polyadenylation signal AAATAAA (SEQ ID NO:28), followed 15 bases further by a poly(A) stretch.
- Ten bp of the 5 '-untranslated sequence has been added for completeness.
- the open reading frame beginning with an ATG at position 1 encodes a protein of 382 amino acid residues.
- the ATG start codon which is flanked by the consensus sequence Pur XXAUG Pur (SEQ ID NO:29), is followed by a predominantly hydrophobic segment of 18 amino acid residues characteristic of a signal peptide.
- a hydropathicity plot of the deduced amino acid sequence revealed a sequence presumed to be the transmembrane domain, encompassing residues 320 to 339.
- the sequence predicts a transmembrane bound protein comprised of a predominantly hydrophilic 103 amino acid extracellular domain, a 20 amino acid transmembrane segment, and a 43 amino acid cytoplasmic domain, with an Mr of 38,340.
- the deduced amino acid sequence revealed a 17 amino acid signal peptide.
- two immunoglobulin (Ig)-line domains an 86 amino acid mucin-like region rich in serine/threonine residues, a 20 amino acid transmembrane domain, and a 43 amino acid charged cytoplasmic domain.
- the sequences of the two N- terminal Ig-like domains are highly conserved (59-65%) with the corresponding receptor-binding Ig domains of mouse MAdCAM- 1.
- No counterpart to the third IgA-like domain of mouse MAdCAM- 1 was present, and instead the serine/threonine-rich mucin domain has been extended as two distinguishable regions, here designated the major and minor mucin domains.
- the major domain is formed from six tandem repeats of an eight amino acid sequence having the consensus DTTSPEP/SP (SEQ ID NO:30), which is similar to the imperfect repeats of the intestinal mucin MUC-2.
- the mucin domains of the MAdCAM-1 human/mouse species homologs are distinct, in accord with the notion that mucin domains are not phylogenetically conserved.
- Human MAdCAM- 1 mRNA transcripts were restricted to small intestine, colon, spleen, pancreas, and brain which is a further indication that the clones encode MAdCAM- 1.
- the extracellular domain comprises two Ig-like domains of 52 and 69 amino acid residues, respectively, each possessing the invariant cysteine residues that stabilize the immunoglobulin loop; with the first domain having doublet cysteines.
- the mucin domain is formed from six tandem repeats of an eight amino acid sequence having the general consensus DTTSPEP/SP (SEQ ID NO:30). The repeats are highly conserved with one another (15-100%), suggesting that they arose by duplication. This domain has 19 potential sites for O-linked glycosylation.
- the mucin-like nature of the region extends to a lesser degree as far as the transmembrane domain, since the serine/threonine/proline content is still quite high (43%).
- a search of the NBRF database revealed that human
- MAdCAM- 1 was most similar to mouse MAdCAM- 1, but striking homologies were also identified with VCAM-1, and ICAM-1. Alignment of the human and mouse sequences (not shown) revealed an overall weak similarity of 39%. However, Ig domains 1 and 2 in particular have been highly conserved, 59 and 65%o, respectively; and similarity increases to 69 and 81%. respectively, when conservative substitutions are included. This is to be expected since these two Ig domains interact to support binding to the LPAM-1 receptor, and both domains are required for full function.
- the membrane-proximal regions of the extracellular domains of human and mouse MAdCAM- 1 are peptide backbones designed for decoration with complex O-linked carbohydrate moieties for presentation to L-selectin, and as such, only the serine/threonine/proline content needs to be conserved. Hence, after the first mucin repeat there is little similarity between the human and mouse sequences, except for transmembrane domain which is 55% identical. The short charged cytoplasmic domains share only 35% identity, and the human sequence extends 24 amino acid residues further than the mouse sequence. Clone HEBBC23X lacks an equivalent of the third Ig domain of mouse MAdCAM- 1.
- mouse MAdCAM- 1 variant has been identified in which exon 4 is spliced out removing both the mucin domain and the third Ig domain (Sampaio et al, J. Immunol. 155: 2477-86 (1995)).
- the third Ig domain of mouse MAdCAM- 1 is strikingly similar to the C ⁇ 2 constant region immunoglobulin loop of human and gorilla IgAl (Briskin et al, Nature 363:461- 64 (1993)). It was suggested that it may be able to interact with IgA-specific Fc receptors or related surface receptors on mucosal T cells, given that the C ⁇ 2 constant regions mediates IgA interactions with the poly-immunoglobulin Fc receptor.
- Human MAdCAM- 1 may have compensated for a lack of a third Ig domain by having two mucin domains to hold the two N-terminal ligand-binding domains above the glycocalyx for presentation to LPAM-1.
- mouse there are two mucin domains to hold the two N-terminal ligand-binding domains above the glycocalyx for presentation to LPAM-1.
- the repeats in the major mucin domain may have been inserted, possibly by a gene conversion event involving a mucin gene, to enrich the overall content of serine/threonine residues (40% in major domain) and to enable better presentation to L-selectin by positioning the major mucin repeat above the glycocalyx.
- a search of the NBRF database with the sequence of the tandem repeats of the major mucin domain revealed most similarity (up to 62% including conservative substitutions) with a region of imperfect repeats in the human intestinal mucin MUC-2.
- MUC-2 contains two distinct regions with a high degree of internal homology. (Toribara et al, J. Clin. Invest. 88: 1005-13 (1991)). There is a region of imperfect repeats that range from 7 to 40 amino acids, with the most common length being 16 amino acids. This 385 amino acid region has a high threonine (47.8%), proline (35.6%) and serine (10.6%) content. It is this region to which MAdCAM- 1 shares similarity (Fig. 2).
- the major MAdCAM- 1 tandem repeat domain is not as rich in such residues, and 22% of the dissimilar amino acids are acidic residues which are totally absent from the imperfect repeats of MUC-2.
- MUC-2 there is also a 3' region composed of 69 bp tandem repeats arranged in an array of up to 115 units, which is not similar to the MAdCAM- 1 mucin region despite having a high serine/threonine/proline content (87%). (Zrihan-licht et al, Eur J. Biochem 224: 787-95 (1994)).
- the human intestinal mucin MUC-1 has a serine/threonine/proline-rich 20 amino acid residue domain (SEQ ID NO:31) PDTRPAPGSTAPPAHGVTSA, repeated up to 200 times, (Gum et al, J. Biol. Chem. 266: 22733-38 (1991)) and rat intestinal mucin has the repeat sequence (SEQ ID NO:32) TTTPDV, (Spicer et al, J. Biol. Chem. 266: 15099-109 (1991)) but neither of these sequences bear similarly to MAdCAM- 1.
- MAdCAM- 1 clone HEBBC23Y appears to be a splice variant in the 3 mucin repeats are missing (amino acid residues 231-254) (Figs. 8A, 10).
- MAdCAM- 1 transcripts were amplified from human fetal brain using sense and antisense PCR primers designed to the start of Ig domain 2 and the cytoplasmic domain, respectively.
- Several novel splice variants were identified including one which lacked almost all of the second Ig domain and all the major mucin repeats; and two others which had lost half of Ig domain 2 and 2 to 3 mucin repeats (Fig. 10A).
- mouse MAdCAM- 1 has been stringently conserved in humans. This includes the tissue distribution of human
- MAdCAM- 1 and the structure of the two Ig ligand-binding domains; yet the 3'-region is quite divergent.
- the function of human MAdCAM- 1 is likely to be regulated by extensive alternative splicing as evidenced by the variant forms described herein.
- the two human genomic libraries screened were a Stratagene 1 FIX II library prepared from human placenta genomic DNA digested with Mbol, and a cosmid library constructed in the vector pAVCV007 from DNA partially digested with Mbo I.
- the cosmid library was replica plated onto Gene-Screen Plus filters (Du Pont, Boston, MA), and screened with the Xho I-EcoR I 32P-labeled 500 bp insert of the MAdCAM- 1 cDNA clone PCR Y. Positive clones HEBBC23591 and GM3 were isolated from the phage and cosmid libraries, respectively.
- DNA sequence was determined by cycle sequencing using an Applied Biosystems 373A automated DNA sequencer (School of Biological Sciences, University of Auckland, Auckland). The entire transcribed regions of the MAdCAM- 1 gene, previously defined by the MAdCAM- 1 cDNA, were identified and sequenced. Exon-intron boundaries were assigned by direct comparison of the cDNA and genomic sequences, and according to the GT/AG rule for splicing. The determined DNA sequence has been submitted to GenBank databank.
- a combination of PCR analysis of a panel of human/rodent somatic cell hybrids and fluorescence in situ hybridization (FISH) to human metaphase chromosomes was used to define the chromosomal location of the MAdCAM- 1 gene.
- FISH fluorescence in situ hybridization
- Fourteen of the cell hybrids contained a single human chromosome, whereas the remaining 10 contained 2 to 3 chromosomes, or 1 to 3 chromosomal fragments.
- Two primers U707 and LI 072 were designed to nucleotide positions
- the precise regional localization of the MAdCAM- 1 gene was determined by single copy gene fluorescence in situ hybridization (FISH) to human male metaphase chromosome spreads. Briefly, a 1.3 kb MAdCAM- 1 cDNA was nick- translated using digoxygenin 1 1-dUTP (Boehringer Mannheim), and FISH was carried out. Individual chromosomes were counterstained with 4'-6-diamidino-2- phenyindole-2HCl (DAPI).
- FISH fluorescence in situ hybridization
- Color digital images containing both DAPI bands and gene signal detected with anti-digoxygenin-tagged rhodamine fluorescent label were recorded using a triple-band pass filter set (Chroma Technology, Inc., Brattleburo, VT) in combination with a charged coupled-device camera (Photometries, Inc., Arlington, AZ) and variable excitation wave length filters. Images were analyzed using the ISEE software package (Inovision Corp., Durham, N.C.).
- a 700 base pair fragment encoding a region immediately 5' of the MAdCAM- 1 gene and including the translational start site was PCR amplified from a Sac I-Pst I subclone of the cosmid clone pGM3 using the T7 forward primer (SEQ ID NO:41) (5'-GTA ATA CGA CTC ACT ATA GG-3'; sense) and the MAdCAM- 1 -specific antisense primer MAD-2 (SEQ ID NO:42) (5'-AGG GCC AGT CCG AAA TCC ATG CTC AGT CCC-3').
- the PCR product was subcloned into the EcoRV site in pBluescript, excised with Hind III and subcloned into the pGL-2 Basic vector (Promega, Madison, WI) which contains a firefly luciferase reporter gene.
- the MAdCAM- 1 gene In order to isolate the MAdCAM- 1 gene, 200,000 colonies of a genomic library in the cosmid vector, pAVCV007, were screened with the MAdCAM- 1 cDNA clone PCR Y that encodes from nucleotide positions 273 to 858. Of two clones obtained, the longest, GM3, contained the entire gene, and 5 '-untranslated region, but did not contain exons encoding the transmembrane and cytoplasmic domains, and 3'-untranslated region. The missing portion of the MAdCAM- 1 gene was located on clone HEBBC23592, isolated by screening plaques from a
- FIX II genomic library with a 1.3 kb MAdCAM- 1 cDNA probe. Southern blot, PCR, and DNA sequence analysis demonstrated that clone HEBBC23592 contained at least exons 3 to 5 of the MAdCAM- 1 gene.
- DNA sequencing revealed that the coding portion of the MAdCAM- 1 gene is contained within 5 exons, with the sequences being identical to the
- MAdCAM- 1 cDNA sequence All intron-exon splice junction sequences are in agreement with the GT/AG rule for splicing.
- the introns are all type I, where interruption occurs after the first nucleotide of a codon.
- the first exon (52 bp) encodes the signal peptide and 5'-untranslated sequence; exons 2 and 3 encode the N-terminal Ig domains; exon 4 encodes the mucin domain; and exon 5 encodes the transmembrane and cytoplasmic domains, and the 3' untranslated region.
- a splice variant of human MAdCAM-1 lacks exon 4 encoding the mucin domain
- Splice variants of human MAdCAM- 1 where the variant forms lack all or part of the second Ig domain, and all or part of the major mucin domain, are described above in Example 5. Comparison with the MAdCAM- 1 genomic sequence confirms that all four splice variants were derived by internal splicing of exons, unlike the single splice variant identified for mouse MAdCAM- 1 which is formed by splicing out exon 4, which encodes the mucin/IgA-like Ig domain. Further splice variants of 250 (minor), 350 (major), and 500 (minor) bp in size, compared to a full-length PCR product of 700 bp, were amplified from human fetal brain.
- a 700 bp 5'-flanking region of the MAdCAM- 1 gene was sequenced, revealing several potential transcriptional regulatory elements. These include two tandem NF-kB binding sites at positions -98 and -110 with respect to the translational start codon; thirteen SP-1 sites at -66, -141, -157, -164, -177, -189,
- pGL-2/B-718+ and pGL-2/B-718 " constructs which contain a 700 bp fragment of the MAdCAM-1 gene 5'-flanking sequence (nucleotide positions -718 to +20 relative to the translational start) fused to the luciferase reporter gene (Figs.HA) were used in transient transfection assays to test for promoter activity.
- Promoter activity was tested in PMA-treated and untreated HMEC cells, a human dermal endothelial cell line which consitutively produces MAdCAM- 1 RNA (Fig.l4B).
- the reporter construct directed a low but consistent level of luciferase activity in unactivated cells as compared to the pGL-2/B basic control vector, and the control pGL-2/-718 " vector containing the promoter in the incorrect orientation.
- the activity of the pGL-2/B-718+ vector was doubled following cell stimulation with PMA, in comparison to the pGL-2/-718 " vector control (Fig.14C).
- PCR primers directed to the MAdCAM- 1 sequence.
- the expected 386 bp PCR fragment was specifically amplified from human DNA, but not from mouse or hamster DNAs, and was specifically obtained from a hybrid cell line (GM10612) containing only human chromosome 19.
- the MAdCAM-1 gene was regionally localized to chromosome 19 by in situ hybridisation of metaphase chromosomal spreads with the 1.3 kb cDNA insert of MAdCAM- 1 cDNA clone HEBBC23X (see Example 5). Approximately thirteen spreads were analyzed by eye, most of which had a doublet signal characteristic of genuine hybridization on at least one chromosome 19. Doublet signals were not detected on any other chromosome.
- the genomic organization of the MAdCAM- 1 gene correlates well with the subdomain structure of the encoded protein.
- the 5 '-untranslated region and signal peptide are encoded by exon 1
- the two N-terminal Ig domains and mucin- like domain are encoded by exons 2, 3, and 4, respectively
- the transmembrane and cytoplasmic domains and 3 '-untranslated region are combined together on exon 5.
- Several features of MAdCAM- 1 have been conserved between humans and mice, including the structure of the two Ig ligand-binding domains, yet the 3'-region is quite divergent.
- the human MAdCAM- 1 gene contains no sequence equivalent to the third IgA-homologous domain of mouse MAdCAM- 1 adjoining the 3'-end of the mucin domain. It is possible that a third Ig domain exists as a separate exon in the large intron separating exons 4 and 5, but given all the available evidence, and in particular sequence analysis of MAdCAM- 1 splice variants from RT-PCR analysis, this seems unlikely. Despite this major difference in gross structure other regions of human and mouse MAdCAM- 1 are highly conserved, including the positions of four of the five intron-exon splice junctions, highlighting the close evolutionary relationship between the molecules.
- TNF- ⁇ and IL-l ⁇ binding sites for TGF- ⁇ -inducible transcription factors (NF 1 and AP 1 ), previously identified in the mouse promoter, were not present.
- Multiple AP-2 sites in addition to the NF-kB sites may be responsible for the increased activity of the promoter in response to PMA.
- CACCTG MyoD site (SEQ ID NO:45), which is found within the muscle creatine kinase enhancer, is interesting, given that the related VCAM-1 is expressed on myoblasts and myotubes in culture and in vivo at sites of secondary myogenesis.
- VCAM-1 and ICAM-2 are located on chromosomes 1 and 17. respectively.
- MAdCAM- 1 mucin-like domain is decorated with carbohydrate moieties recognized b> L-selectin.
- a cluster of three (FUT6-FUT3-FUT5) of five cloned human fucosyltransferase genes responsible for the synthesis of sialyl Lewis x and a, and related fucosylated antigens recognised by selectins is located on 19pl 3.3.
- band 19pl3.3 is frequently involved in structural anomalies of chromosome 19, associated with ovarian cancer, leukemia, and multiple myeloma.
- Genes at 19pl3.3 which have so far been shown to be involved include the insulin receptor, E2A transcription factor, and MLLT1 genes.
- ADDRESSEE STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
- NAME GOLDSTEIN, JORGE A.
- MOLECULE TYPE DNA (genomic)
- GCT GGG TTA AGG GGG ACC GGC CAG GTC GGG ATC AGC CCC TCC 1146
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- FEATURE FEATURE
- AGG CTT CTG CCC CAG GTG TCG GCC TGG GCT GGG TTA AGG GGG ACC GGC 768 Arg Leu Leu Pro Gin Val Ser Ala Trp Ala Gly Leu Arg Gly Thr Gly 225 230 235
- CTCAAAGTCA TCCCTCTGTT CACAGAGATG GATGCATGTT CTGATTGCCT CTTTGGAGAA 939
- CTCAAAGTCA TCCCTCTGTT CACAGAGATG GATGCATGTT CTGATTGCCT CTTTGGAGAA 1080
- MOLECULE TYPE DNA (genomic)
- ACG TAT CAC CTC 816 Ser Ala Val Leu Gly Leu Leu Leu Leu Ala Leu Pro Thr Tyr His Leu 240 245 250 255 TGG AAA CGC TGC CGG CAC CTG GCT GAG GAC GAC ACC CAC CCA CCA GCT 864 Trp Lys Arg Cys Arg His Leu Ala Glu Asp Asp Thr His Pro Pro Ala 260 265 270
- MOLECULE TYPE cDNA
- SEQUENCE DESCRIPTION SEQ ID NO: 11: CGCCCATGGG CCAGTCCCTC CAGGTG 26
- MOLECULE TYPE cDNA
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JP52132098A JP2001503271A (en) | 1996-11-01 | 1996-11-01 | Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and splice variants thereof |
EP96938720A EP0948597A4 (en) | 1996-11-01 | 1996-11-01 | HUMAN MUCOSAL ADDRESSIN CELL ADHESION MOLECULE-1 (MAdCAM-1) AND SPLICE VARIANTS THEREOF |
PCT/US1996/017549 WO1998020110A1 (en) | 1996-11-01 | 1996-11-01 | HUMAN MUCOSAL ADDRESSIN CELL ADHESION MOLECULE-1 (MAdCAM-1) AND SPLICE VARIANTS THEREOF |
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PCT/US1996/017549 WO1998020110A1 (en) | 1996-11-01 | 1996-11-01 | HUMAN MUCOSAL ADDRESSIN CELL ADHESION MOLECULE-1 (MAdCAM-1) AND SPLICE VARIANTS THEREOF |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998020110A1 true WO1998020110A1 (en) | 1998-05-14 |
Family
ID=22256061
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1996/017549 WO1998020110A1 (en) | 1996-11-01 | 1996-11-01 | HUMAN MUCOSAL ADDRESSIN CELL ADHESION MOLECULE-1 (MAdCAM-1) AND SPLICE VARIANTS THEREOF |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0948597A4 (en) |
JP (1) | JP2001503271A (en) |
WO (1) | WO1998020110A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001098481A2 (en) * | 2000-06-16 | 2001-12-27 | Biogen, Inc. | Renal regulatory elements and methods of use thereof |
WO2004081049A1 (en) * | 2003-03-10 | 2004-09-23 | Auckland Uniservices Limited | Monoclonal antibodies that recognise mucosal addressin cell adhesion molecule-1 (madcam-1), soluble madcam-1 and uses thereof |
US7597887B2 (en) | 2002-12-30 | 2009-10-06 | Biogen Idec Ma Inc. | KIM-1 antagonists and use to modulate immune system |
US8206705B2 (en) | 2005-03-02 | 2012-06-26 | Biogen Idec Ma Inc. | KIM-1 antibodies for treatment of TH2-mediated conditions |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994013312A1 (en) * | 1992-12-15 | 1994-06-23 | The Board Of Trustees Of The Leland Stanford Junior University | Mucosal vascular addressin, dna and expression |
MX9706049A (en) * | 1995-02-10 | 1998-02-28 | Leukosite Inc | Mucosal vascular addressins and uses thereof. |
US6037324A (en) * | 1996-01-04 | 2000-03-14 | Leukosite, Inc. | Inhibitors of MAdCAM-1-mediated interactions and methods of use therefor |
-
1996
- 1996-11-01 WO PCT/US1996/017549 patent/WO1998020110A1/en not_active Application Discontinuation
- 1996-11-01 EP EP96938720A patent/EP0948597A4/en not_active Withdrawn
- 1996-11-01 JP JP52132098A patent/JP2001503271A/en active Pending
Non-Patent Citations (5)
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001098481A2 (en) * | 2000-06-16 | 2001-12-27 | Biogen, Inc. | Renal regulatory elements and methods of use thereof |
WO2001098481A3 (en) * | 2000-06-16 | 2003-02-27 | Biogen Inc | Renal regulatory elements and methods of use thereof |
US7179901B2 (en) | 2000-06-16 | 2007-02-20 | Biogen Idec Ma Inc. | Renal regulatory elements and methods of use thereof |
US7597887B2 (en) | 2002-12-30 | 2009-10-06 | Biogen Idec Ma Inc. | KIM-1 antagonists and use to modulate immune system |
WO2004081049A1 (en) * | 2003-03-10 | 2004-09-23 | Auckland Uniservices Limited | Monoclonal antibodies that recognise mucosal addressin cell adhesion molecule-1 (madcam-1), soluble madcam-1 and uses thereof |
US8206705B2 (en) | 2005-03-02 | 2012-06-26 | Biogen Idec Ma Inc. | KIM-1 antibodies for treatment of TH2-mediated conditions |
Also Published As
Publication number | Publication date |
---|---|
EP0948597A4 (en) | 2002-07-10 |
EP0948597A1 (en) | 1999-10-13 |
JP2001503271A (en) | 2001-03-13 |
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