WO1996040881A1 - Fucosyltransferase genes and uses thereof - Google Patents
Fucosyltransferase genes and uses thereof Download PDFInfo
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- WO1996040881A1 WO1996040881A1 PCT/US1996/006427 US9606427W WO9640881A1 WO 1996040881 A1 WO1996040881 A1 WO 1996040881A1 US 9606427 W US9606427 W US 9606427W WO 9640881 A1 WO9640881 A1 WO 9640881A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/1048—Glycosyltransferases (2.4)
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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
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- C12P21/00—Preparation of peptides or proteins
- C12P21/005—Glycopeptides, glycoproteins
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Definitions
- This invention relates to recombinant fucosyltransferases, DNA, and uses thereof.
- the structurally related endothelial cell receptors E-selectin (Bevilacqua et al., Proc. Natl . Acad. Sci . USA 134:9238, 1987; Bevilacqua et al., Science 243:1160 f 1989) and P-selectin (Hsu-Lin et al., J. Biol . Chem . 259;9121. 1984; Stenberg et al., J. Cell Biol .
- P-selectin also mediates monocyte and neutrophil binding to activated platelets (Larsen et al., Cell 59:305, 1989; Hamburger et al., Blood 75:550, 1990).
- the leading candidate ligands for the two receptors are the sialyl-Le x structure for E-selectin (Lowe et al., Cell 62:475, 1990; Phillips et al.. Science 250:1130, 1990); alz et al., Science 250:1132.
- Gal,_ 3GlcNAc respectively (Kukowska-Latallo et al.. Genes D v. 4.:1288, 1990); at least one enzyme, Fuc-TIV, solely forming ⁇ (l,3) linkages, which cannot utilize sialylated substrates (Goelz et al.. Cell 3:1349, 1989; Lowe et al., J. Biol . Chem. 266:17467. 1991); at least two enzymes, Fuc-TV ( eston et al., J. Biol . Chem . 267:4152 f 1992a) and Fuc-TVI (Weston et al., J. Biol . Chem . 267:24575.
- the invention features substantially pure ⁇ (l,3) fucosyltransferase, including an amino acid sequence substantially identical to the sequence shown in Fig. 3 (SEQ ID NO: 2) .
- pure ⁇ (l,3) fucosyltransferase is obtained from a mammal (for example, a murine cell line (e.g., 32D cl3) , or from a human) .
- the invention features a fragment or analog of ⁇ (1,3)fucosyltransferase polypeptide including an amino acid sequence substantially identical to the sequence shown in Fig. 3 (SEQ ID NO: 2) .
- the invention features substantially pure DNA having a sequence substantially identical to the nucleotide sequence shown in Fig. 3 (SEQ ID NO: 1) .
- such DNA is cDNA or is genomic DNA.
- the invention also features a vector and a cell (e.g., a murine cell such as 32D cl3 or a human cell such as human cell line 293) which includes such substantially pure DNA.
- the vector-containing cell is a prokaryotic cell, for example, JL. coli. or, more preferably, is a eukaryotic mammalian cell (e.g. , the murine cell line 32D cl3 or human cell line 293) .
- the invention features a method of fucosylating a polypeptide in vivo involving: (a) providing a cell containing the fucosyltransferase DNA of the invention including a nucleotide sequence which is substantially identical to the sequence shown in Fig. 3 (SEQ ID NO: 1) positioned for expression in the cell; and (b) culturing the transformed cell under conditions for expressing the DNA, resulting in the fucosylation of the protein.
- fucosylation occurs in a mammalian cell, for example, a human cell (e.g., human cell line 293) or a murine cell (e.g., 32D cl3) .
- the cell contains a second fucosyltransferase gene.
- a second gene is substantially identical to the nucleotide sequence shown in Fig. 6A (SEQ ID NO: 3) which encodes a polypeptide including an amino acid sequence substantially identical to the sequence shown in Fig. 6B (SEQ ID NO: 4) .
- the protein which is fucosylated according to the above method is an AGP-antibody fusion protein or is an antibody (e.g., IgG or IgM) .
- the invention features a recombinant polypeptide fucosylated using a cell expressing DNA which is substantially identical to the nucleotide sequence shown in Fig. 3 (SEQ ID NO: 1) .
- the fucosylated polypeptide is an AGP- antibody fusion protein, or is an antibody (e.g., IgG or IgM) .
- the polypeptide is further fucosylated using a second fucosyltransferase.
- a second fucosyltransferase is substantially identical to a polypeptide including an amino acid sequence shown in Fig. 6B (SEQ ID NO: 4) .
- the cell used to fucosylate the polypeptide is a mammalian cell (e.g., the murine cell line 32D cl3 or the human cell line 293) .
- the invention features a polypeptide fucosylated in vitro using a fucosyltransferase having an amino acid sequence substantially identical to the sequence shown in Fig. 3 (SEQ ID NO: 2) .
- the fucosylated polypeptide is further fucosylated using a second fucosyltransferase.
- a second fucosyltransferase includes an amino acid sequence substantially identical to the sequence shown in Fig. 6B (SEQ ID NO: 4) .
- the fucosylated polypeptide is an AGP-antibody fusion protein or is an antibody (e.g., IgG or IgM).
- the invention features a substantially pure polypeptide of the invention which is fucosylated .in vivo or in vitro and which is capable of protecting a mammal against an adverse immune reaction.
- an adverse immune reaction is septic shock or is septicemia.
- the invention features a cell containing at least two recombinant fucosyltransferases, one of the fucosyltransferases being substantially identical to the amino acid sequences shown in Fig 3. (SEQ ID NO: 2) and another of the fucosyltransferases being substantially identical to the amino acid sequence shown in Fig. 6B (SEQ ID NO: 4) .
- a DNA- containing cell is a prokaryotic cell (e.g., ]___. coli) or is a eukaryotic cell, for example, a mammalian cell (e.g., the murine cell line 32D cl3 or human cell line 293) .
- the invention features a method of fucosylating a polypeptide in vitro comprising: (a) providing an ⁇ (l,3) fucosyltransferase of the invention; and (b) contacting the polypeptide with the fucosyltransferase under conditions sufficient for fucosylating the polypeptide.
- polypeptide is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
- substantially identical is meant a polypeptide exhibiting at least 50%, preferably 70%, more preferably 90%, and most preferably 95% homology to a reference amino acid or is meant a nucleic acid sequence exhibiting at least 85%, preferably 90%, more preferably 95%, and most preferably 97% homology to a reference nucleic acid sequence.
- the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids.
- the length of comparison sequences will generally be at least 30 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
- Sequence identity is typically measured using sequence analysis software (e.g.. Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) . Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications.
- Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
- substantially pure polypeptide a fucosyltransferase polypeptide which has been separated from components which naturally accompany it.
- the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally- occurring organic molecules with which it is naturally associated.
- the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, fucosyltransferase polypeptide.
- a substantially pure fucosyltransferase polypeptide may be obtained, for example, by extraction from a natural source (e.g., a murine cell such as 32D cl3); by expression of a recombinant nucleic acid encoding a fucosyltransferase polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
- a protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
- substantially pure polypeptides include, without limitation, those derived from eukaryotic organisms but synthesized in E_i. coli or other prokaryotes, or those derived from a eukaryotic cell which does not normally synthesize such a protein, or those derived from a eukaryotic cell engineered to overexpress such a protein.
- substantially pure DNA DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene.
- the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
- transformed cell is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a fucosyltransferase polypeptide.
- positioned for expression is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a recombinant fucosyltransferase polypeptide or RNA molecule) .
- promoter is meant the minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render transcription controllable for cell- type specific, tissue-specific, or inducible expression; such elements may be located in the 5' or 3' regions of the native gene.
- operably linked is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s) .
- purified antibody is meant antibody which is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated.
- the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., a fucosyltransferase-specific antibody.
- a purified fucosyltransferase antibody may be obtained, for example, by affinity chromatography using recombinantly-produced fucosyltransferase protein or conserved motif peptides and standard techniques.
- telomere binding protein By “specifically binds” is meant an antibody which recognizes and binds a fucosyltransferase protein but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes fucosyltransferase.
- Figure 1 is a flow cytometry profile showing the expression of fucosylated glycans by COS cells transfected with murine myeloid fucosyltransferase. The results are expressed as mean fluorescence intensity in arbitrary units. No expression above background of any carbohydrate epitope except sialyl-Le x was seen.
- Figures 2A-C are panels of autoradiograms showing different nucleic acid blot hybridizations.
- Panel (A) is an autoradiogram showing a DNA blot hybridization of total genomic DNA from mouse kidney tissue.
- Murine genomic DNA (15 ⁇ g) was digested with the indicated restriction enzymes and subjected to fractionation, transfer, and blot hybridization.
- Panel (B) is an autoradiogram showing an RNA blot hybridization of total RNA from different cell lines.
- Total RNA (20 ⁇ g) prepared from each of the cell lines shown was denatured, fractionated by gel electrophoresis, transferred to nylon and hybridized.
- the lineage origins of the cell lines are: YAC-1 (T cell leukemia) , EL4 (thymoma) , RDM4 (T cell leukemia) , CTLL-2 (IL-2 dependent cytotoxic T cell) , 32D cl3 (IL-3 dependent granulocyte precursor) , WEHI-231 (B cell lymphoma, non-secreting, mouse) , WEHI-279 (B cell lymphoma, non-secreting, mouse) , Sp2/0 (plasmacytoma) , WEHI-3B (myeloid (monocytic) leukemia) , P815 (mastocytoma) , Ltk (fibroblast) , and Balb 3T3 (fibroblast) .
- YAC-1 T cell leukemia
- EL4 thymoma
- RDM4 T cell leukemia
- CTLL-2 IL-2 dependent cytotoxic T cell
- 32D cl3 IL-3 dependent granulocyte precursor
- Panel (C) is an autoradiogram showing an RNA blot hybridization of total RNA from skeletal tissue.
- Total RNA (20 ⁇ g) prepared from skeletal muscle (Sk. muscle) was denatured, fractionated by gel electrophoresis, transferred to nylon and hybridized. Arrows denote the location of ribosomal RNAs.
- Figure 3 is the nucleotide (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of the murine myeloid-lineage fucosyltransferase cDNA. Two sites for N-linked glycan addition are underlined in the predicted peptide sequence, as is the upstream ATG in the nucleic acid sequence.
- Figure 4 is a flow cytometry profile showing the expression of fucosylated glycans by 32D cl3 cells stably transfected with the human myeloid fucosyltransferase.
- Pooled products of the transfection of 32D cl3 cells with a human Fuc-TIV myeloid fucosyltransferase expression plasmid bearing a selectable marker were evaluated by indirect immunofluorescence using anti-carbohydrate monoclonal antibodies and flow cytometry. The results are expressed as mean fluorescence intensity in arbitrary units.
- Figure 5 is a bar graph of cell adhesion assays (Panels A-B) .
- Panel (A) shows the adhesion to E-selectin or P-selectin IgG fusion proteins of COS cells transfected with either the human Fuc-TIV (FTIV) or the murine 32D cl3 fucosyltransferase (32DFT) cDNAs in the presence or absence of the P-selectin glycoprotein ligand (PSGL) .
- FTIV human Fuc-TIV
- 32DFT murine 32D cl3 fucosyltransferase
- Panel (B) shows adhesion to E-selectin or P- selectin IgM fusion proteins of 32D cl3 cells (32D cl3) or 32D cl3 cells transfected with the human myeloid Fuc- TIV cDNA (FTIV) .
- TLISA control IgM fusion protein
- E P
- Columns represent average cpm bound of triplicate samples.
- panel A is the nucleotide sequence (SEQ ID NO: 3) and, panel B, the deduced amino acid sequence (SEQ ID NO: 4) of the human myeloid-lineage Fuc-TIV cDNA.
- SEQ ID NO: 3 the nucleotide sequence
- panel B the deduced amino acid sequence (SEQ ID NO: 4) of the human myeloid-lineage Fuc-TIV cDNA.
- YAC-1, EL4, RDM4, CTLL2, Sp2/0, WEHI-231, WEHI-279 and P815 cell lines were cultured in IMDM, 10% FCS, 50 mM mercaptoethanol, 50 U/ml penicillin and 50 ⁇ g/ml streptomycin.
- Balb 3T3, Ltk " and COS-7 m6 cell lines were passaged in DMEM, 10% calf serum (CS) and 25 ⁇ g/ml gentamicin sulfate.
- the IL-3 producing, macrophage-like cell line WEHI-3B was cultured in RPMI- 1640, 10% FCS, 50 U/ml penicillin and 50 mg/ml streptomycin.
- the IL-3 dependent mouse neutrophil progenitor cell line, 32D cl3 was cultured in RPMI-1640, 10% FCS, 10% WEHI 3B-conditioned medium, 50 U/ml penicillin and 50 ⁇ g/ml streptomycin (Kreider et al., Oncogene 7:135, 1992).
- an expression library was prepared from mRNA isolated from the murine cell line 32D cl3, which phenotypically resembles a granulocyte precursor and which binds murine E- and P- selectin (Levinovitz et al., J. Cell Biol . 121:449 f 1993) as follows.
- a cDNA library in the expression vector CDM8 was prepared from 32D cl3 cells as described by Aruffo et al. (Proc. Natl . Acad . Sci .
- the cDNA pellet was resuspended in 225 ⁇ l distilled water, 25 ⁇ l 10X Mung bean incubation buffer (500 mM sodium acetate, 300 mM NaCl, 10 mM zinc sulfate, pH 5.0), and 10 U of Mung bean nuclease (New England Biolabs, Beverly, MA.).
- the reaction was stopped by adding 20 ⁇ l of 1 M Tris-HCl, pH 8.0 and 3 ⁇ l 0.5 M EDTA, pH 8.0.
- the cDNA was phenol extracted, ethanol precipitated and resuspended in 90 ⁇ l distilled H 2 0.
- the ligated cDNA in CDM8 was introduced into electrocompetent MC1061/p3 cells by electroporation in 0.2 cm gap cuvettes (Bio-Rad laboratories, Hercules, CA) at a voltage of 2.5 kV, a capacitance of 25 ⁇ F and a parallel resistance of 400 Ohms.
- Transformed bacteria were plated on 20 dishes, 23 x 23 cm in size (Nunc, Denmark). Bacteria from each dish ( « 1.25 X 10 5 colonies) were harvested and an aliquot stored frozen at -70°C in 40% glycerol.
- Plasmid DNA was isolated from each pool using a commercial kit (plasmid midi prep QIAGEN Inc., Chatsworth, CA) according to the manufacturer's recommendations.
- the library was then divided into 20 pools of 1.25 x 10 5 cells each and between 200 and 500 ng of plasmid DNA from each of the 20 pools was separately transfected into COS-7 m6 cells at approximately 70% confluence in a 10 cm-dish using the DEAE-dextran method described by Seed et al. (Proc. Natl . Acad. Sci USA 84.J3365, 1987).
- the COS cells were stained with the sialyl-Le x antibody and bacteria from positive pools replated at lower density.
- dishes with positive cells were identified by immunocytochemistry using an anti-sialyl- Le x antibody (KM93, mouse IgM; Kamiya Biomedical Company, Thousand Oaks, CA) , and an avidin-biotin complex protocol employing 9-amino-3-ethylcarbazol as a peroxidase substrate kit (Vector Labs, Burlingame, CA) essentially as described Horst et al. (Nucleic Acids Res . 19:4556, 1991; Vector Labs) . Bacteria corresponding to positive pools were subsequently replated at lower density on 10 cm dishes.
- an anti-sialyl- Le x antibody KM93, mouse IgM; Kamiya Biomedical Company, Thousand Oaks, CA
- an avidin-biotin complex protocol employing 9-amino-3-ethylcarbazol as a peroxidase substrate kit (Vector Labs, Burlingame, CA) essentially as described Horst e
- Plasmid DNA from these subpools was transfected into COS cells in 6 cm dishes. The procedure was repeated until a single plasmid was recovered that conferred binding of anti-sialyl-Le x antibody to transfected COS cells. Bacterial cells from the pool giving rise to the highest number of positive transfectants were plated at lower density on agar plates and DNA prepared from the bacteria was transfected into COS cells, allowing pools of successively less sequence complexity to be obtained until finally a single clonally pure plasmid isolate was shown to be capable of directing the appearance of the sialyl Lewis-X epitope in COS cells. Five out of twenty pools contained five or more positive cells.
- Staining of cells for FACS analysis was done by incubating 2 x 10 6 cells on ice for 20 to 30 minutes in 0.5 ml of 3% BSA in PBS with 4 ⁇ g/ml of antibody or in 0.5 ml of hybridoma supernatant for 20 to 30 minutes.
- anti-Le x antibody PM81, mouse IgM; Medarex, Inc., West Lebanon, NH
- anti-sialyl-Le x KM93, mouse IgM
- anti-sialyl-Le a KM231, mouse IgGl; Kamiya Biomedical Co., Thousand Oaks, CA
- anti-Le a antibody T174, mouse IgGl; Signet, Dedham, MA
- anti-CD65 (VIM-2) antibody 88H7, mouse IgM; AMAC, Westbrook, ME
- hybridoma secreting a mouse IgG3 antibody against di- and tri- fucosylated Le x FHCR-1-2075/FH4; ATCC, Rockland, MD
- the cells were resuspended in 0.5 ml of 3% BSA in PBS containing 2 ⁇ g/ml FITC-conjugated anti-mouse IgG or IgM antibody (Organon Teknika Corp., Durham, NC) . Washed cells were immediately analyzed by flow cytometry (Coulter Corp., Hialeah, FA) according to standard methods. Flow cytometric analysis of the transfected cells showed that sialyl-Le x , but neither Le x , CD65, di/trimeric Le x (FH4 epitope) , Le a , nor sialyl-Le a determinants could be detected (Fig. 1) .
- DNA Blot hybridization was performed as follows. Fifteen micrograms of mouse genomic DNA (Adult:, male, Balb/c kidney; Clontech Labs, Palo Alto, CA) was digested overnight in a volume of 300 ⁇ l with 50 U of BamHl, 90 U of EcoRl, 100 U of Hindlll and 100 U of PstI individually. The digests were phenol extracted, ethanol precipitated and separated on a 0.8% agarose gel.
- the gel was denatured by incubation in 0.5 M NaOH, 1.5 M NaCl, at room temperature for 30 minutes, briefly rinsed in distilled water, and neutralized for 30 minutes in 0.5 M Tris-HCl, pH 7.0, 1.5 M NaCl at room temperature. Following an incubation in 2OX SSC for 30 minutes, the DNA was blotted and probed as for the RNA blots described below. The results of this analysis indicated that the fucosyltransferase is encoded by at least a single copy gene (Fig. 2A) .
- RNA Blot Hybridization Analysis To study the expression of the 32D cl3 fucosyltransferase gene, RNA blot hybridization was performed as follows. Total RNA was isolated from YAC-1, EL4, RD-M4, CTLL2, 32D Cl3, WEHI 231, WEHI 279, Sp2/0, WEHI 3B, P815, Ltk- and Balb 3T3 cells using a guanidinium-acid phenol protocol (Chomczynski et al., Analyt . Biochem . 162:156. 1987).
- RNA was isolated from the tissues as described above after homogenization on ice in guanidinium thiocyanate buffer using a handheld homogenizer (Omni International, Waterbury, CT) . Twenty micrograms of total RNA was separated by electrophoresis in a 1.2% agarose/formaldehyde gel and transferred to nylon membranes (Schleicher & Schuell, Keene, N.H.) using a downward transfer system (Schleicher & Schuell) according to the manufacturer's recommendations.
- RNA absorbed to the membrane was crosslinked by UV irradiation (1200 ⁇ J) and detected by hybridization with a randomly primed probe using standard conditions (Ausubel et al.. Current Protocols In Molecular Biology, Wiley Interscience, 1995) .
- RNA blot analysis showed a pattern of highly tissue-restricted expression of a message of 1.9 kb (Fig. 2 B and C) .
- high levels of mRNA were found in 32D cl3 and CTLL-2, an IL-2 dependent cytotoxic T cell line, with the myeloid cell line WEHI 3B and the mastocytoma P815 having significant quantities of the mRNA (Fig. 2B) .
- No message was detected in T-cell lines (YAC-1, EL4 and RDM4) , in B cell or fibroblast lines, or in any of the tissues sampled (Fig. 2B) .
- the cDNA insert consists of 1814 nucleotides, terminating just 3' to a canonical upstream poly(A) sequence motif (Fig. 3) .
- the largest open reading frame begins at a methionine at position 325 which does not meet the sequence requirements for a translational initiation consensus, but is preceded by only one other candidate ATG, similarly lacking a consensus initiation context and giving rise to a translation product which terminates within a few residues.
- the predicted polypeptide contains a long amino-terminal hydrophobic region preceded by arginine residues, similar in structure to the transmembrane domain of type II (amino terminally anchored) integral membrane proteins typical of this class of glycosyltransferase.
- the predicted molecular mass of the encoded protein is 39.4 kilodaltons, with the presence of two N-linked glycan addition sites at residues 81 and 291 suggesting that the mature protein may be larger.
- the Sffv Fuc-TIV plasmid was linearized by digestion with Avr2, phenol extracted, ethanol precipitated, and electroporated into the 32D cl3 cell line as follows.
- the cells (8 x 10 7 ) were resuspended in 0.8 ml RPMI-1640, 10% FBS, 10% WEHI-3B conditioned medium, and transferred together with 40 ⁇ g of linearized plasmid DNA to a 0.4 cm-gap electroporation cuvette (Bio-Rad, Hercules, CA) on ice.
- a single pulse was delivered at a voltage of 250V and a capacitance of 500 ⁇ F.
- the cuvette was put back on ice for 10 minutes before the cells were transferred to a flask containing 50 ml of medium. Puromycin was added to the medium the following day at a concentration of 0.5 ⁇ g/ml. After approximately 2 weeks, with media changes every second to third day, the cells were checked for expression of the sialyl-Le x epitope.
- Fusion Proteins The construction of DNA sequences coding for fusions between E- and P-selectin extracellular domains (for P-selectin only 2 of the complement regulatory domains were included) and the Fc part (hinge, CH2 and CH3) of human genomic IgGl was performed as previously described (Walz et al.. Science 250:1132. 1990; Aruffo et al.. Cell .61:1303, 1990).
- the cDNA sequences for E- and P-selectin extracellular domains were fused to the genomic sequence of human IgM Fc (CH2, CH3 and CH4) by transferring the selectin sequences from an IgG fusion vector to an IgM fusion vector created in this laboratory (Zettlmeissl et al., DNA Cell Biol . 9:347, 1990).
- the PSGL-1 cDNA coding sequence was obtained by PCR from an HL-60 cDNA library and confirmed by DNA sequencing.
- the coding segment for the mature extracellular, transmembrane and intracellular domain was inserted in an expression vector based on CDM8 which lacks the polyoma virus origin of replication and contains the leader sequence for the CD5 antigen positioned just upstream of the coding region for an influenza hemagglutinin peptide epitope tag.
- COS cell supernatants containing soluble E- and P- selectin/IgG and IgM fusion proteins were produced as previously described (Walz et al.. Science ___50:1132, 1990; Aruffo et al.. Cell 61:1303, 1990).
- the concentration of fusion protein in the tissue culture supernatants was determined by a 96-well ELISA assay, in which the fusion proteins were captured with an affinity purified, polyclonal anti-human IgG Fc or anti-human IgM ( ⁇ chain specific) antibody (Organon Teknika, Durham, NC) . Captured fusion proteins were detected with a peroxidase-conjugated, affinity purified, polyclonal anti-human IgG Fc or anti-human IgM ( ⁇ chain specific) antibody (Organon Teknika) using O-phenylenediamine dihydrochloride as substrate (Sigma) . The ELISA was calibrated using purified human IgG or IgM (Sigma) .
- Adhesion assays were performed in 96-well ELISA plates (Becton-Dickinson, Oxnard, CA) as follows. The wells were incubated with 100 ⁇ l of 20 ⁇ g/ml anti-human IgG Fc or anti-human IgM (heavy chain specific) in PBS for 2 hrs in a humid chamber at room temperature. After washing the plate twice with PBS, additional protein- binding sites were blocked by an overnight incubation with 200 ⁇ l 3% BSA in PBS. The plate was washed with PBS four times and incubated with 200 ⁇ l of fusion protein supernatants for 2 hrs.
- Transfected cells used for the assay were lifted off the dish with 0.5 mM EDTA in PBS 48 to 60 hrs after transfection and loaded with 100 ⁇ l Na 2 5 lCr0 4 (1 mCi/ml; DuPont, Boston, MA) in 0.9% NaCl plus 100 ml medium at 37°C for 1 hr. Loaded cells were washed twice in PBS and resuspended in 0.2% BSA, 0.15 M NaCl, 3 mM CaCl 2 .
- Adherent cells were lysed by the addition of 200 ⁇ l 2% SDS and counted in a gamma ray spectrometer.
- the 32D cl3 cell line itself binds both human E- and P-selectin/IgM fusion proteins (Fig. 5B) .
- IgM fusion proteins were used in these experiments to avoid the possible contribution of Fc receptor binding.
- 32D cl3 cells stably expressing the human Fuc-TIV (myeloid) enzyme (Fig. 6B) were evaluated in the selectin adhesion assay.
- the transfectants showed an approximately 10-fold higher binding density to human E-selectin relative to untransfected cells, whereas binding to P-selectin was not significantly affected (Fig. 5) .
- fucosyltransferase oligonucleotide probes, including fucosyltransferase degenerate oligonucleotide probes (i.e., a mixture of all possible coding sequences for a given amino acid sequence) .
- fucosyltransferase degenerate oligonucleotide probes i.e., a mixture of all possible coding sequences for a given amino acid sequence
- isolation of other fucosyltransferase genes is performed by PCR amplification techniques well known to those skilled in the art of molecular biology using oligonucleotide primers designed to amplify only sequences flanked by the oligonucleotides in genes having sequence identity to fucosyltransferase of the invention.
- the primers are optionally designed to allow cloning of the amplified product into a suitable vector.
- Hybridization techniques and procedures are well known to those skilled in the art and are described, for example, in Ausubel et al., supra. and Guide to Molecular Cloning Techniques , supra.
- a combination of different oligonucleotide probes may be used for the screening of the recombinant DNA library.
- the oligonucleotides are labelled with 32 P using methods known in the art, and the detectably-labelled oligonucleotides are used to probe filter replicas from a recombinant DNA library.
- Recombinant DNA libraries may be prepared according to methods well known in the art, for example, as described in Ausubel et al., supra. or may be obtained from commercial sources.
- high stringency conditions may be used; such conditions include hybridization at about 42°C and about 50% formamide; a first wash at about 65°C, about 2X SSC, and 1% SDS; followed by a second wash at about 65°C and about 0.1% SDS, IX SSC.
- Lower stringency conditions for detecting fucosyltransferase genes having about 85% sequence identity to the fucosyltransferase gene described herein include, for example, hybridization at about 42°C in the absence of formamide; a first wash at about 42°C, about 6X SSC, and about 1% SDS; and a second wash at about 50°C, about 6X SSC, and about 1% SDS.
- fucosyltransferase oligonucleotides may also be used as primers in PCR cloning strategies.
- PCR methods are well known in the art and described, for example, in PCR Technology, H.A. Erlich, ed. , Stockton Press, London, 1989; PCR Protocols: A Guide to Methods and Applications , M.A.
- fucosyltransferases may be isolated using the PCR "RACE" technique, or Rapid Amplification of cDNA Ends (see, e.g., Innis et al., supra) .
- RACE Rapid Amplification of cDNA Ends
- oligonucleotide primers based on a fucosyltransferase conserved domain are oriented in the 3' and 5' directions and are used to generate overlapping PCR fragments. These overlapping 3'- and 5'-end RACE products are combined to produce an intact full-length cDNA. This method is described in Innis et al. , supra: and Frohman et al., Proc. Natl . Acad. Sci . USA 85:8998, 1988.
- Fucosyltransferase Polypeptide Expression Fucosyltransferases according to the invention may be expressed or produced by transformation of a suitable host cell with all or part of a fucosyltransferase- encoding cDNA fragment (e.g., the cDNA described herein) in a suitable expression vehicle (e.g., those described herein) .
- a fucosyltransferase- encoding cDNA fragment e.g., the cDNA described herein
- a suitable expression vehicle e.g., those described herein
- a fucosyltransferase may be produced in a prokaryotic host (e.g., E ⁇ . coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae or mammalian cells, e.g., 32D cl3, human cell line 293, COS 1, NIH 3T3, and JEG3 cells) .
- a prokaryotic host e.g., E ⁇ . coli
- a eukaryotic host e.g., Saccharomyces cerevisiae or mammalian cells, e.g., 32D cl3, human cell line 293, COS 1, NIH 3T3, and JEG3 cells
- Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, MD; also, see, e.g., Ausubel et al., supra) .
- transformation and the choice of expression vehicle will depend on the host system selected. Transformation methods are described, e.g., in Ausubel et al. (supra) ; expression vehicles may be chosen from those provided, e.g., in Cloning Vectors : A Laboratory Manual (P.H. Pouwels et al., 1985, Supp. 1987).
- pMAMneo provides: an RSV-LTR enhancer linked to a dexamethasone- inducible MMTV-LTR promotor, an SV40 origin of replication which allows replication in mammalian systems, a selectable neomycin gene, and SV40 splicing and polyadenylation sites.
- DNA encoding a fucosyltransferase polypeptide is inserted into the pMAMneo vector in an orientation designed to allow expression. The recombinant fucosyltransferase is isolated as described below.
- pMAMneo expression vehicle examples include COS cells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61, respectively) . More preferably, fucosyltransferase of the invention is expressed or produced by a stably- transfected mammalian cell line (e.g., 32D cl3, or human cell line 293) using the methods and vectors described herein.
- a stably- transfected mammalian cell line e.g., 32D cl3, or human cell line 293
- a number of other vectors suitable for stable transfection of mammalian cells are available to the public, e.g., see Pouwels et al. (supra) : methods for constructing such cell lines are also publicly available, e.g., in Ausubel et al. (supra) .
- cDNA encoding the fucosyltransferase polypeptide is cloned into an expression vector which includes the dihydrofolate reductase (DHFR) gene.
- DHFR dihydrofolate reductase
- the fucosyltransferase-encoding gene into the host cell chromosome is selected for by inclusion of 0.01-300 ⁇ M methotrexate in the cell culture medium (as described in Ausubel et al., supra) . This dominant selection can be accomplished in most cell types. Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described in Ausubel et al. (supra) ; such methods generally involve extended culture in medium containing gradually increasing levels of methotrexate.
- DHFR-containing expression vectors commonly used for this purpose include pCVSEII-DHRF and pAdD26SV(A) (described in Ausubel et al., supra) .
- Any of the host cells described above or, preferably, a DHFR-deficient CHO cell line e.g., CHO DHFR ⁇ cells, ATCC Accession No. CRL 9096
- a DHFR-deficient CHO cell line e.g., CHO DHFR ⁇ cells, ATCC Accession No. CRL 9096
- the recombinant fucosyltransferase polypeptide is expressed, it is isolated, e.g., using affinity chromatography.
- an anti- fucosyltransferase antibody e.g., produced as described below
- Lysis and fractionation of fucosyltransferase-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra) .
- the recombinant protein can, if desired, be further purified, e.g. , by high performance liquid chromatography (see, e.g..
- Isolation of the fucosyltransferase gene also facilitates the identification of molecules which increase or decrease fucosyltransferase expression, and which are therefore useful as therapeutics, e.g., for treatment of inflammation.
- candidate molecules e.g., peptide or non-peptide molecules found, e.g., in a cell extract, mammalian serum, or growth medium on which mammalian cells have been cultured, or oligonucleotides
- Fucosyltransferase expression is then measured by standard Northern blot analysis (Ausubel et al.
- fucosyltransferase cDNA as a hybridization probe.
- the level of fucosyltransferase expression in the presence of the candidate molecule is compared to the level measured for the same cells in the same culture medium but in the absence of the candidate molecule.
- a molecule which promotes an increase or decrease in fucosyltransferase expression is considered useful in the invention.
- Anti-Fucosyltransferase Antibodies Fucosyltransferases described herein (or immunogenic fragments or analogues) may be used to raise antibodies useful in the invention; such polypeptides may be produced by recombinant or peptide synthetic techniques (see, e.g.. Solid Phase Peptide Synthesis, supra: Ausubel et al., supra) .
- the peptides may be coupled to a carrier protein, such as KLH as described in Ausubel et al, supra.
- the KLH-peptide is mixed with Freund's adjuvant and injected into guinea pigs, rats, or preferably rabbits.
- Antibodies may be purified by peptide antigen affinity chromatography.
- Monoclonal antibodies may be prepared using the fucosyltransferase polypeptides described above and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495. 1975; Kohler et al., Eur. J. Immunol . j5:511, 1976; Kohler et al., Eur. J. Immunol . jS:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas , Elsevier, NY, 1981; Ausubel et al., supra) .
- polyclonal or monoclonal antibodies are tested for specific fucosyltransferase recognition by Western blot or immunoprecipitation analysis (by the methods described in Ausubel et al., supra) .
- Antibodies which specifically recognize fucosyltransferase are considered to be useful in the invention; such antibodies may be used, e.g., in an immunoassay to monitor the level of fucosyltransferase produced by a mammal.
- the invention features genes, enzymes, and methods for fucosylating virtually any protein bearing one or more glycan addition sites, e.g., an N-linked glycan addition site.
- N-linked is meant bonded to the amide nitrogen of an asparagine residue of a protein.
- Such antibodies are useful for disrupting undesirable interactions between cells or proteins, or, generally, for disrupting an interaction between any two molecules, one of which bears a determinant specifically recognized by an antibody.
- the carbohydrate moieties block the immunoglobulin domain which triggers complement fixation and F c receptor binding, such antibodies do not elicit the undesirable side effects (i.e., those resulting from complement fixation and F c receptor binding) frequently associated with antibody-based therapies.
- the carbohydrate groups serve not only to inhibit undesirable complement fixation and F c receptor binding, but also perform the function of competitively inhibiting a carbohydrate ligand-cell adhesion protein interaction.
- the antibody generally does not serve any function arising from its specificity, but serves only as a carrier for the carbohydrate groups.
- the carbohydrate side chain includes the sialyl-Le x determinant.
- Sialyl-Le x normally acts to facilitate interaction between cells which bear it (e.g., neutrophils) and cells which bear the protein, ELAM-1 or E-selectin (e.g., endothelial cells, e.g., those lining the blood vessel walls) .
- ELAM-1 or E-selectin e.g., endothelial cells, e.g., those lining the blood vessel walls
- Disrupting this interaction has therapeutic applications, for example, in minimizing inflammation, such as that which occurs following tissue injury, e.g., myocardial infarction, which is characteristic of diseases such as psoriasis or rheumatoid arthritis, or for preventing or inhibiting septicemia or septic shock which is induced by a microbial- or host-mediated immune reaction.
- the gene encoding a protein bearing a sialyl-Le x determinant e.g., an IgGl antibody or an ⁇ -AGP-antibody fusion
- a vector designed to express the protein in a eukaryotic cell see, e.g., those vectors described in Gillies et al., U.S. Patent No. 4,663,281, hereby incorporated by reference.
- the eukaryotic host cell is preferably a mammalian cell (e.g., 32D cl3, or human cell line 293, or a CHO, or lecll cell) , and the expression vector containing the sialyl-Le x -encoding sequence is introduced into the host cell by transient or stable transfection using standard techniques.
- Such host cells are also transfected (transiently or stably) with a vector capable of expressing an ⁇ (1,3)fucosyltransferase of the invention (i.e., an enzyme capable of attaching one or more sialyl-Le x groups to the protein molecule at sialyl- Le x consensus glycosylation sites (N-X-T/S)) .
- the (1,3)fucosyltransferase gene described herein or a combination of the a (1,3)fucosyltransferase gene described herein and the Fuc-TIV gene may be expressed from a vector distinct from that encoding the protein containing sialyl-Le x addition sites, or, if desired, the genes may be carried on, and expressed from, a common vector.
- Mammalian cells are particularly useful hosts for the synthesis of sialyl-Le x modified proteins because they provide all required precursors for sialyl-Le x production. Proteins (e.g., antibodies, AGP, or AGP-antibody fusions) which are fucosylated according to the methods of the invention have important therapeutic and diagnostic uses.
- antibody fusion proteins may be generated and secreted transiently from transfected mammalian cells (for example, COS cells) .
- mammalian cells for example, COS cells
- cDNA encoding a domain of interest is fused in-frame, for example, to human IgG domains (for example, constant domains) by standard techniques, and the fusion protein is expressed.
- the antibody portion of the molecule facilitates fusion protein purification and also prolongs the plasma half- life of otherwise short-lived polypeptides or polypeptide domains.
- Recombinant plasmids expressing ⁇ 1 -AGP-IgGl fusion proteins are disclosed in Seed et al., USSN 08/472,888, entitled "AGP-Antibody Fusion Proteins and Related Molecules and Methods," filed June 7, 1995.
- Host cells expressing the ⁇ (1,3)fucosyltransferase of the invention or a combination of any a(1,3)fucosyltransferase of the invention and Fuc-TIV (e.g., SEQ ID NO: 4) along with a protein which is to be fucosylated, e.g., IgGl or an AGP- antibody fusion, are grown by standard methods and the fucosylated protein purified by standard techniques (for example, for an antibody or antibody fusion protein, using a Protein A column) .
- any protein e.g., IgGl or an AGP- antibody fusion, bearing sialyl-Le x addition sites may be fucosylated .in vitro using any of the enzymes or any combination of enzymes described herein according to standard methods known in the art. Again, such in. vitro fucosylated proteins can be purified using any standard technique of isolation and purification. Fucosyltransferase Kits Kits for carrying out any of the methods disclosed herein are also included in the invention.
- kits generally include a gene encoding the ⁇ (1,3)fucosyltransferase of the invention (for example, a fucosyltransferase gene encoding a polypeptide including an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 3; SEQ ID NO: 2).
- a kit may also include a gene encoding Fuc-TIV (for example, a human Fuc-TIV polypeptide including an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 6B; SEQ ID NO: 4) and/or a cell useful for expressing one or more fucosyltransferase genes.
- a kit according to the invention may include a transformed cell harboring an (l,3) fucosyltransferase gene described herein, optionally in combination with a Fuc-TIV-encoding gene.
- fucosyltransferases are expressed in the 32D cl3 cell line or human cell line 293.
- a kit may include a fragment of an ⁇ (1,3)fucosyltransferase nucleic acid sequence useful for hybridization purposes, and may also include means for detecting and quantitating ⁇ (1,3)fucosyltransferase RNA hybridization.
- kits according to the invention include substantially pure ⁇ (1,3)fucosyltransferase polypeptide (for example, a fucosyltransferase polypeptide including an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 3; SEQ ID NO: 2).
- Such a kit may also include substantially pure Fuc-TIV polypeptide (for example, a fucosyltransferase polypeptide including an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 6B; SEQ ID NO: 4) .
- Such fucosyltransferase kits are useful for fucosylating a molecule in vitro.
- Other Embodiments Polypeptides according to the invention include the entire murine fucosyltransferase sequence (as shown in Fig. 3; SEQ ID NO: 2) as well as any analog or fragment of the murine fucosyltransferase.
- Polypeptides of the invention also include all mRNA processing variants (e.g., all products of alternative splicing or differential promoter utilization) as well as analogous fucosyltransferases from other mammals, including humans.
- mRNA processing variants e.g., all products of alternative splicing or differential promoter utilization
- analogous fucosyltransferases from other mammals, including humans.
- Specific fucosyltransferase fragments or analogues of interest include full-length or partial (see below) proteins including an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy enzymatic activity (as assayed above or according to any other standard method) .
- conservative amino acid substitutions for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy enzymatic activity (as assayed above or according to any other standard method) .
- Analogs also include fucosyltransferase polypeptides which are modified for the purpose of increasing peptide stability; such analogs may contain, e.g., one or more desaturated peptide bonds or D-amino acids in the peptide sequence or the peptide may be formulated as a cyclized peptide molecule.
- MOLECULE TYPE protein
Abstract
Description
Claims
Priority Applications (3)
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EP96915560A EP0832199A4 (en) | 1995-06-07 | 1996-05-08 | Fucosyltransferase genes and uses thereof |
AU57308/96A AU5730896A (en) | 1995-06-07 | 1996-05-08 | Fucosyltransferase genes and uses thereof |
JP9500522A JPH11512921A (en) | 1995-06-07 | 1996-05-08 | Fucosyltransferase gene and use thereof |
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US08/483,151 | 1995-06-07 | ||
US08/483,151 US5858752A (en) | 1995-06-07 | 1995-06-07 | Fucosyltransferase genes and uses thereof |
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EP (1) | EP0832199A4 (en) |
JP (1) | JPH11512921A (en) |
AU (1) | AU5730896A (en) |
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WO1997032889A1 (en) * | 1996-03-08 | 1997-09-12 | The Regents Of The University Of Michigan | MURINE α(1,3)FUCOSYLTRANSFERASE Fuc-TVII, DNA ENCODING THE SAME, METHOD FOR PREPARING THE SAME, ANTIBODIES RECOGNIZING THE SAME, IMMUNOASSAYS FOR DETECTING THE SAME, PLASMIDS CONTAINING SUCH DNA, AND CELLS CONTAINING SUCH A PLASMID |
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Also Published As
Publication number | Publication date |
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EP0832199A1 (en) | 1998-04-01 |
AU5730896A (en) | 1996-12-30 |
CA2223440A1 (en) | 1996-12-19 |
JPH11512921A (en) | 1999-11-09 |
US5858752A (en) | 1999-01-12 |
EP0832199A4 (en) | 2003-01-22 |
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