Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • 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]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4727—Mucins, e.g. human intestinal mucin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/473—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used alpha-Glycoproteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• This invention relates to P-selectin ligand molecules, DNAs, and uses thereof.
• P-selectin is an integral membrane C-type lectin found within the eibel-Palade bodies of endothelial cells and the alpha granules of platelets (McEver et al., J. Clin. Invest., 84:92-99, 1989; Bonfanti et al., Blood, 73:1109-1112, 1989; Hsu-Lin et al., J. Biol. Chem., 259:9121-9126, 1984; Stenberg et al., J. Cell Biol., 101:880-886, 1985).
• P-selectin supports the attachment of myelomonocytes to platelets or endothelial cells (Larsen et al., Cell, 59:305-312, 1989; Hamburger and McEver, Blood, 75:550-554 1990; Geng et al., Nature, 343:757-760, 1990; Gamble et al., Science, 249:414-417, 1990) .
• its appearance heralds an underlying tissue insult and supports the initial step in leukocyte extravasation, the rolling of neutrophils along the postcapillary venule wall (Lawrence and Springer, Cell, 65:859-873, 1991).
• mice which are homozygously deficient for the P-selectin structural gene exhibit decreased leukocyte rolling and show delayed recruitment of granulocytes to sites of experimentally induced inflammation ( Mayadas et al., Cell, 74:541-554, 1993).
• the mediators which induce P-selectin expression are involved in signaling trauma or wounding.
• One of the first recognized responses to tissue trauma is mast cell activation, which is accompanied by release of histamine, serotonin, and other diffusible mediators.
• Other common events include thrombus formation at sites of vascular rupture and complement alternative pathway engagement by foreign bodies.
• P-selectin expression is induced by signals generated in each of these contexts.
• the invention features an organic molecule to which there is covalently bonded a sialyl-Le x determinant and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule.
• the invention features a P- selectin ligand selected from the group consisting essentially of: (a) amino acids 21-57 of Fig. 8A and (b) amino acids 38-57 of Fig. 8A.
• the invention features fusion proteins that include a P-selectin ligand joined to an antibody domain (for example, one or more of the hinge, CH2, and CH3 domains).
• the invention features purified nucleic acid encoding a protein containing sites for the attachment of a sialyl-Le x determinant and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the protein; purified nucleic acid encoding any one of the P- selectin ligands of the invention; purified nucleic acid encoding a P-selectin-antibody fusion protein; and vectors and recombinant cells including any of these nucleic acids.
• P-selectin ligands or organic molecules bearing such ligands are also included in the invention.
• other proteins such as antibody or ⁇ 1 -acid glycoprotein domains
• the invention features a method of inhibiting the binding of a cell bearing a P- selectin protein to a molecule or cell bearing a sialyl- Le x determinant and a sulfated determinant.
• the method involves contacting the P-selectin protein-bearing cell with either an organic molecule bearing sialyl-Le x and sulfated determinants, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any of the P-selectin ligands of the invention.
• the invention features a method of reducing inflammation in a mammal involving administering to the patient a therapeutically-effective amount of either an organic molecule bearing sialyl-Le x and sulfated determinants, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any one of the P-selectin ligands of the invention.
• the invention features a method of reducing or protecting a mammal against any extravasation-dependent adverse reaction (including, without limitation, extravasation-dependent organ damage and/or clotting associated with adult respiratory distress syndrome, glomerular nephritis, and ischemic myocardial injury) .
• the method involves administering to the mammal a therapeutically-effective amount of either an organic molecule to which there is covalently bonded a sialyl-Le x and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any of the P-selectin ligands of the invention.
• the invention features a method of reducing or protecting a mammal against an adverse immune reaction, involving administering to the mammal a therapeutically-effective amount of either an organic molecule to which there is covalently bonded a sialyl-Le x and a sulfated determinant, at least one of these determinants being positioned at a non-naturally occurring site on the molecule; a P-selectin-antibody fusion protein; or any of the P-selectin ligands of the invention.
• this method involves treating the mammal for an adverse immune reaction w ich is induced by a microbial factor.
• Such microbial factors include, without limitation, gram-negative bacteria lipopolysaccharides (LPS) , peptidoglycans from gram- positive organisms, mannan from fungal cell walls, polysaccharides, extracellular enzymes (e.g., streptokinase) and toxins (e.g., toxic shock enterotoxins of staphylococci) .
• LPS gram-negative bacteria lipopolysaccharides
• peptidoglycans from gram- positive organisms e.g., peptidoglycans from gram- positive organisms
• mannan from fungal cell walls
• polysaccharides e.g., extracellular enzymes (e.g., streptokinase)
• toxins e.g., toxic shock enterotoxins of staphylococci
• the method involves treating a mammal for any adverse immune reaction which is induced by a host factor.
• Such host factors include, without limitation, metabolites of complement, kinin, and coagulation systems, factors released from stimulated cells (e.g., cytokines such as interleukin 1 (IL-1) and tumor necrosis factor-o (TNF)) , enzymes and oxidants from polymorphonuclear leukocytes (PMNs), vasopeptides (e.g., histamine), and products of the metabolism of arachidonic acid.
• cytokines such as interleukin 1 (IL-1) and tumor necrosis factor-o (TNF)
• PMNs polymorphonuclear leukocytes
• vasopeptides e.g., histamine
• the adverse immune reaction is induced by recombinant TNF- ⁇ or is induced by recombinant IL-1.
• the adverse immune reaction is septic shock or is septicemia.
• the organic molecule or protein also inhibits the binding of a cell bearing an E-selectin (ELAM-1) protein to a molecule or cell bearing a sialyl-Le x determinant and thus inhibits E-selectin-mediated inflammation, extravasation-dependent adverse reactions, and adverse immune reactions;
• the sialyl-Le and sulfated determinants are present on a P-selectin ligand consisting essentially of: amino acids 21-57 of Fig. 8A (for example, amino acids 38-57 of Fig.
• the sialyl- Le x determinant is N-linked or o-linked; the molecule or protein contains multiple sialyl-Le x and/or multiple sulfated determinants;
• the organic molecule is a protein (for example, an antibody (for example, IgG or IgM) , a ⁇ - acid glycoprotein (AGP) , or an antibody fusion protein (for example, an AGP-antibody fusion protein) ;
• the protein is an antibody, AGP, or an antibody fusion protein (for example, an AGP-antibody fusion protein) to which any of the P-selectin ligands described herein is appended (for example, at the protein's amino-terminus) ;
• the antibody or antibody fusion protein (for example, the AGP-antibody fusion protein) includes, as an antibody portion, an IgGl CH2, CH3, and/or hinge domain;
• the antibody, AGP, or antibody fusion protein includes one or more of the N-linked g
• P-selectin ligand any amino acid sequence capable of mediating an interaction with the P-selectin receptor and includes those proteins referred to as P-selectin counter- receptors.
• Preferable P-selectin ligands include, without limitation, amino acids 21-57, and more preferably amino acids 38-57, of Fig. 8A.
• P-selectin ligands according to the invention may be used in conjunction with additional protein domains (for example, antibody domains) to produce fusion proteins useful in the invention.
• non-naturally occurring is meant a sialyl-Le x or sulfated determinant that is not one which is naturally bound to the molecule at that amino acid location.
• inflammation is meant a pathologic process consisting of cytologic and histologic reactions that occur in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent. Inflammation, as used herein, includes any acute inflammatory response (for example, during or following adult respiratory distress syndrome or ischemic myocardial injury) as well as any chronic inflammatory response (for example, rheumatoid arthritis, psoriasis, or pemphigus vulgaris) .
• purified nucleic acid is meant 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.
• N-linked is meant bonded to the amide nitrogen of an asparagine residue of a protein.
• O-linked is meant bonded to the hydroxy1- group oxygen of a serine, threonine, or hydroxylysine residue of a protein.
• an extravasation-dependent adverse reaction any reaction which is detrimental to the host and which results directly or indirectly from the inappropriate attachment of neutrophils to endothelium at or proximate to a site of inflammation, tissue damage, or thrombus formation and results in migration of those neutrophils into the attached blood vessel or organ.
• Organs which may be affected by such damage include, without limitation, the heart, lungs, and kidneys.
• an “adverse immune reaction” is meant any reaction mediated by an immune cell (i.e., any B cell, T cell, monocyte/macrophage, natural killer cell, mast cell, basophil, or granulocyte) and which is detrimental to the host.
• an immune cell i.e., any B cell, T cell, monocyte/macrophage, natural killer cell, mast cell, basophil, or granulocyte
• Fig. IA is a schematic representation of the structure of the PSGL-1 deletion mutants. Systematic deletion of the ectodomain of PSGL-1 was accomplished with conventional PCR methods. A representative 10-residue repeat (stippled; SEQ ID NO:l) and the transmembrane domain (hatched) are illustrated.
• Fig. IB is histogram which represents P-selectin binding activity of transfected COS cells expressing the deletions shown in Fig. IA. 51 Cr-labeled cells were allowed to adhere to soluble P-selectin adsorbed to microtiter wells. The cells were washed, the bound cells were then lysed, and 51 Cr levels were counted.
• Fig. 2A is a schematic representation of chimeras of PSGL-1 and CD43. The membrane proximal extracellular domain, transmembrane, and intracellular domains of PSGL-1 were replaced with the cognate sequences of CD43. The resulting molecule lacks cysteines and thus cannot form a disulfide linked dimer.
• Fig. 2B is a histogram representing P-selectin binding activity of transfected COS cells expressing the chimeras shown in Fig. 2A.
• FTVIIh cotransfection with the human FTVII fucosyltransferase.
• Fig. 3A is a schematic representation of chimeric mucins bearing the PSGL-1 apical domain appended to intact or truncated mucin C-termini.
• the PSGL-1 N-terminus (stippled; SEQ ID NO:l) and the transmembrane (TM) domains (hatched) are illustrated.
• the sequence of PSGL-1-NH 2 /CD43 "repeats" are represented by SEQ ID NO:2.
• PSGL-1 was fused to the N-terminus of the predicted mature CD34 and GlyCAM-1 molecules, and to the N-terminus of the repeat region of CD43.
• Fig. 3B is a histogram representing P-selectin binding activity of transfected COS cells expressing the constructs shown in Fig. 3A.
• FTVIIh human FTVII fucosyltransferase.
• Fig. 4A is a schematic representation of PSGL deletion mutants. The amino terminal domain was appended to PSGL molecules having varying numbers of the repeated element.
• Fig. 4B is a histogram representing P-selectin binding activity of transfected COS cells expressing the chimeras illustrated in Fig. 4A.
• Fig. 5 is a photograph of an autoradiogram of mucin:immunoglobulin fusion proteins labeled with 35 S-sul ate and electrophoresed on an 8% denaturing polyacrylamide gel under reducing conditions.
• Lane A supernatant of CDM8 transfected cells
• Lane B supernatant of cells transfected with Ig expression vector (no mucin insert)
• Lane C supernatant of cells expressing PSGL-l:Ig
• Lane D supernatant of cells expressing CD43:Ig
• Lane E supernatant of cells expressing CD34:Ig
• Lane F supernatant of cells expressing GlyCAM-l:Ig.
• Fig. 6A and Fig. 6B are histograms representing binding to immobilized P- and E-selectin of COS cells expressing PSGL-1 with or without fucosyltransferase and in the presence or absence of 10 mM NaCl0 3 .
• Fig. 6A is a histogram representing binding of cells to P-selectin.
• Fig. 6B is a his ogram representing binding of cells to E-selectin.
• Fig. 7 is a photograph of an autoradiogram of PSGL-1:immunoglobulin fusion proteins labeled with 35 S-sulfate in the presence or absence of 10 mM NaC10 3 and electrophoresed on an 8% denaturing polyacrylamide gel under reducing conditions. The photograph indicates that chlorate inhibits incorporation of 35 S-sulfate into soluble mucin chimeras.
• Lane A supernatant of CDM8 transfected cells in the absence of chlorate
• Lane B supernatant of cells expressing PSGL-l:Ig in the absence of chlorate
• Lane C supernatant of CDM8 in the presence of chlorate
• Lane D supernatant of cells expressing PSGL-l:Ig in the presence of chlorate.
• Fig. 8A is a listing of the sequence endpoints of various PSGL-1 deletion mutants (indicated by the arrows) The uppermost sequence is SEQ ID NO:3; the middle sequence is SEQ ID NO:13; the lowermost sequence is SEQ ID NQ:14.
• Fig. 8B is a histogram representing P-selectin binding activity of transfected COS cells expressing the deletion mutants having the endpoints shown in Fig. 8A.
• Fig. 9A is a schematic diagram of the constructs employed to measure the effect of appending wildtype and mutant variants of PSGL-1 residues 38-57 to deleted PSGL-1 or CD43. The inserted sequences are shown at bottom left.
• Fig. 9B is a histogram representing P-selectin binding activity of transfected COS cells expressing the chimeras illustrated in Fig. 9A.
• Fig. 10 is a listing of the nucleotide sequence (SEQ ID NO:8) encoding IgGl (SEQ ID NO:9) and mutations designed to create N-linked glycan addition sites (SEQ ID NO:12).
• Fig. IIA is the nucleotide sequence (SEQ ID NO:10) and Fig. IIB is the amino acid sequence (SEQ ID NO:11) of an AGP-IgGl fusion protein.
• Fig. 12A is a schematic diagram of immunoglobulin fusion proteins consisting of either intact PSGL-1 (SEQ ID NO:4) or 20 residue peptides joined to the hinge, CH2, and CH3 domains of human IgGl. Construct Y/F-hlgG bears SEQ ID NO 5; construct T/AhlgG bears SEQ ID NO:6; construct Y/F-T/A-hlgG bears SEQ ID NO:7.
• Fig. 12B is a photograph of an 8% polyacrylamide gel used to assess incorporation of [ 35 S]cysteine and methionine by the fusion proteins shown in Fig. 12A following transfection into COS cells. Lane A, supernatant of cells transfected with CDM8 control.
• Lane B supernatant of cells transfected with PSGL-1-immunoglobulin fusion protein.
• Lane C supernatant of cells transfected with T-hlgG.
• Lane D supernatant of cells transfected with Y/F-hlgG.
• Lane E supernatant of cells transfected with T/A-hlgG.
• Lane F supernatant of cells transfected with Y/F-T/A- hlgG.
• Fig. 12C is a photograph of an 8% polyacrylamide gel used to assess incorporation of [ 35 S]sulfate by the fusion proteins shown in Fig. 12A following transfection of COS cells.
• a control fusion protein bearing no amino-terminal addition was included (Lane B) .
• Lanes C through G correspond to Lanes B through F in Fig. 12B.
• Fig. 13 is a bar graph of interacting HL-60 cells per video-captured field.
• the cells were infused into a parallel plate flow chamber precoated with either P- selectin-immunoglobulin chimera or a CD4-immunglobulin chimera control.
• the cells were subjected to a shear stress of 0.75 dynes/cm 2 .
• Each bar represents the average number of cells ( ⁇ SEM) per field from eight frames taken at 15 second intervals. Cells rolling or flowing appear as streaks on the video image.
• the bars represent, from left to right: HL-60 cells rolling or flowing over P-selectin-immunoglobin chimera, HL-60 cells pretreated in a sulfate-free medium with 10 mM sodium chlorate, and HL-60 cells flowing over CD4-immunoglobulin chimera.
• Sialyl-Lewis X Sialyl-Le x
• sulfated determinants were shown to interact with P-selectin and facilitate binding by the following experiments. These examples are presented to illustr te, not limit, the invention. The methods used in the following experiments will first be described. Production of Soluble P-Selectin
• P-selectin and E-selectin Ig chimeras were prepared by transient expression in COS cells of an expression plasmid encoding the lectin, EGF-related, and first two short consensus repeat related domains of P-selectin joined to the hinge, CH2, and CH3 domains of human IgGl (Aruffo et al., EMBO J., 6:3313-3316, 1991; alz et al.. Science, 250:1132-1135, 1990).
• the PSGL-1 cDNA coding sequence was obtained by PCR amplification of an HL-60 cDNA library, and the sequence confirmed by DNA sequencing.
• the coding segment for the mature extracellular, transmembrane, and intracellular domain was inserted into 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 (flu) peptide (Field et al., Mol. Cell. Biol. 8:2159-2165, 1988) epitope tag. Construction of PSGL-1 Deletions
• Amino terminal PSGL-1 deletion constructs were prepared by PCR amplification using primers encoding the desired endpoint of the deletion mutant located downstream of an Xbal site in frame two (encodes Leu Asp) .
• the resulting sequences encoded a polypeptide in which the residues listed below immediately followed the aspartic acid (D) of the Xba site: A118, A128, A138, A148, A158, A168, G178, A188, A198, A208, A218, A228, A238, A248, A258, and T268 of the PSGL-1 precursor.
• the PCR fragments were then inserted in the CD5 leader flu tag expression vector used for expression of the intact PSGL-1. The flu tag terminates in an Xbal site in the frame described above.
• CD34, CD43, and GlyCAM-1 mucins were prepared for addition of the PSGL-1 amino-terminal domain by appending an EcoRI site to either the mature amino terminus (CD34 or GlyCAM-1) , or to the beginning of a region of threonine/proline-rich repeats (CD43) .
• the EcoRI site was in the frame glutamic acid phenylalanine (frame 1) .
• the CD34 sequence began at residue F30 of the precursor, the Gly-CAM-1 at precursor L19, and the CD43 at precursor 1135. To each of these was appended the flu-tagged PSGL-1 domain terminating in EcoRI as above.
• PSGL-1 The amino terminus and repeat elements of PSGL-1 were appended to the membrane proximal, transmembrane, and intracellular domains of CD43 through an EcoRI site in the glutamic acid phenylalanine frame positioned immediately upstream of the sequences S225 of the CD43 precursor.
• the complementary fragment from PSGL-1 corresponded to the amino-terminal residues of the precursor up to T267.
• Fine Structure Mapping of the Amino-Terminal Domain A similar strategy was employed for the construction of deletions in the amino-terminal domain, in which PCR generated deletions were formed using primers bearing an Xbal site in the leucine aspartic acid frame (frame 2) .
• the cells were then loaded with 100 ⁇ l 51 Cr0 4 (1 mCi/ l; DuPont, Boston, MA) in 0.9% NaCl plus 100 ml medium by incubating them at 37°C for 1 hour. Loaded cells were washed twice in PBS and resuspended in 0.2% BSA, 0.15 M NaCl, 3 mM CaCl 2 . Variation in labeling rate (counts incorporated per cell) between cells prepared in parallel with the same batch of labeled chromate was typically minimal.
• the labeled cells were incubated in wells of 96-well microculture plates which had been coated with affinity purified goat anti-human IgG antibody (100 ⁇ l of 20 ⁇ g/ml anti-human IgG Fc (heavy chain specific) in PBS) for 2 hours in a humid chamber at room temperature. After the plate was washed 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 hours.
• affinity purified goat anti-human IgG antibody 100 ⁇ l of 20 ⁇ g/ml anti-human IgG Fc (heavy chain specific) in 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 hours
• Adherent cells were lysed by the addition of 200 ⁇ l 2% SDS, and labeled chromate was counted with a gamma ray spectrometer.
• Immunofluorescence Analysis Cells were prepared for cytometry by incubation with the primary monoclonal antibody (a 1:200 dilution of ascites or 5 ⁇ g/ml of purified antibody is suitable) in PBS containing 3% BSA for 30 to 45 minutes. The cells were washed twice with PBS and incubated with 2 ⁇ g/ml FITC-conjugated affinity purified antibody to either mouse IgG (12CA5) or mouse IgM (CSLEX-1) for 30 to 45 minutes in PBS/3% BSA.
• transfected cells were fixed with 4% freshly depolymerized paraformaldehyde, washed, exposed to BSA at 3% in PBS for 30 minutes, and then incubated with primary antibody (ascites, 1:250) for 30-45 minutes.
• primary antibody ascites, 1:250
• the cells were then washed twice with PBS and incubated for 30-45 minutes with FITC-conjugated affinity-purified antibody to mouse IgG (Cappel1; 2 /ng/ml in PBS containing 3% BSA) .
• Serum was not added, and radionuclide was typically present at a concentration of 200 ⁇ Ci/ml. After a labeling interval of 12 to 16 hours, the supernatants were harvested, and the fusion proteins were collected by adsorption to goat anti-human IgG agarose (Cappel) . Adsorbed proteins were subjected to denaturing electrophoresis on 8% polyacrylamide gels under reducing conditions. Chlorate Inhibition of Adhesion
• COS cells were transfected with DEAE dextran and incubated immediately in DMEM containing 10% calf serum and 10 mM sodium chlorate. One day after transfection the cells were trypsinized and incubated in fresh dishes in the same medium for 6 hours. The medium was then removed, the cells were washed with PBS, and then incubated for 18 additional hours in a custom prepared DMEM medium (Life Technologies) lacking sulfate and containing 2% of the conventional levels of cysteine and methionine with 10% dialyzed fetal bovine serum in the presence of 10 mM sodium chlorate (Baeuerle and Huttner, Biochem. Biophys. Res. Comm., 141:870-877, 1986).
• HL-60 cells were washed once with PBS and grown for 18 hours in sulfate-free medium containing 2% of the normal levels of cysteine and methionine, 10 mM sodium chlorate, and dialyzed serum as described above.
• IO 6 cells were suspended in 1 ml of 0.15 M NaCl, 3 mM CaCl 2 and drawn through the chamber. Glass coverslips were coated with affinity-purified goat anti-human IgG antibody at a concentration of 10 ⁇ g/ml in 50 mM Tris-HCl (pH 9.0) for 2 hours, washed twice with PBS, and blocked overnight with 0.2% BSA in PBS.
• truncated cDNAs were inserted downstream of a secretory peptide sequence which had been fused to a short oligopeptide tag derived from influenza hemagglutinin (HA) .
• Expression plasmids encoding the truncated molecules (Fig. IA) were transfected into COS cells in the presence of a specific myeloid fucosyltransferase, designated FTVII, which directs the expression of sLe x determinants exclusively (Sasaki et al., J. Biol.
• Table 1 shows the mean fluorescence intensity (MFI) of COS cells that were cotransfected with human FTVIIh and the deletion constructs (shown in Fig. IA) , and subjected to indirect immunofluorescence with antibody against the amino terminal flu peptide or sLe x .
• PSGL-1 In the Context of Large. Sulfated Mucins. the Amino Terminus of PSGL-1 is Sufficient for P-Selectin Binding
• PSGL-1 sequences other than those found in the first 100 N-terminal amino acids (i.e., the apical domain) of PSGL-1 were required for binding to P-selectin, the transmembrane and cytoplasmic regions of PSGL-1 were replaced with those of the CD43 antigen (Pallant et al., Proc. Natl. Acad. Sci., 86:1328- 1332, 1989; Shelley et al., Proc. Natl. Acad. Sci., 86:2819-2823, 1989).
• the predicted first 100 amino acids of PSGL-1 were then genetically grafted onto the amino termini of ucin-like repeat elements of several unrelated mucins to determine whether or not the PSGL-l apical domain is sufficient for P-selectin ligand (i.e., counterreceptor) activity (Fig. 3A) .
• Certain of these chimeric mucins were able to support P-selectin binding in this setting.
• CD34 and CD43 two relatively large mucins found predominantly on human hematopoietic cells, were both able to support binding.
• the apparent molecular masses of CD43 and CD34 expressed in COS cells are reported to be 100-130 kD (Shelley et al., Proc. Natl. Acad. Sci., 86:2819-2823, 1989) and 100 kD (Simmons et al., J. Immunol., 148:267- 271, 1992), respectively; the PSGL-1 monomer exhibits an effective molecular mass of 110 kD (Sako et al., Cell, 75:1179-1186, 1993). GlyCAM-1, in its native (untethered) state comigrates with 50 kD proteins, suggesting that it is substantially smaller (Lasky et al., Science, 258:964- 969, 1992).
• COS cells were cotransfected with PSGL-1 and FTVII, or transfected with PSGL-1 and FTVII separately.
• the cells were incubated in a modified DMEM medium lacking sulfate and containing 10 mM sodium chlorate, a relativelly selective inhibitor of sulfation (NaC10 3 ) .
• NaC10 3 a relativelly selective inhibitor of sulfation
• a soluble PSGL-1 immunoglobulin chimera synthesized under comparable conditions showed essentially complete inhibition of 35 S-sulfate incorporation (Fig. 7) , under conditions in which protein synthesis as measured by [ 35 S]cysteine and methionine incorporation was not inhibited.
• the 20 amino acid region which is necessary for P-selectin binding contains three potential tyrosine sulfation sites and two threonine residues for O-linked glycosylation. To assess the importance of these residues, the tyrosines were converted to phenylalanine (Fig. 9A) . In a second peptide, the threonines were converted to alanines. In addition, a third peptide, containing a quintuple mutation, was prepared such that both conversions were made in a single peptide.
• Each mutated peptide was then positioned, separately, downstream of the flu tag and upstream of either (1) the truncated PSGL-1 lacking the apical domain, or (2) the CD43 repeat elements and transmembrane domain.
• Cells expressing the resulting chimeras were tested for their ability to bind to immobilized P-selectin (Fig. 9A) .
• Conversion of the tyrosines to phenylalanines resulted in a loss of binding activity to P-selectin.
• Replacement of the threonine residues with alanine diminished binding, but did not abolish it entirely.
• Expression of the flu tag or sLe x epitope was not affected in these cells.
• Binding mediated by the apical 20 residues was, like that of native PSGL-1, dependent on the presence of calcium. These data indicate that sulfation of tyrosines at positions 46, 48, and 51 is required for P-selectin binding activity. E-selectin binding was unaffected under the same condition. In addition, these data indicate that the threonines at positions 44 and 57 are required. These threonine residues can serve as sites for O-linked glycan addition. These experiments, in conjunction with our experiments showing that FTVII expression is necessary for P-selectin binding, provide evidence that P-selectin binding requires sLe x at threonines 44 and 57. In sum, the above-described experiments demonstrate that amino acids 38-57, containing three residues for sulfation and two residues for sLe x addition, are sufficient to confer P-selectin binding activity.
• fusion proteins consisting of the native or mutant peptide sequences joined to human immunoglobulin Gl (IgGl) (Fig. 12A) .
• the resulting fusion proteins were expressed in COS cells, and their ability to assimilate inorganic sulfate was assessed (Fig. 12B) .
• Immunoglobulin chimeras bearing the native peptide sequences were capable of incorporating sulfate, whereas those bearing phenylalanine substituted for tyrosine were not (Fig. 12C) . Replacement of threonine with alanine had no effect on sulfate incorporation (Fig. 12C) .
• HL-60 cells To explore whether inhibition of sulfation would compromise a physiologically relevant adhesion, we subjected HL-60 cells to growth in medium containing chlorate and examined the ability of the resulting cells to attach and roll on coverslips coated with P-selectin- immunoglobulin chimeras under conditions of defined fluid shear stress (Lawrence et al.. Blood, 75:227-237, 1990). HL-60 cells were capable of attaching to and rolling upon coverslips precoated with P-selectin-immunoglobulin chimeras, whereas no such interaction was observed with coverslips coated with a CD4-immunoglobulin chimera (Fig. 13) . Growth of HL-60 cells in chlorate dramatically reduced the frequency of cell interaction with the substrate (Fig. 13).
• the invention features an antibody bearing sialyl-Le x and sulfated determinants.
• an antibody may be created by introducing sulfation sites (i.e., a tyrosine in an acidic context) into an existing antibody molecule in the vicinity of an introduced or existing sialyl-Le x addition site (for example, by standard site-directed mutagenesis) .
• appropriate sialyl-Le x and/or sulfation sites may be added by appending any P-selectin ligand sequence (for example, any P-selectin domain described herein) to a naturally-occurring antibody sequence (for example, IgG or IgM) by standard recombinant DNA techniques to produce a P-selectin-antibody fusion protein.
• the P-selectin ligand sequence is appended to the amino-terminus of the antibody molecule.
• Such antibodies are useful for disrupting undesirable interactions between cells or proteins, or, generally, for disrupting any interaction between two molecule ⁇ , one of which bears a determinant carried by the antibody.
• one or more sialyl-Le x moieties which mask the CH2 portion of the immunoglobulin molecule and thus inhibit complement fixation and F c receptor binding may also be incorporated into the antibody sequence.
• 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 an E-selectin and/or P-selectin mediated intracellular interaction.
• sialyl-Le x determinants may be added to the antibody molecule at any appropriate site.
• N-linked glycan addition sites are well known to be: N X S/T (where N is asparagine, S is serine, T is threonine, and X is any amino acid except proline) .
• an exemplary molecule may be designed that includes several such sites for attachment of sialyl-Le x side chains. Inspection of the IgGl sequence (Fig. 10) reveals at least five sites at which N-linked glycan addition sites may be introduced into the molecule in advantageous locations, where complement fixing and F c receptor binding ability will be impaired by the process. These sites include amino acid residues 274, 287, 295, 322, and 335.
• the site ⁇ are, preferably, located in the CH2 region of the immunoglobulin molecule, i.e., in the portion of the molecule responsible for complement fixation and F c receptor binding; (2) the site ⁇ are located in regions of the sequence, predicted by their hydrophilic nature, to be present on the outside of the immunoglobulin molecule and therefore accessible to the enzymes responsible for attachment of carbohydrate side chains; (3) the sites are located in a region which is minimally disruptive to the primary amino acid sequence and, thus, the predicted secondary amino acid structure.
• N-linked glycan addition site For example, a naturally-occurring site which differs from an N-linked glycan addition site by a single amino acid would be preferable to a site requiring two alterations in the amino acid sequence. Moreover, it is preferable to create an N-linked glycan addition site by substituting amino acids of similar charge or polarity (e.g., substitution of one uncharged amino acid for another) .
• One or more N-linked glycan addition site substitutions may be engineered into a particular IgGl-encoding sequence; such sequences (i.e., those which encode an antibody molecule to which sialyl-Le x moieties are attached) are termed IgGl-sialyl-Le x or IgGl-Le x .
• a particular IgGl molecule bearing sialyl-Le x moieties i ⁇ produced a ⁇ follow ⁇ .
• the IgGl gene i ⁇ publically available, and it ⁇ ⁇ equence i ⁇ shown in Fig. 10.
• the gene is mutagenized by standard methods of in vitro site-directed mutagenesis in order to introduce one or more N-linked glycan addition sites (e.g., those described above and shown above the naturally-occurring sequence in Fig. 10) .
• the gene is then inserted into 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., a CHO or lecll cell), and the expression vector containing the mutated IgGl-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 expres ⁇ ing an ⁇ (1,3)fucosyltransferase capable of attaching the sialyl-Le x groups to the antibody molecule at the glycosylation site ⁇ .
• the ⁇ (1,3)fucosyltransferase gene may be expres ⁇ ed from a vector di ⁇ tinct from that encoding IgGl-Le x , or both genes may be carried on, and expressed from, a common vector.
• Mammalian cells are particularly u ⁇ eful hosts for the synthesis of IgGl-Le x because they provide all required precursors for sialyl- Le x production.
• the gene encoding the antibody sequence is preferably expressed in a cell which also expresses an ⁇ (1,3)fucosyltransferase that exclusively catalyzes ⁇ (1,3)fucose linkages; ⁇ uch an enzyme i ⁇ described in Walz et al., Science 250:1132-1135 (1990) and in Seed, U.S.S.N. 08/483,151, entitled “Fucosyltransferase Genes and Use ⁇ Thereof," filed June 7, 1995 (hereby incorporated by reference).
• Le ⁇ preferably, the ⁇ (1,3)fucosyltransfera ⁇ e cDNA described in Lowe et al.
• Thi ⁇ fuco ⁇ yltransferase recognize ⁇ a sialylated precursor molecule and adds either an ⁇ (l,3)- or an ⁇ (1,4)-linked fucose moiety to N-acetylglucosamine ⁇ ide chain ⁇ .
• sialyl-Le x determinant i ⁇ characterized by an ⁇ (l,3)- linkage, and, a ⁇ such, the ⁇ (1,3)fucosyltransferase enzyme of Lowe (supra) produces both the desired ⁇ ialyl- Le x -modified molecules and products bearing ⁇ (1,4)-linked fucose which, although not active in binding to P- selectin and E-selectin, do not interfere with the action of the sialyl-Le -modified molecules nor produce other undesirable side effects.
• Host cells expressing ⁇ (1,3)fucosyltransferase and the antibody to be modified are grown by standard methods, and the antibody i ⁇ purified from a cell ly ⁇ ate ba ⁇ ed on its affinity for a Protein A column or any other standard technique of antibody isolation and purification.
• o ⁇ -Acid Glycoprotein-Antibody Fusion Proteins Bearing Sialyl-Le and Sulfated Determinants are grown by standard methods, and the antibody i ⁇ purified from a cell ly ⁇ ate ba ⁇ ed on its affinity for a Protein A column or any other standard technique of antibody isolation and purification.
• antibody fusion proteins modified by sulfation and sialyl-Le x addition have important therapeutic and diagnostic uses.
• DNA encoding an AGP and a P- selectin ligand domain are fused in-frame to human IgG domains (for example, constant domains) by standard techniques, and the fu ⁇ ion protein i ⁇ expressed, also by standard techniques.
• antibody portion of the molecule facilitates fusion protein purification and also prolongs the plasma half-life of otherwise short-lived polypeptides or polypeptide domains.
• antibody fusion proteins are expressed according to the methods disclosed in Seed et al., U.S.S.N. 08/483,151 entitled “Fucosyltransferase Genes and Use ⁇ Thereof," filed June 7, 1995 (which i ⁇ hereby incorporated by reference), e.g., u ⁇ ing IgG or IgM antibodie ⁇ or portion ⁇ thereof (see also Zettlemeisl et al., DNA Cell Biol. 9:347 (1990) for IgM fusion protein ⁇ ) .
• Recombinant plasmids expressing particular AGP- antibody fusion proteins have been con ⁇ tructed a ⁇ follows.
• a cDNA encoding the acute phase ot j -AGP gene was cloned from,a human liver cDNA library by polymerase chain reaction (PCR) using oligonucleotide primers corresponding to the 5' and 3' coding regions of o ⁇ -AGP (Board et al.. Gene 4_: 121 , 1986) according to standard techniques.
• the 5' AGP primer was designed to contain a Hindlll restriction site and the 3' primer was designed to contain a BamHI restriction site rather than the AGP stop codon.
• the PCR-amplified product was digested with Hindlll/BamHI and cloned into a Hindlll/BamHI-cut plasmid expression cas ⁇ ette (see Aruffo et al.. Cell, 61:1303. 1990) containing constant domains of human IgGl (i.e., Hinge-CH2-CH3 or CH2-CH3) .
• a nucleotide sequence and amino acid sequence of this AGP-IgG fusion protein are shown in Fig. IIA and Fig. IIB, respectively.
• sites for sulfation and, if necessary, sialyl-Le x addition are introduced into the antibody fusion protein sequence (for example, the antibody fusion proteins described above) .
• Such site ⁇ may be incorporated into an exi ⁇ ting fusion molecule, for example, by introducing one or more sulfation sites (i.e., a tyrosine in an acidic context) in the vicinity of an introduced or existing sialyl-Le x addition site (for example, by standard techni .ies of ⁇ ite-directed mutagenesis) , or a P-selectin ligand sequence (for example, any of the P-selectin ligand sequences described herein) may be appended to the antibody fusion protein sequence using standard techniques of recombinant DNA technology.
• sulfation sites i.e., a tyrosine in an acidic context
• sialyl-Le x addition site for example, by standard techni .ies of ⁇ ite-directed mutagenesis
• P-selectin ligand sequence for example, any of the P-selectin ligand sequences described herein
• the P-selectin-AGP-antibody fusion genes are then introduced into expres ⁇ ion plasmids, and the plasmids are transfected into any appropriate fucosyltransferase- expressing cell for the production of soluble antibody fusion proteins.
• sialyl-Le x con ⁇ en ⁇ u ⁇ glyco ⁇ ylation ⁇ ite ⁇ N-X- T/S
• N-X- T/S sialyl-Le x con ⁇ en ⁇ u ⁇ glyco ⁇ ylation ⁇ ite ⁇
• any number of recombinant P-selectin-AGP-antibody fusion proteins may be designed having long pla ⁇ ma half-live ⁇ and the ability to inhibit unde ⁇ irable cell-cell interaction ⁇ (for example, the interaction ⁇ between leukocytes and selectin-bearing cells) .
• candidate molecules are designed and screened using the assays described above.
• molecules may be screened for their ability to incorporate ⁇ ialyl-Le x and ⁇ ulfated determinants and block the binding of neutrophils to activated endothelial cell ⁇ ; ⁇ uch molecule ⁇ find u ⁇ e in the inhibition of ⁇ electin-dependent inflammatory reactions and tis ⁇ ue injury inflicted by invading leukocytes.
• molecules for example, proteins
• molecules may be constructed that include both a P-selectin ligand domain (i.e., a domain bearing sialyl-Le x and ⁇ ulfated moietie ⁇ ) and an E- selectin ligand domain (i.e., a domain bearing a sialyl- Le x moiety) .
• Such a molecule may be constructed by combining domain ⁇ , for example, by appending a P-selectin ligand domain to a sialylated molecule (for example, a sialylated antibody or antibody fusion protein described herein) .
• a sialylated molecule for example, a sialylated antibody or antibody fusion protein described herein
• sialyl-Le x -modified and/or ⁇ ulfated molecule to interfere with intracellular interaction ⁇ may also be tested a ⁇ de ⁇ cribed in Walz et al., ⁇ upra. or by any standard technique, for example, by assaying the ability of increasing concentrations of the determinant-bearing molecule to inhibit adherence of T lymphocytes or myeloid cells to immobilized P-selectin and/or E-selectin.
• the pharmaceutically-pure protein or molecule is suspended in an acceptable carrier, e.g., physiological saline, and is delivered to the patient by any appropriate route (for example, intravenously) in a single dose or in multiple doses.
• an acceptable carrier e.g., physiological saline
• a sufficient quantity of the therapeutic is provided to saturate all P-selectin and, for a dual function molecule, all E-selectin binding site ⁇ on an endothelial cell.
• thi ⁇ may be achieved with doses of 0.1 mg/kg or greater.
• the preferred dosage is in the range of 0.1-2.0 mg/kg.
• sialyl-Le x -modified and sulfated molecules and proteins of the invention may be used, in one example, for the treatment of extravasation-dependent organ damage and/or clotting.
• the molecules and proteins of the invention provide useful therapeutics for blocking such interactions.
• P-selectin likely mediates the migration of neutrophils into the lung following adult respiratory distress syndrome and into the heart following ischemic myocardial injury (i.e., infarction) , and may play a role in glomerular damage to the kidneys under certain conditions.
• ischemic myocardial injury i.e., infarction
• a sialyl-Le x -modified and sulfated molecule or protein of the invention may be administered to a patient suffering from such a disease or condition.
• Such treatment attenuates extravasation-dependent damage by competitively inhibiting the interaction between the invading neutrophils and the endothelial cells of the blood vessel or organ.
• P-selectin ligand-AGP fusion proteins and P-selectin ligand-AGP-antibody fusion proteins may also be used, as described above, for the treatment of septic shock or septicemia.
• antibodie ⁇ or antibody fu ⁇ ion proteins according to the invention may be used in conventional techniques of antibody-based therapies or jn vivo diagnostics, taking advantage of the antibody's ⁇ pecificity to target therapeutic or diagno ⁇ tic sites.
• the P-selectin ligand domain of an antibody fusion protein according to the invention target ⁇ that protein to a ⁇ ite of inflammation and provide ⁇ both a therapeutic (u ⁇ eful for blocking deleteriou ⁇ P- ⁇ electin-mediated intracellular interaction ⁇ ) and a diagno ⁇ tic (u ⁇ eful for tagging the site of inflammation) .
• sialyl-Le x determinants may be used to mask the CH2 domain of the antibody and block the undesirable effects of complement fixation and F c receptor binding.
• any other appropriate carrier molecule to which a ⁇ ialyl-Le x and a sulfated determinant may be attached may be utilized in the invention.
• proteins are preferred becau ⁇ e of their relatively long half-live ⁇ in serum.
• carrier proteins are serum proteins such as albumin (e.g., bovine serum albumin or human serum albumin) , transferrin, or ⁇ -2 macroglobulin.
• the carrier proteins may contain endogenous sulfation and glycan addition site ⁇ in addition to which ⁇ ites are introduced into the DNA sequence of the carrier protein (as described above) by, for example, site-directed mutagenesi ⁇ .
• the carrier molecule, le ⁇ preferably, may be a lipid.
• the lipid, with one or more attached ⁇ ialyl-Le x and sulfated determinants is delivered a ⁇ a liposome to a target cell wall (e.g., an endothelial cell wall).
• the liposome may block a cell or protein interaction or may be u ⁇ ed to deliver a drug to it ⁇ appropriate ⁇ ite of action.
• Production of carrier molecule ⁇ bearing ⁇ ialyl-Le x and sulfated determinants may be carried out in a cell, preferably, a eukaryotic cell other than yea ⁇ t.
• Mammalian cells e.g., mammalian cell lines, provide particularly ⁇ uitable hosts. These cell ⁇ generally synthesize the neces ⁇ ary precursor molecules and produce or can be engineered to produce the enzymes responsible for sulfation and carbohydrate attachment.
• mammalian cell lines such as CHO and lecll are particularly suitable.
• either or both of the sialyl-Le x and sulfated determinants may be attached to a carrier molecule in vitro, i.e., extracellularly.
• ⁇ (1,3)fucosyltransferase would be bound to a solid support (e.g., a column) and a sulfated carrier molecule pas ⁇ ed over the bound fuco ⁇ yltran ⁇ fera ⁇ e enzyme, under condition ⁇ which facilitate attachment of sialyl- Le x groups to their appropriate site( ⁇ ) on the carrier molecule.
• the invention also encompasses the use of sulfated and sialyl-Le x -modified AGP-antibody fusion proteins for protecting against, inhibiting, or treating a shock- inducing event, the clinical manifestations of shock, or both which are caused by microbial factors (e.g., lipopolysaccharides (LPS)), microbial toxins (e.g., toxic shock enterotoxins) , host mediators (e.g., cytokines), or anti-tumor therapies (e.g., administration of tumor necrosis factor (TNF) or interleukin-l (IL-1)) , or any combination thereof.
• microbial factors e.g., lipopolysaccharides (LPS)
• microbial toxins e.g., toxic shock enterotoxins
• host mediators e.g., cytokines
• anti-tumor therapies e.g., administration of tumor necrosis factor (TNF) or interleukin-l
• an antibody fusion protein can be administered to a human patient to alleviate the effects of septic shock induced by microbial LPS.
• the ability of an antibody fusion protein to protect against, treat, or inhibit the effects of shock is evaluated according to standard methods known in the art (e.g., those described in Libert et al. (1994) J. Exp. Med. 180: 1571-1575).
• MOLECULE TYPE DNA (genomic)
• AGCACAGCCT ACATGGAGCT GAGCAGCCTG AGATCTGAGG ACACGGCCGT GTATTACTGT 360
• GGCACCACCT CTCTTGCAGC CTCCACCAAG GGCCCATCGG TCTTCCCCCT
• CTCCTCCCAG ATTCCAGTAA CTCCCAATCT TCTCTCTGCA GAGCCCAAAT CTTGTGACAA 1380
• TCCCCCCAAA ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG 1620 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG GACGGCGTGG 1680
• CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA TGAGTGCGAC 2280
• MOLECULE TYPE DNA (genomic)
• GAGATCCAAG CAACCTTCTT TTACTTCACC CCCAACAAGA CAGAGGACAC GATCTTTCTC 240
• GCCAAGAGCC ATATCCGGGA GGACCCTGCC CCTGACCTAA GCCCACCCCA AAGGCCAAAC 900
• MOLECULE TYPE protein

Priority Applications (5)

Applications Claiming Priority (2)

Publications (1)

Family

ID=21690430

Family Applications (1)

Country Status (14)

Cited By (2)

Families Citing this family (1)

Citations (1)

Family Cites Families (3)

• 1996
  • 1996-06-11 AU AU62729/96A patent/AU6272996A/en not_active Abandoned
  • 1996-06-11 TR TR97/01620T patent/TR199701620T1/xx unknown
  • 1996-06-11 BR BR9608918A patent/BR9608918A/pt not_active Application Discontinuation
  • 1996-06-11 KR KR1019970709424A patent/KR19990022954A/ko not_active Application Discontinuation
  • 1996-06-11 IL IL12259096A patent/IL122590A0/xx unknown
  • 1996-06-11 EP EP96921519A patent/EP0833650A4/en not_active Withdrawn
  • 1996-06-11 HU HU9901131A patent/HUP9901131A2/hu unknown
  • 1996-06-11 WO PCT/US1996/010043 patent/WO1997000079A1/en not_active Application Discontinuation
  • 1996-06-11 JP JP9503258A patent/JPH11508131A/ja active Pending
  • 1996-06-11 CA CA002224625A patent/CA2224625A1/en not_active Abandoned
  • 1996-06-11 CZ CZ974014A patent/CZ401497A3/cs unknown
  • 1996-06-13 ZA ZA965032A patent/ZA965032B/xx unknown
  • 1996-06-13 AR ARP960103147A patent/AR003436A1/es unknown
• 1997
  • 1997-12-12 NO NO975862A patent/NO975862L/no unknown

Patent Citations (1)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events