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  • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• • A—HUMAN NECESSITIES
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• • C—CHEMISTRY; METALLURGY
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  • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
  • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
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  • 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
  • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
  • C07K14/70564—Selectins, e.g. CD62
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  • C12N9/10—Transferases (2.)
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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to the biology of cell adhesion and, in particular, to molecules that are involved in the expression of surface ligands, particularly CDX, a glycoprotein involved in leukocyte binding to the adhesion molecule, ELAM1.
• Inflammation characteristically involves, among other things, the adhesion of leukocytes (white blood cells) to the endothelial wall of blood vessels and the infiltration of leukocytes into the surrounding tissues. (Harlan, 1985.) In normal inflammation, the infiltrating leukocytes phagocytize invading organisms or dead cells and play a role in tissue repair. However, in pathologic inflammation, infiltrating leukocytes can cause serious and sometimes deadly damage.
• ELAMl appears to be a major mediator of PMN adhesion to the inflamed vascular wall in vivo.
• ELAMl is a 116 kD cell surface glycoprotein.
• HUVECs human umbilical vein endothelial cells
• HUVECs human umbilical vein endothelial cells
• CDX is present on leukocyte cell types known to adhere to ELAMl and is absent from leukocyte cell types and other cell types that do not adhere to ELAMl.
• CDX is a molecule expressed on certain leukocytes that plays an important role in ELAMl-mediated leukocyte- endothelial cell adhesion.
• ELAMs may play important roles in a wide range of pathological states involving cell-cell recognition, including tumor invasion, metastasis and viral infection.
• CDX and ELAMl play important roles in inflammation and, perhaps, other pathologies.
• the isolation of molecules that contribute directly or indirectly to their expression will be an important step in the development of therapies aimed at preventing cell adhesion during inflammation or at limiting ELAMl and CDX involvement in other pathological states.
• This invention provides DNA sequences encoding molecules that cause several cell lines, including COS, CHO and Rl.l, both to express surface glycoproteins that are recognized by anti-CDX ( ⁇ -CDX) antibodies and to bind to ELAMl.
• This invention provides, in particular, clone 7.2 and clone 1, and protein 7.2 and protein 1, respectively. These proteins appear to be 1,3-fucosyl transferases.
• This invention also provides the glycoproteins, Pseudo-X and Pseudo-X , which cause COS cells and CHO cells, respectively, to bind ELAMl and to be recognized by ⁇ :-CDX antibodies.
• Figure 1 depicts the sequence of cDNA coding for protein 7.2 and the deduced amino acid sequence of protein 7.2 derived from pSQ219 and CDX pCDM8 clone 7.2.
• the nucleotides are numbered 1-2175.
• Figure 2 depicts the sequence of cDNA coding for protein 1 derived from clone 1.
• the nucleotides are numbered 1-2861.
• the coding DNA sequence of this figure as the DNA sequence for clone 1.
• the polypeptide comprising the amino acid sequence depicted in this figure as protein 1.
• Expression control sequence A DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
• a DNA sequence is operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
• the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
• Standard hybridization conditions salt and temperature conditions substantially equivalent to 5 x SSC and 65°C for both hybridization and wash.
• DNA sequences of this invention will hybridize to other DNA sequences having sufficient homology, including homologous sequences from different species. It is understood that the stringency of hybridization conditions is a factor in the degree of homology required for hybridization.
• DNA sequences of this invention refer to DNA sequences prepared or isolated using recombinant DNA techniques. These include cDNA sequences, DNA sequences isolated from their native genome, and synthetic DNA sequences. The term as used in the claims is not intended to include naturally occurring DNA sequences as they exist in Nature. Expression of recombinant DNA molecules according to this invention may involve post- translational modification of a resultant polypeptide by the host cell. For example, in mammalian cells expression might include, among other things, glycosylation, lipidation or phosphorylation of a polypeptide, or cleavage of a signal sequence to produce a "mature" protein.
• the term "protein” encompasses full-length polypeptides and modifications or derivatives thereof, such as glycosylated versions of such polypeptides, mature proteins, polypeptides retaining a signal peptide, truncated polypeptides having comparable biological activity, and the like.
• the molecules of the present invention are involved in the expression of the glycoprotein, CDX, on the surface of certain leukocytes. CDX appears on SDS- PAGE as a single, diffuse band of about 150 kD. A 90 kD protein band was sometimes observed in the bound proteins from HL-60 cells and always in the proteins from neutrophils. We believe this 90 kD band represents a CDX degradation product.
• 1,3-fucosyl transferases are highly specific enzymes that function in the Golgi apparatus and endoplasmic reticulum to attach fucosyl moieties to appropriate acceptor carbohydrates through a 1,3 glycosidic linkage. The genetic structure of these sequences is consistent with that of other, known glycosyl transferases.
• CHO cells transfected with clone 7.2 express fucosyl transferase activity.
• COS 7 cells When COS 7 cells were transfected with either of these two clones, they behaved like cells expressing CDX, that is, they became "visible" to ELAMl in that they were able to produce a surface glycoprotein to which ELAMl binds and which are recognized by the ⁇ -CDX monoclonal, SGB 3 B 4 .
• ⁇ -CDX monoclonals we immunoprecipitated a 130 kD glycoprotein from transfected COS cells, which we have designated
• Pseudo-X CHO cells transfected with clone 7.2 also became visible to ELAMl and ⁇ -CDX. They express a 140 kD glycoprotein which we have designated Pseudo-X . Neither Pseudo-X nor Pseudo-X are CDX.
• Pseudo-X has a molecular weight of about 130 kD and Pseudo-X , of 140 kD.
• CDX has a molecular weight of 150 kD.
• N-glycanase or hydrofluoric acid which removes all carbohydrate
• Pseudo-X shifts to 110 kD.
• Pseudo-X shifts to approximately 120 kD.
• CDX shifts to about 70 kD. Neither migrates at 46 kD or 59 kD, the predicted molecular weights of protein 7.2 and protein 1.
• Pseudo-X and CDX also have different V8 and chymotrypsin digestion patterns.
• DNA sequences disclosed herein may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
• Such operative linking of a DNA sequence of this invention to an expression control sequence includes the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence, if it is not already part of the DNA sequence or the expression vector.
• Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
• Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA, e.g., M13 and Filamentous single stranded phage DNA; yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasm
• any of a wide variety of expression control sequences sequences that control the expression of a DNA sequence operatively linked to it — may be used in these vectors to express the DNA sequences of this invention.
• useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5) , the promoters of the yeast ⁇ - mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
• a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
• These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli. Pseudomonas, Bacillus. Streptomyces. fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10) , insect cells (e.g., Sf9) , and human cells and plant cells in tissue culture. It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention.
• Suitable expression control sequence a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures.
• Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products. It will also be recognized that expression of the DNA sequences of the present invention may have different effects in different hosts.
• clone 7.2 expressed in COS cells leads to the appearance of an ELAMl-binding surface molecule
• expression of clone 7.2 in, e.g., prokaryotic host cells may have no similar effect, since prokaryotes lack internal cell structures (e.g., Golgi apparatus) that may be necessary for the biological functionality of protein 7.2.
• prokaryotes lack internal cell structures (e.g., Golgi apparatus) that may be necessary for the biological functionality of protein 7.2.
• host cells in which protein 7.2 does not have a function in the cellular biochemistry such as the catalytic role of a glycosyl transferase
• the practitioner will be able to select the appropriate host cells and expression mechanisms for a particular purpose.
• Several strategies are available for the isolation and purification of protein 7.2 and protein 1 after expression in a host system.
• One method involves expressing the proteins in bacterial cells, lysing the cells, and purifying the protein by conventional means.
• Colley et al. (1989) describe purifying a sialyltransferase by engineering the cleavable signal peptide of human gamma-interferon onto the DNA sequence for the transferase.
• Larsen et al. (1990) fused the DNA sequence for protein A to the amino-terminal end of a fucosyl transferase gene and expressed it as an excreted fusion protein.
• the 1,3-fucosyl transferases of this invention are useful for enzymatic synthesis of carbohydrates in vitro. Specifically, they are useful for catalyzing the linkage of fucose to appropriate acceptors through a 1,3 glycosidic bond.
• suitable conditions for this catalysis in Example I, relating to an assay for fucosyl transferase activity.
• One skilled in the art will recognize other suitable conditions under which the 1,3 fucosyl transferases described herein may be advantageously employed.
• CDX is important in ELAMl-mediated cell adhesion.
• a molecule comprising the carbohydrate moiety of CDX, Pseudo-X or Pseudo-X , or a fucose-containing portion of that moiety may be sufficient to function as an ELAMl ligand.
• Such molecules may be useful in methods, including therapies, directed to inhibiting ELAM1- mediated cell adhesion.
• This invention is also directed to small molecules that inhibit the activity of the 1,3-fucosyl transferases described herein, including synthetic organic chemicals, natural fermentation products, peptides, etc. These molecules may be useful in therapies aimed at inhibiting ELAMl-mediated cell adhesion.
• a test mixture by contacting together an inhibitor candidate, a fucose acceptor and a 1,3-fucosyl transferase.
• the fucose acceptor is, preferably,
• the 1,3-fucosyl transferase preferably is derived from an extract from a cell transformed with clone 7.2 or clone 1. Then one assays the test mixture for 1,3-fucosyl transferase activity, such as described in Example I.
• CDX clone 1 We isolated several long inserts from the HL-60 library, transfected them into COS 7 cells, and selected clones that bound to ELAMl and ⁇ -CDX. In this way we identified a 2.9 kb insert that could have come from the 3.0 kb message. We called it CDX clone 1.
• the transfected COS cells also formed rosettes around Sepharose beads coated with recombinant soluble ELAMl (rsELAMl) ; and the rosetting was cation dependent and was inhibited by both BB11 (anti-ELAMl antibody) and ⁇ -CDX.
• COS cells and CHO cells transfected with pCDM8 alone (without the inserted clone) did not rosette rsELAMl beads.
• the COS and CHO cells transfected with clone 7.2 did not rosette to beads coated with bovine serum albumin.
• Clone 1 encodes a polypeptide of 530 amino acids (encoded by nucleotides 174-1763 of Figure 2) .
• Clone 7.2 encodes a 405-amino acid polypeptide (encoded by nucleotides 66-1280 in Figure 1) .
• UWGCG Sequence Analysis Software Package version 6.1, Aug. 1989
• NBRF Protein database release 23, Dec. 1989
• FASTA FASTA for homology to other proteins.
• GenBank release 63, Mar. 1990
• EMBL release 19, May 1989
• the portion of the nucleotide sequence of clone 7.2 from nucleotide 9 to nucleotide 2162 ( Figure 1) is identical to the portion of the sequence of clone 1 from nucleotide 492 to nucleotide 2645 ( Figure 2).
• the first methionine of protein 7.2 corresponds to the methionine at amino acid 126 of protein 1.
• the two inserts represent different transcripts from the same DNA segment. As we stated earlier, these clones do not code for CDX, Pseudo-X or Pseudo-X — the polypeptides they encode are not the correct size.
• clone 7.2 and clone 1 encode 1,3-fucosyl transferases that glycosylate other proteins, such as CDX, Pseudo-X and Pseudo-X , in a way that makes them "visible” (i.e., recognized by or able to bind to) ELAMl or ⁇ -CDX.
• DNA sequences of clone 1 and clone 7.2 share several structural features with the DNA sequences of known glycosyl transferases. For example, genes encoding known glycosyl transferases commonly have consecutive methionine start sites and are capable of producing more than one mRNA transcript.
• clone 1 contains two codons that can serve as transcription start signals.
• the clones have multiple SP1 enhancer sites. The nucleotide sequences for these sites are GGGCGG or CCGCCC; clone 1 has five such sites.
• clones 7.2 and 1 are rich in guanine (G) and cytosine (C) . For example, clone 1 is 75% GC rich in the 5' region of the gene and 60% GC rich in the 3 ' region of the gene.
• Glycosyl transferases in addition are typically class II membrane proteins, in which the membrane-spanning domain is near the amino terminus and the extracellular portion is near the carboxy terminus.
• Clone 1 and clone 7.2 encode a polypeptide having a hydrophobic region near the amino terminus.
• Glycosyl transferases also tend to have molecular weights between 40 kD to 60 kD; clone l encodes a polypeptide of about 59 kD and clone 7.2 encodes a polypeptide of about 46 kD.
• known glycosyl transferases usually have one to three N-glycosylation sites; clone 1 and clone 7.2 both encode two such sites.
• enzyme assays performed on extracts from CHO cells transfected with clone 7.2 revealed the presence of fucosyl transferases not expressed in untransformed cells.
• the assays tested the ability of the enzyme to link radioactively labelled fucose to an acceptor molecule.
• We prepared the enzyme by isolating about 1.5 million CHO cells transfected with clone 7.2 and lysing them by sonication for 15 seconds in 150 ⁇ l ice-cold 1% Triton X-100 in water.
• the cocktail contained 75 ⁇ M 14 C-GDP fucose, 100 mM ATP, 500 mM L-fucose, 1 M MnCl and 1 M cacodylate at pH 6.2.
• 10X acceptor contained, variously, 200 mM LacNAc, Lac-N-biose, or lactose, 250 mM phenyl-3-D-galactoside, or 50 mM 2 '-fucosyllactose.

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• 1990
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