WO2002089819A1 - Glycoconjugues et utilisations de ceux-ci - Google Patents

Glycoconjugues et utilisations de ceux-ci Download PDF

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Publication number
WO2002089819A1
WO2002089819A1 PCT/NL2001/000345 NL0100345W WO02089819A1 WO 2002089819 A1 WO2002089819 A1 WO 2002089819A1 NL 0100345 W NL0100345 W NL 0100345W WO 02089819 A1 WO02089819 A1 WO 02089819A1
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agp
glycoconjugate
lactosamino
linked
glycan structure
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PCT/NL2001/000345
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English (en)
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Willem Van Dijk
Dennis Cornelis Wilhelmus Poland
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Vereniging Voor Christelijk Wetenschappelijk Onderwijs
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Priority to PCT/NL2001/000345 priority Critical patent/WO2002089819A1/fr
Publication of WO2002089819A1 publication Critical patent/WO2002089819A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • A61P35/00—Antineoplastic agents

Definitions

  • cytokines such as IL-1 and TNF
  • inflamed tissue Koj A., et al., in Acute phase proteins: Molecular biology, biochemistry and clinical applications, Mackiewicz, A., Kushner, I. and Baumann, H., eds., Boca Raton: CRC press. (1993) 275- 87) .
  • complement factors Activation of complement factors is regarded to contribute to the excessive attraction of PMN, for example along the chemo-attractive activity of activated C5a (Niessen H.W.M., et al . , Cardiovascular Research, (1999) 41:603-10). Therefore, inhibiting or modifying the complement response is one of the approaches to prevent sustained inflammation.
  • Intravenous injection of specific glycoforms of recombinant human cti-acid glycoprotein (AGP) having an increased fucosylation prior to restoration of perfusion in a rat ischaemic model has been shown to ameliorate both neutrophil- and complement-induced damage in lungs and intestinal tissue.
  • AGP recombinant human cti-acid glycoprotein
  • Human AGP also known as orosomucoid, is a heavily glycosylated plasma protein, of which the hepatic synthesis and secretion is increased as part of the hepatic inflammatory response. It contains five N-linked glycans comprising about 45 % of its apparent molecular weight of 43 kD. The fucosylation of the glycans increases strongly upon inflammation, resulting in high plasma levels of AGP glycoforms with varying amounts of sLe x groups (de Graaf T.W. et al., J. Exp. Med., (1993) 177:657-66; Van Dijk W., et al . , TIGG. (1998) 10:235- 45) .
  • Extrahepatic production of AGP by a variety of tissues may contribute to the pool of AGP present in plasma under normal as well as inflamed conditions.
  • AGP glycoforms comprising highly fucosylated glycan structures
  • PMN classical complement activation route and therewith reducing the damaging effect during inflammatory response being induced by the classic complement activation.
  • substantial inhibition of the classic complement activation was reached at a concentration of the AGP glycoform as low as 0,1 ⁇ M.
  • glycoconjugates comprising polyfucosylated glycan structures have the above mentioned activities as identified for the said AGP in highly fucosylated form and expressed by PMN in late acute fase of inflammation or in chronic inflammatory disease.
  • glycoconjugates comprising such glycan structures can very well be used as active ingredient in the preparation of a medicament for treatment of conditions wherein the classical complement route plays an important role.
  • "Glycoconjugate” is defined as a polypeptide or another organic molecule to which one or more glycans are convalently linked.
  • Glycoform is defined as an isoform of a specific glycoconjugate of which the aglycon (i.e. the carrier molecule of the glycans) is identical but the glycans differ in composition and/or in number) .
  • glycoconjugates specifically binds with the receptors E-selectin and P-selectin, in particular with the E-selectin receptor, that are expressed by e.g. endothelial cells at the site of inflammation.
  • Glycoconjugates comprising the identified glycan structure can therefore advantageously be used as inhibitors for binding of PMN to selectin-expressing endothelial cells, and as drug targeting vehicle to E-selectin and/or P-selectin expressing cells.
  • such glycoconjugates can be used to inhibit binding of tumour cells expressing sLe x moiety on their cell surface (Kawana, T. et al.
  • N-glycosylated regulatory proteins for the classical route of complement activation has been described so far: C4b- binding protein, dv cay accelerating factor (DAF, CD55), CD59, and membrane co-factor protein (MCP, CD46) (Ninomiy H. et al [1992] J. Biol. Chem. 267: 8404-8410). Only the N-glycosylation of MCP was found to be essential for the inhibition of the classical route of complement activation.
  • the aminoterminus of MCP consists of four complement control protein (CCP) repeats, three of which (CCP-1, -2 and -4) posses N-glycans followed by alternative spliced region for extensive O-glycosylation.
  • CCP complement control protein
  • the alternative route does not require complexed antibody, but is generally promoted by specific sugar components present on or released from the cell membrane of microorganisms that have infected the body (e.g. sepsis).
  • specific sugar components present on or released from the cell membrane of microorganisms that have infected the body.
  • sepsis e.g. sepsis
  • the invention therefore relates to the use of a compound comprising at least one glycan structure, said glycan structure comprising:
  • AGP synthesised by neutrophils is as well highly fucosylated and contains polylactosamino-substituted glycans.
  • Such AGP-glycoforms are found to be at least as active as strongly fucosylated serum AGP in the inhibition of complement activation.
  • the highly fucosylated glycoconjungate function as an endogenous feedback inhibitor for excessive recruitment of neutrophils by activated complement factors.
  • a glycoconjugate of the above- defined type can be used as active ingredient in the preparation of a medicament for anti-metastatic therapies, in particular when invasion and/or growth of tumor cells involve sLe x ⁇ dependent binding to E- selectin. Str ⁇ gly fucosylated and sLe x containing AGP glycoforms can bind to E-selectin in vitro (Havenaar, E.G. et al . Glycoconjugate J. (1997) 14:S85) . Further such a glycoconjungate can also be used as targeting vehicle to target a drug to E-selectin expressing cells.
  • the local concentration of drugs or antibiotics in inflamed areas can be increased taking advantage of their strong binding properties of e.g. non-steroid anti-inflammatory drugs (NSAIDs) , antibiotics and anti-clotting drugs (e.g. warfarin).
  • NSAIDs non-steroid anti-inflammatory drugs
  • antibiotics and anti-clotting drugs e.g. warfarin
  • this will prevent unwanted side effects (particularly those leading to gastric ulcers) induced by the inhibition of cyclooxygenase 1 (COX-1) of the decreased formation of prostaglandins and thromboxanes (Insel P.A., in The Pharmacological
  • Glycan structures present in the glycoconjugates can be branched into a di-, tri-, or tetraantennary structure each antenna comprising at least one lactos amino group (in the art also abbreviated as GalG k NAc, but may also comprise linear polylactosaminyl stretches comprising two or more repeating lactosamino groups, therewith providing more possibilities for attachment of fucose residues to the glycoconjugate. Further, fucose, linked to a terminal lactosamino group may be in the form of a sialyl Lewis x group.
  • a minimal molecular weight of the glycoconjugate of at least 20 KD is preferred.
  • Glycan structures of the glycoconjugate of the invention have suitably a molecular weight of about 5 kD per glycan, based on the molecular weight of the sugars.
  • the glycan structure comprises a core region comprising the structure GlcNAc 2 Man 3 .
  • the glycan is linked via the core region GlcNAc 2 Man 3 to the carrier molecule.
  • GlcNAc 2 Man 3 is an abbrevation common in the art for mannose- ⁇ l, 6 (mannose- ⁇ l, 3) - mannose ⁇ l, 3N-acetylglucosamine ⁇ l, 4N- acetylglucosamine ⁇ l- .
  • the core region is defined as the oligosaccharide moiety of the glycan structure, which is covalently linked to the carrier molecule.
  • said glycan structure is therefore preferably linked to a carrier molecule.
  • Carrier molecules for the purpose of the invention are known in the art such as albumin and polyacrylamide of different molecular sizes.
  • the glycan structures of the glycoconjugate are linked to a peptide or protein.
  • protein includes single-chain polypeptide molecules as well as multiple- polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means .
  • polypeptide includes peptides of two or more amino acids in length, typically having more than 5, 10 or 20 amino acids.
  • the glycan is linked to the peptide or protein, to an amino acid with a free amino group or amide group.
  • the glycan is linked to an asparagines residue of the peptide or protein, via a so called N-glycosidic bond to the innermost N-acetylglucosamine of the core region.
  • the "innermost N- acetylglucosamine residue” refers to the N-acetylglucosamine residue of the core regison, preferably GlcNAc 2 Man 3 , that forms the covalent linkage with the carrier molecule.
  • the N-glycosidic bond is also referred to as "N-linkage", and is the naturally occurring linkage of this type of glycans to a protein.
  • the peptide or protein comprises at least 60 amino acids therewith substantially inhibiting renal passage of the glycoconjugate.
  • the peptide preferably comprises a fragment of at least 5 amino acids, being at least 80%, preferably 90% and more preferably 95% or more, homologous with an asparagine-X- serine or asparagine-X-threonine containing fragment of AGP, X being any natural amino acid, i.e. any amino acid, encodable by RNA triplets, the said asparagine, in native AGP, being linked to the glycan structure via an N-glycosidic bond.
  • AGP is the abbreviation of ⁇ l-acid glycoprotein, also known as orosomucoid (Dente L. et al . , (1987) EMBO J. 6: 2289-2296).
  • amino acid sequences for use in the invention are not limited to AGP-sequences or fragments thereof but also include homologous sequences obtained from any source, for example related mammalian proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof.
  • the present invention covers variants, homologues or derivatives of the amino acid sequences of the AGP in the glycoconjugate used in the present invention.
  • a homologous sequence is taken to include an amino acid sequence which is at least 60, preferably 70, more preferably 80 or even more preferably 90% identical, most preferably at least 95 or 98% identical at the amino acid level over at least 5, preferably 10, more preferably 20, or even more preferably 90, most preferably the total sequence of 181 amino acids with a native human AGP-sequence for example as described by Dente et al., supra.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions) , in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences .
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids) . Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed.
  • GCG Bestfit program Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details) . It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • variant or derivative in relation to the amino acid sequences of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence has similar function as the corresponding native human AGP-fragment .
  • amino acid sequence may be modified for use in the present invention. Typically, modifications are made that maintain the AGP- function of the sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the AGP-function. Amino acid substitutions may include the use of non-naturally occurring analogues, for example to increase blood plasma half-life of a therapeutically administered polypeptide.
  • Proteins of the invention are typically made by recombinant means, for example as described in Maniatis et al . , Molecular Cloning, A laboratory Manual, Cold Spring Harbour Laboratory, USA, 1982. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Proteins of the invention may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST) , 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase.
  • GST glutathione-S-transferase
  • 6xHis 6xHis
  • GAL4 DNA binding and/or transcriptional activation domains
  • ⁇ -galactosidase ⁇ -galactosidase
  • fusion protein may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences .
  • the fusion protein will not hinder the AGP-function of the protein or peptide of interest sequence.
  • Proteins of the invention may be in a substantially isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated.
  • a protein of the invention may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90V,, e.g. 95>, 98?, or 99'., of the protein in the preparation is a protein of the invention.
  • the peptide comprises an AGP fragment, preferably a human AGP fragment, comprising an attachment site for the glycan structure.
  • the said attachment site comprises, in AGP in its natural environment, a glycan structure.
  • the attachment site comprises an asparagine residue.
  • glycan structures may also be attached to a lysine residue via the free aminogroup.
  • AGP- based fragments, based on the ORMl or ORM2 sequence to be used according to the invention should include at least the aspargine of one of the above mentioned positions.
  • AGP fragments encompass the amino acid sequences 1-167, 1-89, 1-20, 33-58, 70-150, 80-90, although the skilled person will, based on the teaching herein, be able to identify additional suitable AGP-fragments . It is to be understood that said fragments may also be composed of a sequence homologous to the AGP sequence, as defined herein, as long as the glycan binding position and function of such a homologous sequence is similar to that of a native AGP sequence. Further, the AGP fragment may, when comprising a cysteine residue, be linked, via sulphur bridges, to one or more additional cystein comprising peptides .
  • the glycoconjugate may comprise two or more AGP fragments or fragments homologous thereto, linked to each other via sulphur bridges.
  • the glycoconjugate may comprise the AGP fragment of positions 70-90, including one or two glycan structures, linked to an AGP fragment of positions 135-155, via the cysteine residues on positions 72 and 147 respectively.
  • the AGP sequence of ORMl, ORM2 and of examples of suitable AGP fragments are depicted in figure 8.
  • the peptide comprises the complete sequence of AGP, preferably a human AGP.
  • the glycan structure of the glycoconjugate is branched to a tri- or tetra-antennary structure, each antenna comprising an extension of at least one lactosamino group .
  • at least one of the fucose-substituted lactosamino groups is sialylated in the configuration of a sialyl Lewis x (sLe x ) group. It has been shown that glycan structures comprising sialyl Lewis x groups show a strong inhibitory effect of the classical complement route.
  • the glycan structure specifically binds to the endothelial PMN receptor E-selectin, and to a minor extend also to P-selectin, as is discussed above.
  • PMN bind to E-selectin through cell surface glycans of similar structure as those present on PMN produced AGP- glycoforms
  • said glycoconjugates have shown to be an E-selectin ligand having . similar, binding qualities as the cell surface PMN glycans.
  • the glycan structure of the glycoconjugate comprises a linear polylactosaminyl stretch of at least two repeating lactosamino groups.
  • the glycan structure is in this way "elongated" and has more possibilities for • binding fucose residues through an ⁇ -1, 3-linkage with the N- acetylglycosamine moiety of the lactosamino groups.
  • the said linear polylactosamino stretch comprises at least two fucose residues, each of the fucose residues being linked to the N-acetyl glucosamine moiety of a lactosamino group of the polylactosamine stretch.
  • the polylactosamino stretch is 2-6 lactosamino groups long, in that case it is possible to link up to 6 fucose residues per antenne of the glycan structure.
  • a single glycan structure being branched to- a tetra-antennary structure, wherein each antenna comprises six lactosamino groups, 24 fucose residues may be linked to the structure (at least two of the four terminal lactosamino groups being in the form of a sialyl Lewis x group) .
  • the glycan structure of the glycoconjugate comprises at least five lactosamino groups and five fucose residues, linked via an ⁇ -1,3 linkage to the N-acetylglucosamine moiety of a lactosamino group.
  • a compound may comprise one glycan structure, harbouring all five lactosamino groups (e.g. a tetraantennary structure, of which one of the antennae comprises a polylactosamino stretch of two lactosamino groups, wherein all N-acetylglucosamines of the lactosamino groups are substituted with a fucose residue) .
  • lactosamino groups e.g. a tetraantennary structure, of which one of the antennae comprises a polylactosamino stretch of two lactosamino groups, wherein all N-acetylglucosamines of the lactosamino groups are substituted with a fucose residue
  • the compound it is also possible for the compound to comprise two or more glycan structures, at least one thereof having three fucose residues.
  • the glycoconjugate comprises 3-5 glycan structures . It has been found that at least 3 of the 5 glycans of the AGP-glycoforms with the highest complement inhibitory activity, produced by PMN, were highly fucosylated and contained polyactosaminogroups.
  • each of the glycan structures of the glycoconjugate is branched to a tri- or tetra-antennary structure, each glycan structure comprising at least three fucose residues, linked to the N- acetyl glucosamine moiety of a lactosamino group.
  • each antennary structure of the glycoconjugate comprises at least 2, preferably at least 3, fucose residues.
  • the complement involved conditions are, according to the invention, preferably chosen from the group, consisting of: sterile acute or chronic inflammations, especially rheumatoid arthritis, systemic lupus erytematosus, as well as myocardial infarcts, reperfusion damage and sepsis.
  • Activation of complement factors is generally occurring in acute and chronic inflamed tissue and reperfusion damage and can occur systemically in sepsis.
  • Ongoing activation of complement will, in addition to cell damage, contribute to the excessive attraction of neutrophils to the sites of inflammation, for example along the chemo-attractive activity of C5A. Therefore, inhibiting or modifying the classical complement response would be one of the approaches to prevent sustained inflammation.
  • the invention relates to a glycoconjugate, comprising at least one glycan structure, said glycan structure comprising: at least three lactosamino groups, each comprising a galactose and a N-acetylglucosamine, at least three fucose residues, each linked to the N-acetylglucosamine moiety of the lactosamino groups via an ⁇ -1,3 linkage, a linear polylactosamine stretch of at least two repeating lactosamino groups, the polylactosamine stretch comprising at least two fucose residues, each of the fucose residues being linked to the N-acetylglucosamine moiety of a lactosamino group of the polylactosamine stretch, a core region having the structure GlcNac 2 Man 3 , the glycan structure being linked, via a N-linkage of the innermost N-acetylglucosamine of the Glc
  • glycoconjugates according to the present invention are potent inhibitors of in particular the classical complement response, but are also more powerful inhibitors of the alternative complement response than the glycoconjugates, known in the art (e.g. Williams et al . , supra)
  • At least one glycan structure of the glycoconjugate according to the invention comprises at least 5 lactosamino groups, each comprising a galactose and a N-acetylglucosamine, and at least 5 fucose residues, each linked to the N-acetylglucosamine moiety of the lactosamino groups via an ⁇ -1,3 linkage. It has been found that a glycoconjugate comprising such highly fucosylated glycan structures show an even improved inhibitory effect on the complement-mediated reactions as outlined above.
  • the glycoconjugate according to the invention has a molecular weight of at least 20 kD.
  • the glycan structure of the glycoconjugate according to the present invention preferably comprises a core region having the structure of GlcNAc 2 Man 3 .
  • the glycoconjugate preferably comprises the glycan structure, linked to a peptide or protein.
  • the linkage of the glycan structure to the amino acid sequence of the peptide or protein may be via an N-glycosidic bond of the innermost N-acetylglucosamine residue of the glycan structure with a lysine or asparagine residue.
  • the peptide or protein comprises at least 60 amino acids.
  • the glycan structure of the glycoconjugate. is branched to a tri- or tetra-antennary structure, each antenna comprising at least one lactosamino group.
  • the glycoconjugate comprises at least one fucosylated lactosamino group in the form of a sialyl-Lewis x group.
  • the glycoconjugate comprises 3-5 glycan structures.
  • Each of the glycan structures are preferably branched to a tri- or tetra-antennary structure, each glycan structure comprising at least three fucose residues, linked to the N-acetyl glucosamine moiety of a lactosamino group.
  • Each antennary structure preferably comprises at least 2, preferably at least 3, fucose residues.
  • the peptide or protein of a preferred embodiment of the glycoconjugate according to the invention comprises a fragment of at least 20 amino acids, being at least 80%, preferably 90% and more preferably 95% or more, homologous with an asparagine containing fragment of AGP, the said asparagine, in native AGP, being linked to the glycan structure via an N-glycosidic bond.
  • the peptide or protein preferably comprises an AGP fragment, more preferably a complete sequence of AGP.
  • the following examples and figures demonstrate the expression, secretion and the glycosylation structure of highly fucosylated glycoprotein (AGP) in polymorphonuclear leukocytes (PMN) and its potency to inhibit e.g. the classical route of complement activation. This potency is also shown for compounds comprising such gycan structures.
  • AGP highly fucosylated glycoprotein
  • PMN polymorphonuclear leukocytes
  • This potency is also shown for compounds comprising such gycan structures.
  • the subcellular localisation of AGP in PMN and bone marrow myelocytes was also investigated. It is e.g. demonstrated that the glycosylation of PMN AGP differs from plasma AGP with respect to the presence of polyfucosylated lactosaminoglycans .
  • CAIE crossed affino immunoelectrophoresis
  • HPAEC-PAD high pH anion exchange chromatography with pulsed amperometric detection
  • PNGase-F digestion The glycosylation structure was elucidated by crossed affino immunoelectrophoresis (CAIE) , high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and partial PNGase-F digestion.
  • CAIE of AGP from PMN showed an elevated degree of branching in combination with an increased amount of fucosylated glycoforms as compared to normal plasma AGP.
  • Figure 1 Localisation of AGP in PMN.
  • Ultrathin cryosections of neutrophils from peripheral blood were immunogold labelled with antibodies against AGP; or double labelled with anti-AGP and respectively anti-lactoferrin (Lf) , anti- myeloperoxidase (MPO) or anti-albumin (Alb) as indicated.
  • Lf anti-lactoferrin
  • MPO anti- myeloperoxidase
  • Alb anti-albumin
  • FIG. 1 Localisation of AGP in PMN myelocytes.
  • FIG. 3 Western blot analysis of AGP isolated from PMN (A) and cell culture medium (B) .
  • PMN were isolated, stimulated, subjected to SDS- PAGE with subsequent blotting as described in material and methods.
  • Lane 1 no stimulation
  • lane 2 CytoB/fMLP stimulation
  • lane 3 PAF/fMLP stimulation
  • lane 4 fMLP stimulation
  • lane 5 plasma AGP.
  • Figure 4 PCR analysis of cDNA isolated from bone marrow myelocytes.
  • Figure 5 Reactivity of AGP from plasma (A,B,C) and PMN (D,E,F) with ConA (A,D), AAL (B,E) or without lectin (C,F).
  • AGP was subjected to CAIE as described in example 4. Only the second dimension gels are shown. The lower right corner of each pattern (see black dot) corresponds to the site of application in the first dimension gel. Electrophoreses was performed from right to left through in the first dimension and from bottom to top in the GaH-AGP containing second dimension gel.
  • CO ConA non-reactive fraction
  • Cw ConA weak-reactive fraction
  • Cs ConA strong-reactive fraction.
  • A0 AAL non-reactive fraction
  • Aw AAL weak-reactive fraction
  • As AAL strong-reactive fraction.
  • Figure 7 Dose response of normal plasma and PMN AGP in inhibiting the classical pathway of complement activation.
  • Values represent means ⁇ SD of assays performed in 3 replicates using three different preparations of erythrocytes in the presence of normal plasma AGP (H) , plasma AGP isolated from a patient with severe trauma (A) , and AGP isolated from PMN (•) .
  • CH 50 assay was performed as described in example 6.
  • Figure 8 Amino acid sequence of ORMl and ORM2 according to Dente, L. et al. , supra.
  • Amino acids -18 - -1 indicate the signal peptide, which is cleaved of during the maturation of the AGP protein.
  • the underlined amino acids N represent the glycosylation sites, whereto, in nature, the glycan structures are linked. Cysteine residues involves in sulphur bridge formation are in bold and marked with an asterisk. Residue 5 bridges with residue 164 and residue 72 with residue 147.
  • the lines below the sequences represent selections of AGP- glycopeptides according to the invention. Said peptides and compass residues 1-167, 1-89, 1-20, 33-58, 70-150 and 70-90 respectively.
  • Figure 9A depicts the cDNA sequence for ORMl (Dente, L. et al., Nucl. Acid Res. 13 (11), 3941-3952 (1985)).
  • Figure 9B depicts the cDNA sequence encoding ORM2 (Dente, L. et al., N. Bol. J. 6 (8), 2289-2296 (1987)).
  • the underlined sequences indicate the primer annealing sequences used in Example 4.
  • the ATG- startcodon is in bold.
  • Concanavalin A (Con A) (TypeV) and Coomassie Brillant Blue R250 were from Sigma (St. Louis, MO, USA) .
  • Aleurxa Aurantia lectin (AAL) was obtained from Biomed Labs (Newcastle u/o Tyne, UK) .
  • Both the FPLC system, the 5 ml HiTrap desalting columns and the 1 ml affinity HiTrap columns were obtained from Pharmacia (Uppsala, Sweden) .
  • the 1 ml mab-AGP-HiTrap colums were prepared by immobilizing 6 mg mAb-AGP (IgG 3 ) (producing cell line was a kind gift of Dr. H.B. Halsall,
  • PNGase-F was obtained from New England BioLabs Inc. (Beverly, MA, USA) .
  • Polyacryla ide and agarose M were from BioRad (Richmond, CA, USA) and human plasma protein calibrator (HSPC) and monospecific rabbit anti-human AGP
  • Rh-AGP Alkaline phosphatase- conjugated goat anti-rabbit IgG (GaR-IgG) were obtained from Zymed (San Francisco, CA, USA) and monospecific goat anti-human AGP (GaH- AGP) polyclonal antibodies from Dr. A Mackiewicz (Poznan, Poland) .
  • Bone marrow was obtained from healthy volunteers . Bone marrow-aspiration was performed by iliac crest or sternal puncture after informed consent. The aspirates were mixed with 3 ml McCoy's medium (Sigma, St Louis, Mo) containing 100 IU of heparin and 75 U of Varidase. The marrow cells were layered on 12 ml of isotonic Percoll with a specific gravity of 1.068g/ml. After centrifugation (20 min, 1000 x g, 20°C) the cells on top of the Percoll layer were collected.
  • Bone marrow cells and PMN from peripheral blood cells were fixed for 2 hours in a mixture of 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) and then processed for ultrathin cryosectioning as previously described (Calafat J, et al . , Blood. (1997) 90:1255-66). 45-nm cryosections were cut at -125°C using diamond knives (Drukker Cuijk, The Netherlands) in an ultracryo- microtome (Leica Aktiengesellschaft, Vienna, Austria) and transferred with a mixture of sucrose and cellulose onto formvar-coated copper grids (Liou W. et al . , Histochem. Cell. Biol.
  • the antibodies used were rabbit anti-AGP with respectively rabbit anti-human lactoferrin from Cappel Laboratories (Cochranville, PA) , rabbit anti-human albumin and mouse monoclonal anti-human myeloperoxidase both from CLB (Amsterdam, The Netherlands) .
  • the cryosections were embedded in a mixture of methylcellulose and uranyl acetate and examined with a Philips CM 10 electron microscope (Eindhoven, The Netherlands) .
  • the primary antibody was replaced by a nonrelevant murine or rabbit antiserum.
  • AGP colocalises with all these markers indicating that it is present in all these granules and in the secretory vesicles .
  • AGP was also synthesised during PMN maturation by investigating the distribution of AGP in the PMN progenitors from bone marrow. Cryosections then were double labelled with anti-AGP and anti-lactoferrin respectively anti-myeloperoxidase . A few promyelocytes containing only myeloperoxidase-positive granules were detected and some of them were also positive for AGP (not shown) .
  • Myelocytes were abundant and contained specific granules double labelled for lactoferrin and AGP; both labels were also found on endoplasmic reticulum and Golgi stacks (Fig. 2) . These results strongly suggest that AGP is synthesised during PMN maturation.
  • AGP and lactoferrin were both detected in the specific and azurophilic granules and the secretory vesicles. The same distribution of AGP was also found in PMN progenitors from bone marrow. AGP and lactoferrin were both found on endoplasmic reticulum and Golgi stacks. Since de novo synthesis and glycosylation takes place in the endoplasmic reticulum and Golgi respectively, AGP synthesis during PMN maturation is clearly demonstrated. Other granule originated complement inhibiting substances have already been described before. Van den Berg et al. showed an inhibitory effect of azurophilic granule originated human neutrophil defensins on the classical pathway of complement activation (Van den Berg RH, et al., Blood (1998) 92:3898-903).
  • PMN stimulation experiments 100 x 10 6 PMN per incubation were used. PMN were resuspended in Hepes medium (consisting of 132 mM NaCl, 6 mM KCL, 1 mM CaCl 2 , 1 mM MgS0 4 , 1.2 mM potassium phosphate, 20 mM HEPES, 5 mM glucose, 0.5% (w/v) human serum albumin, pH 7.4) .
  • Affinity chromatography was used to isolate AGP from PMN stimulation supernatant, PMN cell lysate, normal human plasma and plasma from patients suffering from severe trauma (acute-phase plasma) (Brinkman-van der Linden ECM, et al . Glycoconjugate Journal (1998) 15:177-82).
  • AGP was bound on two 1 ml mab-AGP HiTrap columns containing 6 mg mAb-AGP each.
  • the buffer used for AGP binding was PBS, elution of the bound AGP was performed with PBS + 1 M NaCl. Demineralized and filtered water (milli Q water) was used for desalting of the samples by two 5 ml desalting HiTrap columns.
  • cDNA was synthesised from 4 ⁇ g of total RNA by using the 3 ' RACE system from Gibco, Life Technologies. cDNA corresponding to 80 ng of RNA template was used for PCR analysis.
  • PCR reactions were performed in a final volume of 50 ⁇ l containing: Taq buffer (Promega) , 1.5 mM MgCl 2 , 0.2 mM dNTPs each (Boehringer) , 1.25 units of TaqBeads Hot Start polymerase (Promega) and 50 pmol of each primer (Isogen) .
  • the sense primer GAC CAG TGC ATC TAT AAC ACC ACC and for the ORM2 sense primer sequence TGA TGT TTG GTT CCT ACC TGG AC were used.
  • As an antisense primer sequence for both ORMl and ORM2 GTT CCA AAC ACA GAA GCT TTA TTG A was used (see also Figure 9) .
  • actine the sense primer TGA CGG GGT TCA CCC ACA CTG TGC CCA TCT and the antisense primer AGT CAT AGT CCG CCT AGA AGC ATT TGC were used as a positive control.
  • Amplifications were carried out for 40 cycles using the following conditions: 1 min at 60°C, 1.5 min at 72°C and 1 min at 94°C PCR products were fractionated by 1.5% agarose electrophoresis and the gels were photographed using KODAK DC260/265 photo camera from BioRad (Richmond, CA, USA) .
  • Different forms of AGP can be distinguished in serum, not only on bases of its glycosylation but also on the encoding gene.
  • Three genes, clustered in one locus encode two variants of AGP, of which the AGP-A gene encodes the variant ORMl and the AGP-B/B' genes encode the variant ORM2.
  • the AGP-B/B' genes are identical; the AGP-A gene is structurally similar, but contains several base substitutions, resulting in 21 amino acid differences (Dente L, et al . , EMBO J. 6. 1987;6:2298-6) .
  • CAIE crossed affinoimmunoelectrophoreses
  • AGP concentrations were determined by single radial i munodiffusion, according to Mancini et al . , using monospecific GaH- AGP polyclonal antiserum for precipitation (Mancini G, et al . ,
  • AGP glycosylation of AGP was visualised by crossed affino immunoelectrophoresis (CAIE) as described earlier (Graaf T.W., et al., J. Exp. Med. (1993) 177:657-66).
  • CAIE crossed affino immunoelectrophoresis
  • approximately 0.8 ⁇ g AGP was subjected to electrophoresis through a lectin containing first dimension 1% agarosegel. This resulted in the separation of a non-retarded glycoform and various retarded glycoforms differing in degree of branching or extent of fucosylation when Con A, respectively AAL was used as the lectin.
  • the separated glycoforms were electrophoresed against a monospecific GaH-AGP polyclonal antiserum, the resulting precipitation lines were subsequently stained by Coomassie Brillant Blue. Lysates of non-stimulated PMN were shown to contain 0.42 ⁇ g per 10 6 cells by radial immunodiffusion according to Mancini using serum AGP as a standard. 400 x 10 6 PMN, obtained from 2 L of pooled human blood from 20 healthy donors, were used to isolate sufficient amounts of PMN AGP for the analysis of its glycosylation by various analytical techniques. The preparation was apparently pure according to SDS-PAGE analysis (data not shown) .
  • the degree of branching and fucosylation of the glycans were assessed by CAIE with respectively Con A and AAL as affinity components in the first dimension gel. Marked differences were detected between the CAIE patterns of PMN and normal plasma AGP (Fig. 5E) . All glycoforms of PMN AGP were very strongly retarded by AAL, which can be attributed to a high degree of fucosylation (Fig. 5E) . This is in sharp contrast to serum AGP from which it is known that about half of the glycoforms are not fucosylated at all, and the remainder are fucosylated to various extents (De Graaf, T.W. et al . , supra) (Fig. 5B) .
  • the electrophoretic mobility of PMN AGP appeared to be lower than of serum AGP, as is shown by comparing the patterns in Fig. 5C and 5F, where the lectins were omitted from the first dimension gel.
  • CAIE in the presence of Con A showed a lack of reactivity of PMN AGP with this lectin (Fig. 5D) .
  • AGP isolated in triplicate from the PMN or normal human plasma was treated with PNGase-F to release the N-linked glycans from the protein part of the glycoprotein.
  • Incubations were carried out under reducing conditions in 50 mM sodium phosphate buffer, containing 1 % NP-40 and 1000 U PNGase-F (total volume of 100 ⁇ l) for 1 h at 37°C
  • Incubation with RaH- AGP (1:500, v/v) and alkaline phosphatase conjugated GaR-IgG (1:2500, v/v) were used for detection of the AGP-containing bands.
  • PNGase-F treatment of AGP isolated from PMN showed the presence of 5 N-linked glycans as shown by the Western blot analysis in Fig. 6 lanes 5 - 8.
  • the band appearing at 58 kD is non-digested AGP and contains 5 glycans
  • the band detectable at 23 kD is totally digested AGP and contains no glycans
  • the bands in between are partial digested AGP with respectively 4, 3, 2, and 1 glycan.
  • As a control AGP isolated from normal plasma was also PNGase-F digested and subjected to Western blot analysis (Fig 6 lanes 1-4) .
  • Non-digested AGP could be detected at 43 kD, totally digested AGP was found at 23 kD and in between partial digested AGP was demonstrable with 4, 3, 2 and 1 glycan, respectively.
  • AGP isolated from normal plasma and PMN, turned out to have a molecular weight of 23 kD.
  • the hydrolysed samples were subjected to high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Dionex Carbopack PA 1 column (0.4 x 25 cm) .
  • the column was washed for 20 min with 0.02 M NaOH at 1 ml/min prior to sample injection. Elution was isocratic with 0.1 M NaOH at 1 ml/min for 30 min.
  • the acid hydrolysed glycans were subjected to HPAEC-PAD analysis using a Dionex Carbopack PA-100 column (0.4 x 25 cm) which was run in 0.1 M NaOH at 1 ml/min. The column was washed for 5 min with 0.1 M NaOH/0.5 M sodium acetate, and subsequently for 15 min with 0.1 M NaOH prior to sample injections. Elution was isocratic with 0.1 M NaOH for 10 min after which a gradient was applied of sodium acetate by increasing the concentration from 0-0.25 M in 100 min. The data obtained by the HPAEC-PAD analysis were corrected for monosaccharide degradation during hydrolysis.
  • the carbohydrate composition of PMN AGP is given in Table 1 in comparison to the composition of normal and acute-phase human plasma AGP.
  • the average structure of the N-linked glycans were estimated by expressing the results per 3 mannose residues. This clearly demonstrated that the glycans of PMN AGP contained much more fucose residues than, plasma AGP, 6.6 vs. 0.8 residues per. glycans, confirming the results obtained with CAIE in the presence of AAL (cf. Fig. 5) .
  • the number of sialic acid residues per glycan were found to be higher for PMN AGP than for plasma AGP.
  • glycosylation of glycoproteins isolated from human PMN differs from hepatic glycoprotein synthesis with respect to the presence of polyfucosylated lactosaminoglycans (Fukuda M, et al., J. Biol. Chem. (1985) 260:12957-67, Fukuda M, et al . , Fukuda M, et al . , J. Biol.
  • Table 1 Carbohydrate composition of plasma and PMN AGP. Neutral sugars and sialic acid were determined as described above.
  • CH 50 assay To demonstrate the effect of normal and acute-phase plasma AGP and PMN AGP on the classical route of the complement activation, an in vitro classical pathway 50% hemolysis (CH 50 ) assay was performed (Mayer MM. Experimental Immunochemistry. 2nd ed. (E.A. Kabat and M.M. Mayer, eds.) Thomas, Springfield, 111:133-239; Rapp HJ and Borsos T. Molecular basis of complement action. Appleton-Century-Crofts, New
  • a dilution range of AGP in veronal-saline buffer (VBS; 5 mM barbital, 150 mM NaCl, 0.03 mM CaCl 2 , 0.1 mM MgCl 2 , pH 7.4), standardised normal human serum and rabbit anti-sheep erythrocyte sensitised sheep red blood cells (both kindly provided by the CLB, Amsterdam, The Netherlands) were added together in a 96 wells plate.
  • PMN AGP is a very strong inhibitor of the classical route of complement activation in vitro whereas plasma AGP is not.
  • AGP released by the PMN, was able to strongly inhibit the classical route of complement activation in vitro (CH 50 assay) in a concentration of 100 nM, 190 times below normal physiological concentrations of plasma AGP.
  • Williams et al. described a reduction of the alternative route of complement activation and PMN activity by sLe x -bearing recombinant AGP, at normal physiological concentrations, after intestinal ischemia in the rat (Williams JP, et al., Am. J. Physiol. (1997) 273 :G1031-G1035) .
  • C3b For the classical route of complement activation several N-glycosylated regulatory proteins has been described so far: C4b-binding protein, decay accelerating factor (DAF, CD55) , CD59, and membrane co-factor protein (MCP, CD46) (Hardig Y, et al., Biochem. J. (1995) 308:795- 800, Suzuki H, et al., FEBS Lett. (1996) 399:272-6, Akami T, et al., Transplant. Proc.
  • DAF decay accelerating factor
  • MCP membrane co-factor protein
  • MCP complement control protein
  • AGP has a cAMP-dependent effect on human endothelial cells, inhibits the action of histamine and forms an important component of . the capillary barrier it may also contribute to the maintance of endothelial permeability after release by degranulation of PMN.
  • PMN AGP Another important feature of PMN AGP could be the binding to E-selectin on the endothelial cells via its sLe x -groups and prevent further extravasation of PMN into the inflamed area. Masking of the E-selectin will eventually result in a local decrease in PMN concentration and so prevent further PMN related tissue damage.

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Abstract

L'invention concerne une nouvelle utilisation de glycoconjugués hautement fucosylés comme ingrédient actif dans la préparation d'un médicament pour traiter des états classiques liés au complément, dans le cadre de thérapies antimétastatiques et comme véhicule de ciblage pour cibler un médicament sur des cellules, exprimant la E-sélectine et/ou la P-sélectine. L'invention concerne également de nouveaux glycoconjugués hautement fucosylés.
PCT/NL2001/000345 2001-05-07 2001-05-07 Glycoconjugues et utilisations de ceux-ci WO2002089819A1 (fr)

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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019501A1 (fr) * 1990-06-15 1991-12-26 Cytel Corporation Mediateurs d'adherence intercellulaire
WO1992019634A1 (fr) * 1991-05-06 1992-11-12 The Biomembrane Institute Glycosphingolipides a chaine de type 1 prolongee utilises comme antigenes associes a des tumeurs
WO1994011498A1 (fr) * 1992-11-16 1994-05-26 Board Of Regents Of The University Of Oklahoma Ligand clycoproteique pour la p-selectine et son procede d'utilisation
WO1996009309A1 (fr) * 1994-09-20 1996-03-28 Pharmacia & Upjohn Company Structure oligosaccharide d'un ligand de la selectine e et p
US5639734A (en) * 1994-12-20 1997-06-17 Esko; Jeffrey D. Disaccharide inflammation inhibitors and uses thereof
US5753631A (en) * 1990-06-15 1998-05-19 Cytel Corporation Intercellular adhesion mediators
WO1999012944A2 (fr) * 1997-09-05 1999-03-18 Glycim Oy POLYLACTOSAMINES SYNTHETIQUES CONTENANT UN sLex DIVALENT ET SES METHODES D'UTILISATION
US5929036A (en) * 1989-03-08 1999-07-27 The Board Of Regents Of The University Of Oklahoma Ligand or GMP-140 selectin and methods of use thereof
US6083929A (en) * 1991-05-06 2000-07-04 The Biomembrane Institute Extended type 1 chain glycosphingolipids as tumor-associated antigens
US6124267A (en) * 1991-02-05 2000-09-26 Southpac Trust Internationals, Inc. O-glycan inhibitors of selectin mediated inflammation derived from PSGL-1

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5929036A (en) * 1989-03-08 1999-07-27 The Board Of Regents Of The University Of Oklahoma Ligand or GMP-140 selectin and methods of use thereof
WO1991019501A1 (fr) * 1990-06-15 1991-12-26 Cytel Corporation Mediateurs d'adherence intercellulaire
US5753631A (en) * 1990-06-15 1998-05-19 Cytel Corporation Intercellular adhesion mediators
US6124267A (en) * 1991-02-05 2000-09-26 Southpac Trust Internationals, Inc. O-glycan inhibitors of selectin mediated inflammation derived from PSGL-1
WO1992019634A1 (fr) * 1991-05-06 1992-11-12 The Biomembrane Institute Glycosphingolipides a chaine de type 1 prolongee utilises comme antigenes associes a des tumeurs
US6083929A (en) * 1991-05-06 2000-07-04 The Biomembrane Institute Extended type 1 chain glycosphingolipids as tumor-associated antigens
WO1994011498A1 (fr) * 1992-11-16 1994-05-26 Board Of Regents Of The University Of Oklahoma Ligand clycoproteique pour la p-selectine et son procede d'utilisation
WO1996009309A1 (fr) * 1994-09-20 1996-03-28 Pharmacia & Upjohn Company Structure oligosaccharide d'un ligand de la selectine e et p
US5639734A (en) * 1994-12-20 1997-06-17 Esko; Jeffrey D. Disaccharide inflammation inhibitors and uses thereof
WO1999012944A2 (fr) * 1997-09-05 1999-03-18 Glycim Oy POLYLACTOSAMINES SYNTHETIQUES CONTENANT UN sLex DIVALENT ET SES METHODES D'UTILISATION
US6191271B1 (en) * 1997-09-05 2001-02-20 Glycim Oy Synthetic divalent sLex containing polylactosamines and methods for use

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