WO2000021552A1 - Antigenes de glycoproteines, leurs anticorps specifiques et procede de fabrication correspondant - Google Patents

Antigenes de glycoproteines, leurs anticorps specifiques et procede de fabrication correspondant Download PDF

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WO2000021552A1
WO2000021552A1 PCT/US1999/023478 US9923478W WO0021552A1 WO 2000021552 A1 WO2000021552 A1 WO 2000021552A1 US 9923478 W US9923478 W US 9923478W WO 0021552 A1 WO0021552 A1 WO 0021552A1
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group
antibody
oligosaccharide
galnac
antigen
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WO2000021552A9 (fr
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Brad K. Bendiak
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University Technology Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum

Definitions

  • the present invention relates to the field of oligosaccharide antigens and, as such, broadly relates to the fields of carbohydrate chemistry, immunology and medicinal chemistry.
  • glycoproteins comprise one of the major components of the exterior surface of cells and are proteins that are linked to oligosaccharides.
  • the type of glycoproteins present on the cell surface are often characteristic of cell type. By virtue of their location at the outer cell surface, glycoproteins can function as a protective layer and as highly specific recognition molecules, thereby playing a key role in the physiological properties of the cell.
  • glycoproteins are believed to be related to many of the abnormal properties characteristic of cancer cells, such as rapid and uncontrolled cell growth, altered cell adhesion, avoidance of detection by the immune system and metastatic spread (for a review, see Hounsell, E. F., et al., Clinical Science, 93: 287-293, 1997).
  • Glycoproteins fall into two primary classes depending upon the atom involved in the linkage between the oligosaccharide and the protein.
  • the linkage between the oligosaccharide and an amino acid from the protein involves the anomeric center of the monosaccharide and a functional group in the side chain of the amino acid involved in the linkage.
  • N-linked glycoproteins N-acetyl- ⁇ -D- glucosamine is linked to the amide group of the amino acid asparagine;
  • O-linked glycoproteins the linkage involves the hydroxyl group of either serine, threonine, hydroxylysine or hydroxyproline.
  • the O-linked glycoproteins have been further subclassified into five groups, including a group called the mucins.
  • Mucins are high molecular weight glycoproteins (often exceeding 1 x
  • Mucins form a viscous fluid lining on the epithelium of the gastrointestinal, respiratory, and genitourinary tracts. They function to lubricate the surfaces of these tracts and to protect the epithelial cells from the harsh environment of the lumen. Mucins have a very high carbohydrate content, which can account for 50 to 80% of the total weight of the mucin.
  • the carbohydrates vary in size from monosaccharides to oligosaccharides containing approximately 20 residues and are characterized by significant structural heterogeneity.
  • the polypeptide backbone of the mucin can be attached to several hundred oligosaccharide chains.
  • Mucins are characterized by a linkage involving N-acetyl- ⁇ -D-galactosamine and the hydroxyl group of either serine or threonine located in the protein backbone. This N-acetyl- galactosamine and the monosaccharides attached to it form the "core” or “core structure” of the oligosaccharides attached to the protein backbone.
  • mucin antigens can serve as tools in the diagnosis and treatment of human cancer (see for example, Johnson, V.G., et al., Cancer Res., 46: 850-857, 1986; Burchell, J., et al., J. Immunol, 131: 508-513, 1983).
  • breast cancers are second in terms of the number of structural changes in mucin oligosaccharides that have been characterized as tumor-associated antigens (TAA).
  • TAA tumor-associated antigens
  • One breast mucin TAA is believed to have a carbohydrate epitope that includes sialic acid (Pal S., et al., Int. J. Cancer, 50: 759- 65, 1995).
  • N- glycolylneuraminic acid (Neu5Gc) which previously had been thought not to exist in humans (Devine, P.L., et al., Cancer Res., 51 :5826-5836, 1991 ; and Hanisch, F-G., et al., Eur. J. Biochem., 236: 318-327, 1996).
  • the monoclonal antibody, PD41 shows a very high specificity toward prostrate carcinoma, exhibits cross reactivity with bovine submaxillary mucin and appears to recognize a carbohydrate or glycopeptide epitope involving a N-acetyl galactosamine moiety (Beckett, M.L. and Wright, G.L., Jr., Int. J. Cancer, 62: 703-
  • PD41 appears to be distinct from monoclonal antibodies P25.48 and P25.91, which also exhibit a high specificity toward prostrate carcinoma (Bazinet, M., et al., Cancer Res., 48: 6938-6942, 1988).
  • Lung cancer is yet another example of a correlation between mucin antigens and cancer. Fairly recent reports have indicated that a Le a -X epitope is predominately associated with large glycoproteins and mucins synthesized by squamous lung carcinomas. Cells expressing this epitope form a mucin gel which seems to prevent antibodies from interacting with the underlying cells, thereby apparently providing an immunoprotective effect (Stranahan, P.L., et al., Cancer Res.,
  • mucins In addition to their function as markers of tumor and cancerous cells, mucins also play an important role in cell recognition and cellular differentiation.
  • specific carbohydrate antigens which are expressed at the cell surface vary depending upon the particular stage of cellular differentiation (for a general review, see Richa, J. and Solter, D., "Role of Cell Surface Molecules in Early Mammalian Development,” in Experimental Approaches to Mammalian Embryonic Development (Ro repet, J. and Pederson, R.A., Eds.) pp. 293-320, Cambridge
  • the present invention provides eight novel mucin oligosaccharide alditols and the oligosaccharide structures corresponding to these oligosaccharide alditols.
  • These newly identified and purified oligosaccharide alditols include: (a) Fuc ⁇ l ⁇ 6GalNAc-ol, (b) GalNAc ⁇ l ⁇ 3GalNAc-ol, (c)
  • the core structures corresponding to these alditols and the oligosaccharides of the invention include: (a) Fuc ⁇ l ⁇ GalNAc, (b) GalNAc ⁇ l ⁇ 3GalNAc, (c) GalNAc ⁇ 1 ⁇ 3 [GlcNAc ⁇ l ⁇ 6]GalNAc, (d) GlcNAc ⁇ l ⁇ 3GalNAc ⁇ l ⁇ 6GalNAc, (e) Fuc ⁇ l ⁇ 4GalNAc, (f) GalfNAc ⁇ l ⁇ GalNAc, (g) Fuc ⁇ l ⁇ 4GalNAc and (h) Fuc ⁇ l— »6GalNAc, respectively, and derivatives thereof.
  • These oligosaccharide core structures can function as the haptens of antigens.
  • the present invention further provides antigens based upon these new oligosaccharide structures.
  • these antigens comprise one of the oligosaccharides of the present invention that have been conjugated to a carrier molecule. More specifically, the antigens have the following structures:
  • the antigens of the invention further include derivatives of the foregoing antigens, provided that it is possible to raise antibodies against the derivatives that are capable of specifically binding to the non-derivatized antigens provided herein.
  • X can include any molecule which can serve as a linker between the oligosaccharide and R.
  • linkers includes polypeptides, oligosaccharides and cross-linking agents.
  • X is serine or threonine.
  • R can be any of a number of moieties that increase the immunogenicity of the oligosaccharides provided in the present invention.
  • R can include, but is not limited to, a polypeptide, a lipid, a polysaccharide, a hpopolypeptide, a hposaccharide, or certain water-soluble polymers.
  • R is a polypeptide
  • a variety of polypeptides can be utilized including natural or synthetic polypeptides.
  • the polypeptide can be an animal serum albumin, an animal serum globulin, an animal thyroglobulin, an animal hemoglobulin or an animal hemocyanin.
  • the animal protein source can include, but is not limited to, equine, bovine, rabbit, murine, human and ovine sources.
  • R when R is a lipid, it can include, for example, ceramide, n- octadecylamine or n-hexyldecylamine, or monophosphoryl lipid A (also known as Ribi adjuvant).
  • R can be a water-soluble polymer such as polylysine, polyglutaminic acid, tripalmitoyl-S-glycerylcysteinyl-seryl-serine and various copolymers.
  • a second embodiment of the invention includes antibodies, preferably monoclonal antibodies, that specifically bind to the antigens described in the present invention.
  • the antibodies specifically bind to the oligosaccharide haptens, i.e., the oligosaccharide structures provided by the current invention.
  • the invention further provides a method of preparing antibodies, particularly monoclonal antibodies to the antigens of the present invention. In its most general form, the method involves immunizing a host with an immunogenic composition that includes an antigen of the present invention.
  • the immunogenic composition can also be varied to further include an excipient or an adjuvant to increase the immunogenicity of the immunogenic composition.
  • Certain methods include: (a) creating hybridomas by fusing splenocytes from an immunogenized host with myeloma cells, (b) culturing the hybridoma cells on selective media to identify those cells in which a splenocyte and a myeloma cell have effectively fused, (c) selecting from the hybridoma cells identified in step (b) those which secrete antibodies specific for an antigen of the present invention, (d) cloning the hybridoma cells selected in step (c), (e) culturing the cloned hybridoma cells and (f) recovering the antibody from the cultured hybridoma cells.
  • the present invention provides hybridomas capable of producing antibodies that specifically bind to the antigens described herein.
  • the present invention also provides a method for screening a biological sample for the antigens of the present invention.
  • the biological sample can include, for example, blood, serum, urine, tissue slices and tissue homogenates.
  • the screening method comprises the steps of: (a) contacting the biological test sample with the antibodies of the present invention to allow formation of an antigen-antibody complex is formed, and (b) detecting the formation of the antigen-antibody complex. Detection of the formation of the antigen- antibody complex can be accomplished using a variety of known techniques, including for example, radioimmunoassay, enzyme linked immunosorbent assay and immunoprecipitation.
  • FIG. 1 A is the elution profile of the neutral oligosaccharide-alditols released from bovine submaxillary mucin on a semipreparative column of Glycopak N, eluting with isocratic CH 3 CN/H 2 O, 74/26, at 10.0 mL/min.
  • FIG. IB is the elution profile of the primarily neutral disaccharide alditols released from BSM (i.e., of fraction A, FIG. 1A) on an analytical column of Glycopak N, eluting with isocratic CH 3 CN/H 2 O, 85/15, at 1.0 mL/min. The plot of fraction A3 is shown at 25 -fold intensity.
  • FIGS. 1C-1E are the elution profiles of fractions A4, A6, and A7, respectively, from FIG. IB, on a column of DC-613 (NaAorm), eluting with isocratic
  • FIG. IF is the elution profile of fraction A6d from FIG. ID on a column of DC-613 (Li + form), eluting with an isocratic mixture of CH3CN/H2O,
  • FIG. 1G is the elution profile of fraction A7f from FIG. IE on a column of DC-613 (Li + form), eluting with an isocratic mixture of CH3CN/H2O,
  • FIG. IH is the elution profile of fraction A3(3) from FIG. IB on a column of DC-613 (Na + form), eluting with isocratic CH 3 CN/H 2 O, 88/12, at 0.6 mL/min.
  • FIG. II is the elution profile of fraction A3(3)g, from FIG. IH, on a column of DC-613 (Cs + form), eluting with isocratic CH 3 CN/H 2 O, 86/14, at 0.6 mL/min.
  • FIG. 1 J is the elution profile obtained when fraction A3(3)e , from FIG. IH, was chromatographed on a DC-613 column (Li + form), eluting isocratically with CH 3 CN/H 2 O, 86/14, at 0.6 mL/min.
  • FIG. IK is the elution profile obtained when fraction A3(3)f, from FIG. IH was chromatographed on a DC-613 column (Cs + form), eluting isocratically with CH 3 CN/H 2 O, 86/14, at 0.6 mL/min.
  • FIG. 2 A is the elution profile of the neutral tri- and fucosylated tetrasaccharide-alditols released from bovine submaxillary mucin (i.e., of fraction B,
  • FIG. 1A on an analytical column of Glycopak N, eluting with isocratic CH 3 CN/H 2 O, 82/18, at 1.0 mL/min.
  • FIGS. 2B-2G are the elution profiles of fractions B2, B3, B4, B5, B6, and B7, respectively, from FIG. 2A, on a column of DC-613 (Na + form), eluting with CH 3 CN/H 2 O at 0.6 mL/min at the following CH 3 CN/H 2 O ratios: FIG. 2B. 80/20;
  • FIGS. 2H-2J are the elution profiles of fractions B2b, B2c, and B2d (the foregoing fractions obtained as shown in FIG. 2B), respectively. Columns were eluted with CH 3 CN/H O, 80/20, at a flow rate of 0.6 mL/min.
  • FIG. 2K is the elution profile of fraction B3g, from FIG. 2C, eluting with CH 3 CN/H O, 78/22, at a flow rate of 0.6 mL/min.
  • FIG. 2L is the elution profile of fraction B5e, from FIG. 2E, on a column of DC-613 (Li + form), eluting with CH 3 CN/H 2 O, 78/22, at 0.6 mL/min.
  • FIG. 3A is the ⁇ -NMR spectrum (600 MHz, 300 °K) of Fuc ⁇ l- 6GalNAc-ol (fraction A3(3)g3).
  • the N-acetyl and fucose methyl signals are shown at about 1/3 to 1/5 intensity.
  • FIG. 3B is the H-NMR spectrum (500 MHz, 305 °K) of GalNAc ⁇ 1- 3GalNAc-ol (fraction A6d3) showing an expansion of the exocyclic and ring proton region.
  • FIGS. 4A-4I are the electrospray MSMS data and fragmentation schemes of the di- and trisaccharide-alditols described herein. The MSMS spectra of
  • FIG. 4A Fuc ⁇ l-6GalNAc-ol (fraction A3(3)g3)
  • FIG. 4B GalNAc ⁇ l-3TheNAc-ol, derived from both GalNAc ⁇ 1- 3GalNAc-ol (fraction A6d3), and, independently, from GalNAc ⁇ 1-3 [GlcNAc ⁇ 1- 6]GalNAc-ol (fraction B3g3) by Pb(OAc) 4 oxidation (-30° C)/NaBH 4 reduction;
  • FIG. 4C GalNAc ⁇ l-3GalNAc-ol (fraction A6d3);
  • FIG. 4D GalNAc ⁇ 1-3 [GlcNAc ⁇ l-6]GalNAc-ol (fraction B3g3);
  • FIG. 4E GlcNAc ⁇ l-3GalNAc ⁇ l-6GalNAc-ol (fraction B5e3).
  • FIG. 4F Fuc ⁇ l-4GalNAc-ol (fraction A3(3)el);
  • FIG. 4G GalfNAc ⁇ l-6GalNAc-ol (fraction A3(3)e2);
  • FIG. 4H Fuc ⁇ l-6GalNAc-ol (fractions A3(3)f2 and A3(3)gl);
  • FIG. 41 Fuc ⁇ l-4GalNAc-ol (fraction A3(3)f3).
  • FIGS. 5A-5C are H-NMR spectra of products derived by Pb(OAc) oxidation (-30° C)/NaBH 4 reduction of some of the di- and trisaccharide-alditols reported herein, showing assignments where indicated.
  • FIG. 5 A is the spectrum for Fuc ⁇ l-ethanediol, derived from Fuc ⁇ l- 6GalNAc-ol (fraction A3(3)g3).
  • FIG. 5B is the spectrum of GalNAc ⁇ l-3TheNAc-ol, derived independently from GalNAc ⁇ 1-3 GalNAc-ol (fraction A6d3) and GalNAc ⁇ 1 - 3 [GlcNAc ⁇ l-6]GalNAc-ol (fraction B3g3).
  • FIG. 5C is the spectrum of GlcNAc ⁇ 1-ethanediol, derived from GalNAc ⁇ 1-3 [GlcNAc ⁇ 1 -6] GalNAc-ol (fraction B3g3). N-acetyl and fucose methyl signals are shown at about 1/3 to 1/5 intensity.
  • FIG. 6 A is the 1-D ⁇ NMR spectrum (600 MHz 300 °K) of GalNAc ⁇ 1-3 [GlcNAc ⁇ 1 -6] GalNAc-ol (fraction B3g3) showing an expansion of the exocyclic and ring proton region with some assignments.
  • FIG. 6B is the 1-D ' H-NMR spectrum (600 MHz 300 °K) of
  • GlcNAc ⁇ 1 -3 GalNAc ⁇ l-6GalNAc-ol fraction B5e3 showing an expansion of the exocyclic and ring proton region.
  • FIG. 7A is the ⁇ -NMR spectrum (600 MHz, 300 °K) of Fuc ⁇ 1- 4GalNAc-ol (fraction A3(3)el) showing an expansion of the exocyclic and ring proton region.
  • FIG. 7B is the 1H-NMR spectrum (600 MHz, 300 °K) of GalfNAc ⁇ l- 6GalNAc-ol (fraction A3(3)e2) showing an expansion of the exocyclic and ring proton region.
  • FIG. 8 is the H-NMR spectrum (500 MHz, 300 °K) of Fuc ⁇ l- 4GalNAc-ol (fraction A3(3)f3) showing an expansion of the exocyclic and ring proton region.
  • FIG. 9 is the H-NMR spectrum (500 MHz, 300 °K) of Fuc ⁇ l- 6GalNAc-ol (fractions A3(3)f2 and A3(3)gl) showing an expansion of the exocyclic and ring proton region.
  • FIG. 10 illustrates a synthetic scheme for the synthesis of oligosaccharides wherein a GalNAc residue is linked to a core GalNAc residue to form a ⁇ l-3 linkage.
  • FIG. 11 illustrates a method for synthesizing oligosaccharides wherein a fucosyl residue is attached to a core GalNAc residue to form an ⁇ or ⁇ 1-6 linkage.
  • FIG. 12 shows a synthetic scheme for preparing an oligosaccharide wherein a ⁇ GalNAc and a ⁇ GlcNAc are attached to C-3 and C-6 of a core GalNAc residue, respectively.
  • FIG. 13 A and 13B depict a method for synthesizing oligosaccharides wherein a fucosyl residue is attached to a core GalNAc residue to form an ⁇ or ⁇ 1-4 linkage.
  • FIG. 14A and 14B illustrate one synthetic scheme for preparing the trisaccharide GlcNAc ⁇ l ⁇ 3GalNAc ⁇ l ⁇ 6GalNAc ⁇ l ⁇ R, where R represents an aglycon moiety.
  • a “mucin” is a high molecular weight protein (often exceeding 1 x 10" in relative molecular mass) that is typically characterized by a high degree of O- linked glycosylation through a linkage involving N-acetyl- ⁇ -D-galactosamine and the hydroxyl group of either serine or threonine located in the protein backbone. Mucins comprise a major component of epithelial secretions.
  • the "core structure" of a mucin refers to the basic carbohydrate structure, not including peripheral substitutions, that is directly attached to the protein moiety of a mucin.
  • the core structure refers to the N-acetyl- galactosamine that is directly attached to the protein moiety and the monosaccharides directly attached to N-acetyl-galactosamine.
  • the term as used herein, however, can also include linear trisaccharides, wherein the N-acetyl-galactosamine attached to the protein is attached to a second sugar residue which is connected to yet a third sugar residue.
  • polypeptide refers to a polymer of amino acid residues.
  • the term also applies to amino acid polymers in which one or more amino acids are chemical analogues of a corresponding naturally-occurring amino acid.
  • Amino acids are referred to using standard single or three letter codes. (For example, in the structures listed below, Ser refers to the amino acid serine and Thr refers to the amino acid threonine).
  • isolated means an object species (e.g., an oligosaccharide alditol of the invention) is the predominant macromolecular species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.
  • an isolated, purified or substantially pure composition will comprise more than 80 to 90 percent of all macromolecular species present in a composition.
  • the object species is purified to essential homogeneity
  • composition consists essentially of a single macromolecular species.
  • antibody refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-te ⁇ ninus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.
  • Antibodies exist as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various pepridases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab which itself is a lighr chainjoined to VH-CHl by a disulfide bond.
  • the F(ab)' 2 can be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1 93), for a more detailed description of other antibody fragments).
  • antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab 1 f agments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • antibody as used herem also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
  • Antibodies also include single chain antibodies, such as single chain Fv (scFv) antibodies, in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
  • a single chain Fv (“scFv” or "scFv”) polypeptide is a covalently linked VH::VL heterodimer which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker. Huston, et al. Proc. Nat Acad. Sci. USA, 85:5879-5883 (1988).
  • Specific binding between an antibody or other binding agent and an antigen means that the dissociation constant for the interaction between the antibody and antigen is less than 10 '6 M.
  • Preferred antibody/ antigen complexes have a dissociation constant of less than about 10 " ⁇ M, and preferably 10 " ⁇ M to 10 " M or 10-10 M
  • epitope means an antigenic determinant capable of specific binding to an antibody.
  • epitope means an antigenic determinant capable of specific binding to an antibody.
  • naturally occurring as applied to an object refers to the fact that an object can be found in nature.
  • patient includes human and veterinary subjects.
  • Gal D-galactose
  • GalNAc N-acetyl-D-galactosamine (pyranose form)
  • GalfNAc N-acetyl-D-galactosamine (furanose form)
  • GlcNAc N-acetyl-D-glucosamine
  • GalNAc-ol the alditol formed when GalNAc is cleaved from the mucin protein moiety and reduced under alkaline borohydride conditions.
  • TheNAc-ol 2-acetamido-2-deoxy-L-threitol
  • 1-D and 2-D one dimensional and two dimensional, respectively
  • BSM bovine submaxillary mucin
  • DSS sodium 4,4-dimethyl-4-silapentane-l -sulfonate
  • EIMS electron-impact mass spectrometry
  • GC-EIMS gas chromatography-EIMS
  • GCMS gas chromatography-mass spectrometry
  • HMBC heteronuclear multiple-bond correlation spectroscopy
  • HMQC heteronuclear multiple quantum coherence
  • MAb monoclonal antibody
  • TAA tumor associated antigen
  • the present invention provides novel oligosaccharide alditols and oligosaccharide core structures that are derived from glycoproteins such as mucins.
  • oligosaccharides and alditols can be used as standards in the analysis of complex oligosaccharides and glycoproteins and in a variety of binding studies.
  • antigens that include the oligosaccharides of the invention and antibodies that specifically bind to the antigens.
  • These antigens and antibodies can be used in various types of pharmaceutical compositions that can be administered to treat various diseases such as cancer and diseases associated with tumors, particularly cancers and tumors whose cells include or express an oligosaccharide core structure of the invention.
  • the invention also provides therapeutic and prophylactic methods for treating such diseases.
  • the current invention provides antigens that include the newly identified oligosaccharide core structures and derivatives thereof. These oligosaccharides are novel oligosaccharides that are not included in either the
  • the oligosaccharides were released from the protein backbone of BSM by alkaline borohydride treatment and isolated as oligosaccharide-alditols. As described in more detail below (Example I), the released oligosaccharide-alditols were initially separated into an acidic fraction (i.e., a fraction containing negatively charged sialic acid residues) and a neutral fraction using ion exchange chromatography. The neutral faction was then chromatographed on semipreparative and analytical columns of Glycopack N to obtain isomerically pure fractions of various di- and tri-saccharide alditols (see Example I for additional details).
  • an acidic fraction i.e., a fraction containing negatively charged sialic acid residues
  • the neutral faction was then chromatographed on semipreparative and analytical columns of Glycopack N to obtain isomerically pure fractions of various di- and tri-saccharide alditols (see Example I
  • the structure of the oligosaccharide associated with each fraction was determined through a combination of various techniques, including: various 1- and 2- dimensional NMR techniques, several different mass spectroscopy methods, permefhylation analyses and the analysis of products formed after highly regioselective oxidation of GalNAc-ol with lead tetraacetate.
  • the oligosaccharide core structures corresponding to the newly isolated and purified oligosaccharide alditols, and the corresponding oligosaccharide structures as linked to the mucin protein moiety, are shown in the following table. These oligosaccharides and derivatives thereof can serve as carbohydrate antigens, particularly as the haptens of carbohydrate based antigens.
  • the present invention further provides antigens in which the oligosaccharides of the present invention are conjugated to a carrier molecule. More specifically, the antigens of the present invention have the following general structures:
  • the antigens of the invention also include derivatives of the foregoing structures, provided that it is possible to raise antibodies to the derivatives that are capable of specifically binding to the non-derivitized antigens.
  • Linkers A variety of species can be used as X; it is only necessary that the linker X be a molecule that can be attached to the oligosaccharide and the molecule represented by R at separate locations on the linker. Suitable linkers include, but are not limited to, polypeptides, oligosaccharides, amino acids, and a variety of commercially available cross-linking agents. Threonine and serine are useful as linkers as these are the natural linkers of the oligosaccharides to the mucin backbone.
  • the linker is a polypeptide
  • the polypeptide generally contains less than 25 residues and can be a polypeptide that is naturally occurring or is chemically synthesized.
  • the linker is a polysaccharide, typically it contains 20 residues or less, but the polysaccharide can be longer.
  • the particular sugar residues included in the chain are not critical.
  • a non-exhaustive list of potential sugar residues that can be incorporated into the sugar chain include, D-galactose, D-glucose, D-mannose, L- fucose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, N-acetyl-D-neuraminic acid, N-glycolyl-D-neuraminic acid.
  • the sugars can be either the ⁇ or the ⁇ anomer.
  • a number of commercially-available cross-linking reagents can also be used to link the oligosaccharide and the R group.
  • Such linkers typically include a functional group at each end (i.e., the linkers are bifunctional) that is capable of reacting with a functional group on the R group and the C-l hydroxyl group of the oligosaccharide.
  • the linkers may include the same functional group at each end (homobifunctional linkers) or different functional groups (heterobifunctional linkers) or different functional groups (heterobifunctional groups). Examples of functional groups on the linkers include, for example, amine, carboxyl, hydroxyl and thio groups.
  • R is a polypeptide
  • a linker is selected that can react with an amine or sulfhydryl group, for example.
  • R is an oligosaccharide
  • a linker is chosen that can react with a hydroxyl group on the oligosaccharide, for example.
  • R is a lipid, then a linker capable of reacting with an amide or ester is suitable.
  • homobifunctional linkers that can react with amine groups include, for example, disuccinimydl suberate (DSS), disuccinimydl tartarate (DST) and bis (sulfosuccinimydl) suberate (BS 3 ), all of the foregoing linkers being available from Pierce.
  • DSS disuccinimydl suberate
  • DST disuccinimydl tartarate
  • BS 3 bis (sulfosuccinimydl) suberate
  • linkers can be directly attached to the oligosaccharide or to a serine or threonine that is already attached to the oligosaccharide, or to an amino group on a carrier protein.
  • heterobifunctional cross-linking reagents can also be employed including, but not limited to, m-maleimidobenzoyl-N-hydroxy succinimide ester (MBS), sulfo-MBS, N- succinimydl (4-iodoacetyl) aminobenzoate (SIAB), sulfo-SIAB and succinimydl 4- (N-maleimido-methyl) cyclohexane-1 -carboxylate (SMCC).
  • MBS m-maleimidobenzoyl-N-hydroxy succinimide ester
  • SIAB N- succinimydl (4-iodoacetyl) aminobenzoate
  • SMCC succinimydl 4- (N-maleimido-methyl) cyclohexane-1 -carboxylate
  • R can include a variety of different carrier molecules; it is only necessary that R increase the immunogenicity of the oligosaccharide core structure.
  • R can include, for example, polypeptides, lipids and soluble polymers.
  • R is a polypeptide
  • a variety of polypeptides are suitable.
  • the polypeptide can be a naturally occurring or synthetic polypeptide. In general, the polypeptide is typically from 2 to 10 4 amino acids in length; in other instances from 20 to 10 amino acids long; and in still other instances from 20 to 10 2 amino acids long.
  • the polypeptide can include, but is not limited to, an animal serum albumin, an animal serum globulin, an animal thyroglobulin, an animal hemoglobulin or an animal hemocyanin.
  • the animal protein source can include, but is not limited to, equine, bovine, rabbit, mouse, human and ovine sources.
  • the polypeptide is one that can elicit the formation of Ig G antibodies in mammals.
  • Other specific polypeptides that are suitable include human serum albumin, bovine serum albumin (BSA), keyhole-limpet hemocyanin, tetanus toxin/toxoid, diphtheria toxin/toxoid, Pseudomonas aeruginosa exoprotein A.
  • the polypeptide can also be a lipopeptide.
  • Methods specifically designed to produce conjugates containing mucin antigens and lipopep tides are set forth, for example, by Toyokuni, T. et al., (Bioorg. Med. Chem., 11 :1119-1132, 1994).
  • R is a lipid
  • the lipids typically include a primary amino group. Examples of such lipids include, n-octadecylamine and n- hexadecylamine, as well as ceramides. Ceramides are useful because they are frequently attached to haptens in glycolipids.
  • the use of monophosphoryl lipid A also known as Ribi adjuvant
  • R Various soluble polymers can also be used as compounds for R.
  • Such polymers are readily soluble in aqueous solution and often are polymers containing lysine and/or glutamic acid.
  • R can include polylysine, polyglutamic acid, a lysine-glutamic acid copolymer and other related polymers and copolymers.
  • Tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P 3 CSS) has been used with other synthetic carbohydrate antigens and can also be used with the oligosaccharide antigens of the present invention (see Kjeldsen, et al., U.S. Patent 5,660,834).
  • the oligosaccharides and antigens of the invention include derivatives of the oligosaccharide structures: (a) Fuc ⁇ l ⁇ GalNAc, (b) GalNAc ⁇ 1 ⁇ 3 GalNAc, (c) GalNAc ⁇ 1 ⁇ 3 [GlcNAc ⁇ l ⁇ 6]GalNAc, (d) GlcNAc ⁇ l ⁇ 3GalNAc ⁇ l ⁇ 6GalNAc, (e) Fuc ⁇ l ⁇ 4GalNAc, (f)
  • the term derivative when used in reference to the oligosaccharides or antigens of the invention means structures that include additions, substitutions or deletions from one or more of the sugar moieties of the foregoing oligosaccharide structures, provided that antibodies formed (or capable of being formed) to such derivatives can specifically bind to the oligosaccharides of the invention that are naturally expressed on glycoproteins, i.e., the non-derivative structures. Tests for determining this are known in the art (see, for example, U.S.
  • Such derivatives can include, for example, substitutions at one or more of the hydroxyl groups on any of the monosaccharide units of the oligosaccharides.
  • substitutions involve relatively small moieties, such as an amino or halide.
  • a halide substitution is made, typically a fluorine atom replaces the hydroxyl group as such substitutions are relatively stable and such substitutions with other antigens have shown that antibodies prepared to such derivatives maintain binding specificity to the naturally occurring antigen.
  • Chloro substitutions are possible in some instances.
  • substitutions with somewhat larger groups are also possible in some instances including, for example, methoxy, acetoxy, phosphate, sulfate and urea groups.
  • the derivatives of the invention can also include oligosaccharides wherein the N-acetyl group is replaced with various groups including, but not limited to, for example, N-formyl or an N-fluoroacetate (e.g., a mono or difiuoroacetate).
  • derivatives in which one or more of the ring or glycosidic oxygens is replaced with a sulfur atom, a -NH- group, or a -CH 2 - group.
  • the derivatives may include various combinations of the foregoing alterations. As indicated above, alterations can also be made at a plurality of sites within the oligosaccharide. Guidance regarding how to make such derivatives is provided, for example, by Tsuchiya, T., Advances in Carbohydrate Chemistry and Biochemistry 48:91-277, 1990; and Horton, D., "The Amino Sugars, Chemistry and Biology of Compounds Containing Amino Sugars," in Monosaccharide Amino Sugars, vol. 1 A, (Jeanloz, R.W., and Balazs, E.A., eds.), Academic Press, New York, 1969, pp. 1-211.
  • the antigens of the present invention can be prepared in various ways, including for example, by chemical synthesis, or by chemically or enzymatically treating a mucin sample to expose the oligosaccharide core structures of the present mvention.
  • the oligosaccharides of the invention can be synthesized using methods that are well known in the art. Examples of such methods are described in greater detail in Example IV below.
  • the synthesized oligosaccharides can then be directly attached to a carrier molecule using any of a variety of known conjugation reagents. Methods for conjugating oligosaccharides to carrier molecules such as proteins are well known in the art. For example, conjugation of carbohydrates to a protein carrier is described by Longenecker et al. (Vaccine Res., 2: 151-162, 1993).
  • a linker molecule can be used to connect the oligosaccharide to a carrier molecule.
  • the linker includes a functional group at each end that is capable of reacting with a functional group on the R group and the C-l hydroxyl group of the oligosaccharide.
  • the linker can be joined to the R group and the oligosaccharide using known reactions appropriate for the functional groups, located on the oligosaccharide, R group and linker.
  • serine or threonine are used as a linker or as a portion of a larger linker molecule. Coupling of the serine hydroxyl group to C-l of the reducing end of an oligosaccharide has been described previously and can be performed such that the ⁇ -linkage at C-l of the first residue is maintained (Paulsen and Holek, Carb. Res., 109:89-107, 1982; and Grundler and Schmidt, Lieb.
  • Coupling of serine or threonine to the oligosaccharides of the invention can be accomplished in a similar manner.
  • the resulting compound can then be conjugated to a carrier molecule, or an additional linker can be added to the serine or threonine before conjugating to a carrier molecule.
  • the serine or threonine attached to GalNAc can be joined to a carrier molecule such as a protein using standard chemical methods.
  • such methods involve blocking the amine group of serine or threonine with standard blocking reagents such as t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 9- fluorenylmethylcarbonyl (Fmoc) or allyloxycarbonyl (Aloe) and then coupling the carboxyl group to an amine group in the carrier molecule using standard coupling reagents such as l-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), benzotriazole-1 -yl-oxy-tris-(dimethylamino)- phosphonium hexafiuorophosphate (BOP), benzotriazole-1 -yl-oxy-tris-pyrrolidino- phosphonium hexafiuorophosphate (ByBOP), 2-ethoxy-l
  • the antigens described herein can also be formed by selectively exposing the oligosaccharide core structures as attached to various mucins.
  • the antigens described herein can also be directly isolated from bovine submaxillary mucin (BSM) or desialytated BSM (A-BSM).
  • BSM bovine submaxillary mucin
  • A-BSM desialytated BSM
  • BSM can be purchased from many chemical supply companies including , for example, Sigma. It can also be isolated from bovine submaxiUary glands according to the procedure described by Tettamanti and Pigman (Arch. Biochem. Biophys., 124:41- 50, 1968).
  • Some of the oligosaccharides have also been isolated from other sources, including humans. For example, GalNAc ⁇ 1 ⁇ 3GalNAc ⁇ 1 ⁇ O-Ser/Thr and
  • GalNAc ⁇ l ⁇ 3[GlcNAc ⁇ l ⁇ 6]GalNAc ⁇ l ⁇ 0-Ser or Thr have been shown by the present inventor to be present in human colonic mucin (unpublished observations).
  • specific sugar residues can be selectively removed from the non-reducing terminal end of the oligosaccharides attached to the mucin protein backbone.
  • Influenza virus sialidase for instance, can be used to eliminate terminally located ⁇ 2 ⁇ 3 sialyl residues, and Clostridium perfringense sialidase can be used to eliminate all sialic acid residues.
  • Additional enzymatic modification can be accomplished, for example, using ⁇ -galactosidase, ⁇ -fucosidase, and N-acetylhexosaminidase.
  • ⁇ -galactosidase ⁇ -galactosidase
  • ⁇ -fucosidase ⁇ -fucosidase
  • N-acetylhexosaminidase N-acetylhexosaminidase.
  • Chemical methods can also be used to expose the core structures on mucins (e.g., BSM or A-BSM).
  • mucins e.g., BSM or A-BSM.
  • One example includes the "Smith Degradation” which involves periodate oxidation followed by reduction with sodium borohydride and treatment with weak acid (Spiro, G. Methods Enzymol 28: 3-43, 1972). This method eliminates non-reducing terminals of carbohydrate residues except sialic acid, which can be cleaved using sialidase as described above.
  • the present invention also provides various compositions for the preparation of antibodies and for the treatment of various diseases such as cancers and diseases associated with tumor formation, particularly those associated with the alteration of mucin-type glycoproteins (i.e., cells expressing an oligosaccharide core structure of the invention).
  • various compositions for example, can be used in the treatment of various gastrointestinal, respiratory and genitourinary cancers and tumors.
  • Illustrative examples include, but are not limited to, colon, breast, stomach, prostrate and lung cancers and tumors.
  • the antibodies of the present invention specifically bind to the antigens of the present invention and a method for producing such antibodies.
  • the antibodies bind to the oligosaccharide structures provided in the present invention.
  • These antibodies can be used, for example, to screen samples in order to identify those samples that express the antigens of the present invention and in various pharmaceutical compositions.
  • the antibodies can be either polyclonal or monoclonal antibodies.
  • Antibodies of the present invention can be prepared using methods that are well known in the art. General methods for antibody production are set forth, for example, by Kohler, G. et al. (Nature, 256: 495-496, 1975), Clausen, H. et al. (Biochem., 24:6190-6194, 1985); Harlow and Lane, (Antibodies: A Laboratory
  • the method of producing antibodies involves immunizing a host with an immunogenic composition that includes an antigen of the present invention. More specifically, the immunogenic composition typically includes an antigen of the invention and a suitable diluent or excipient.
  • An adjuvant including, for example, incomplete or complete Freund's adjuvant, Ribi adjuvant, or other adjuvant capable of stimulating T-cells can also be included in the immunogenic composition.
  • the antibodies are typically produced by diluting a predetermined amount of antigen with physiological saline or other diluent to a suitable concentration capable of generating antibodies, such a concentration is defined as a "immunogenically effective dose.”
  • Certain methods for producing antibodies include: (a) creating hybridomas by fusing splenocytes from the immunogenized host with myeloma cells, (b) culturing the hybridoma cells on selective media to identify those cells in which the splenocytes and the myeloma cells have fused, (c) selecting from the hybridoma cells identified in step (b) those which secrete antibodies specific for an antigen of the present invention, (d) cloning the hybridoma cells selected in step (c), (e) culturing the cloned hybridoma cells and (f) recovering the antibody from the cultured hybridoma cells.
  • the present invention further includes hybridomas capable of producing the monoclonal antibodies of the invention.
  • hybridomas can be prepared according to methods well known in the art such as described by Kohler, G. et al. (Nature, 256: 495-496, 1975) and Young et al. (J Exp. Med, 150: 1008-1019,
  • Vaccines are one example of the immunogenic compositions of the invention.
  • the vaccines of the invention are capable of inducing a patient's immune system to mount a response against target cells or pathogens that bear the oligosaccharides or antigens of the invention.
  • These vaccines can be used, for example, for active preventative specific immunotherapy of cancers or tumors wherein the tumor or cancer cells include or express the oligosaccharide antigens of the invention.
  • a vaccine of the invention generally comprises an antigen of the invention and a pharmaceutically acceptable excipient (carrier).
  • the antigen can be prepared by purification from natural sources (e.g., BSM), by enzymatic methods, chemical synthesis, or combinations thereof.
  • BSM natural sources
  • excipients or carriers can be utilized in the vaccines including, for example, adjuvants and compounds that stimulate T-cells, such as Freund's adjuvant, Ribi adjuvant (Ribi, et al., Clin. Immunol. Newsletter, 6:33-36, 1985) and BCG.
  • Other excipients include Montanide ISA51 and Alum adjuvants (previously approved for use in primates and humans, respectively).
  • Suitable adjuvants include aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), and N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L- alanine-2-(r-2'-dipalmitoyl-sn-glycero-3-hydroxy-phosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE).
  • the effectiveness of an adjuvant can be determined by measuring the amount of cross-reactive antibodies directed against the immunogenic antigen.
  • cyclophosphamide an inhibitor of suppressor T-cell response, is included with the vaccine.
  • the vaccines of the invention can be prepared using methods that are well known in the art and closely parallel methods for preparing antibodies generally.
  • the invention further provides methods for inducing a therapeutic or protective immune response in a patient and methods for treating diseases such as those described above. Such methods include administering to a patient a therapeutically effective amount of a vaccine of the invention.
  • a therapeutically effective amount of the vaccine is an amount sufficient to elicit a therapeutic or protective immune response, such as inducing the formation of antibodies and/or other cellular immune responses (e.g., the induction of helper T cells, cytotoxic killer T-cells, anomalous killer cells (AK cells) and/or antibody-dependent cytotoxic cells).
  • a therapeutic or protective immune response such as inducing the formation of antibodies and/or other cellular immune responses (e.g., the induction of helper T cells, cytotoxic killer T-cells, anomalous killer cells (AK cells) and/or antibody-dependent cytotoxic cells).
  • Doses, methods of administrating, and suitable pharmaceutical carriers can be determined readily by the skilled artisan. For example, appropriate doses can be extrapolated from dose-response studies in animals, including non-human primates. In mice, 20-100 g of BSM or A-BSM in Ribi adjuvant was able to induce high antibody and T-cell responses.
  • mice 75-100 g of BSM or A-BSM was required for optimal protection against, for example, a tumor challenge.
  • U.S. Patents 5,747,048 and 5,660,834 to Kjeldsen et al. provide further guidance regarding appropriate doses for mucin-type antigens.
  • An appropriate immunization schedule can be determined by a skilled artisan but generally depends upon the susceptibility of the host or patient to immunization with the antigens of the invention and is typically continued until sufficient antibody is detectable in whole serum.
  • the vaccines can be administered in a variety of different ways including, for example, by oral, intranasal, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal and transdermal methods. It has been found in some instances that although similar immunologic responses are generated by either intraperitoneal or subcutaneous administration that the latter form of administration is capable of inducing higher T-cell responses.
  • compositions containing antibodies of the invention comprise an antibody of the invention and a pharmaceutically acceptable excipient. These pharmaceutical compositions are also effective for treating the diseases set forth above.
  • variable regions from non-human sources can be cloned and expressed as human antibodies using standard immunological techniques. Such "humanized” antibodies can then be administered to human patients.
  • compositions containing antibodies is selected so as not to affect the biological activity of the combination and include, for example, distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, and the like.
  • the compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, detergents and the like.
  • compositions containing the antibodies can be administered for prophylactic and/or therapeutic treatments.
  • the antibody in the pharmaceutical composition typically is present in a therapeutic amount, which is an amount sufficient to remedy a disease state or symptoms or otherwise prevent, hinder, retard, or reverse the progression of disease or any other undesirable symptoms in any way whatsoever.
  • concentration of the antibody in the pharmaceutical composition can vary widely, i.e., from less than about 0.1% by weight, usually being at least about 1 % by weight, to as much as 20% by weight or more.
  • compositions are administered to a patient already suffering from a disease, as just described, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.
  • an appropriate dosage of the antibody-containing compositions can be readily determined according to any one of several well- established protocols, such as extrapolation from animal studies. What constitutes an effective dose also depends on the nature and severity of the disease or condition, and on the general state of the patient's health.
  • compositions containing antibodies of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease or infection. Such an amount is defined to be a "prophylactically effective" amount or dose. In this use, the precise amounts again depends on the patient's state of health and weight, but can be readily determined by the skilled artisan.
  • the antibody-containing compositions can be administered in a variety of different ways. Examples include administering the antibody-containing composition via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.
  • the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • the active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
  • inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
  • additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink.
  • Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • the composition typically contains 10-95% of the active ingredient, that is, an antibody of the invention, and more typically 25-75% by weight. In some instances, the antibody-containing compositions are formulated in an enteric-coated or otherwise protected form.
  • the formulation can be mixed or simply coadministered with a protectant, such as a liquid mixture of medium chain triglycerides, or the formulation can be filled into enteric capsules (e.g., of soft or hard gelatin, which are themselves optionally additionally enteric coated).
  • enteric capsules e.g., of soft or hard gelatin, which are themselves optionally additionally enteric coated.
  • solid formulations comprising the composition can be coated with enteric materials to form tablets.
  • the thickness of enteric coating on tablets or capsules can vary. Typical thickness range from 0.5 to 4 microns in thickness.
  • the enteric coating can comprise any of the enteric materials conventionally utilized in orally administrable pharmaceutical formulations. Suitable enteric coating materials are known, for example, from Remington's Pharmaceutical
  • lipid-associated structures e.g., liposomes, or other lipidic complexes
  • the complex containing the composition may subsequently be targeted to specific target cells by the incorporation of appropriate targeting molecules (e.g., specific antibodies or receptors).
  • compositions prepared for intravenous administration typically contain 100 to 500 ml of sterile 0.9%> NaCl or 5% glucose optionally supplemented with a
  • a typical pharmaceutical composition for intramuscular injection contains, for example, 1 ml of sterile buffered water and 1 to 10 mg of an antibody of the invention.
  • Methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for example, Remington's
  • compositions are to be used in vivo
  • the components used to formulate the pharmaceutical compositions of the present invention are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade).
  • compositions intended for in vivo use are usually sterile.
  • the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process.
  • Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.
  • Methods of detecting the presence of an antigen provided by the invention in a biological test sample are also provided.
  • the method typically comprises the steps of: (a) contacting the biological test sample with the antibodies of the present invention under conditions suitable for the formation of an antigen- antibody complex, and (b) detecting the formation of the antigen-antibody complex.
  • a non-exhaustive list of biological test samples include: blood, serum, urine, tissue slices and tissue homogenates.
  • Detection of the formation of the antigen-antibody complex can be accomplished using any of the techniques which are known in the art, such as radioimmunoassay, enzyme-linked immunosorbent assay, sandwich assays and immunoprecipitation.
  • the oligosaccharides and oligosaccharide alditols of the present invention can be used as standards in the structural analysis of complex oligosaccharide structures. More specifically, the oligosaccharides and alditols of the invention can be used as standards in a variety of chromatographic analyses. For example, the structures of unknown oligosaccharides or oligosaccharide-alditols or components thereof can be estimated or determined by comparing the elution positions of the unknown compounds with the chromatograms of the oligosaccharides or oligosaccharide-alditols provided in the present invention.
  • oligosaccharides and alditols of the invention can be used as standards in a variety of chromatographic techniques including, for example, thin- layer chromatography, gel-permeation chromatography, anion-exchange high- performance liquid chromatography, high-pH anion-exchange chromatography, either normal- or reversed-phase high-performance liquid chromatography (HPLC), gel electrophoresis, and capillary electrophoresis.
  • chromatographic techniques including, for example, thin- layer chromatography, gel-permeation chromatography, anion-exchange high- performance liquid chromatography, high-pH anion-exchange chromatography, either normal- or reversed-phase high-performance liquid chromatography (HPLC), gel electrophoresis, and capillary electrophoresis.
  • Kobata in "Methods in Enzymology” (Lennarz, W.J. and Hart, G.W., ed.), Vol. 230, p.200
  • the oligosaccharides and oligosaccharide-alditols of the invention can be labeled with a variety of labels, including for example, radioisotopes (e.g., 3 H or l 4 C), fluorophores, chromophores, mass labels, and electron dense reagents.
  • suitable fluorophores include 2-aminopyridine (Hase, S. in "Methods in Enzymology" (Lennarz, W.J. and Hart, G.W., ed.), Vol. 230, p.225-237. Academic Press, San Diego, CA, 1994), 2-amino benzamide (Bigge, J.C.
  • the oligosaccharides of the invention can also be used in testing the binding specificities of a variety of carbohydrate-binding proteins by determining the binding of the oligosaccharides in solution with a carbohydrate-binding protein. Inhibition experiments can be conducted in the presence of known carbohydrate- binding proteins to more fully analyze such binding interactions. In a related fashion, the oligosaccharides of the invention can be used in studies on binding interactions between cancer cells suspected of binding to specific carbohydrate ligands and various test oligosaccharide ligands. The oligosaccharides of the invention can serve as potential inhibitors in such studies.
  • the oligosaccharides of the invention can also be used in enzymatic studies, particularly with the glycosyltransferases, a complex group of enzymes that recognize specific oligosaccharide substrates and produce specific glycosidic linkages in products (See, e.g., Schachter, H. and Brockhausen, I., "The Biosynthesis of Serine (Threonine)-N-acetylgalactosamine-linked carbohydrate moieties" in:
  • Glycoconjugates Composition, Structure and Function (Allen, J.H., and Kisailus, E.C., Eds.) pp 263-332).
  • the oligosaccharides of the invention can be used as substrates or inhibitors for these enzymes.
  • BSM prepared essentially according to the method of Tettamanti and Pigman (Arch. Biochem. Biophys., 124:41-50, 1968) was purchased from Sigma (Type I-S). Mucin (10 g) was treated with a 500 mL solution of 0.05 M NaOH, 1.0 M NaBH for 16 h at 45 °C, as described by Carlson, D.M. (J. Biol. Chem., 241 : 2984-2986, 1966). The solution was brought to pH 5 by the addition of 4 M acetic acid, passed through a column (1500 mL) of Dowex AG50W (H + form), and the column washed with 6 L water. The eluate was lyophilized.
  • sialic acid-detectable material behaved as a neutral compound (i.e. , eluting through the column, as detected by a periodate-resorcinol assay (Jourdian, G.W. , et al. , J. Biol. Chem., 246: 430-435, 1971)). This material did not bind to the column in a second pass-through.
  • the sample after methanol evaporations, was dissolved in 20 mL water and taken to pH 11 with 4 mM NaOH. It was left overnight at room temperature to delactonize, diluted to 600 mL and passed through 2 columns in tandem: Dowex AG1 (OAc ⁇ form, 220 mL), then Dowex AG50W (H + form, 30 mL). The columns were washed with 1 L water to collect the neutral fraction free of Na + .
  • Dowex AG1 OAc ⁇ form, 220 mL
  • Dowex AG50W H + form, 30 mL
  • Sialylated oligosaccharide-alditols were eluted from the Dowex AG1 column directly, bypassing the AG50W column, by washing with 1 L freshly -prepared 0.5 M N-methylmorpholinium acetate, pH 7.0. Fractions (25 mL) were collected and aliquots (15 ⁇ L) were analyzed for sialic acid using the method described by Jourdian, G.W. , et al. (J. Biol. Chem., 246: 430-
  • HPLC HPLC was carried out on a Waters system equipped with a multiple wavelength ultraviolet detector used at 200 nm. Columns used were polymeric, including a Waters
  • Glycopak N (semipreparative, 2.15 x 60 cm, and analytical, 7.8 x 300 mm), and a
  • Shodex DC-613 column (6 x 150 mm, Na + form).
  • the DC-613 column was exchanged to the Li + or Cs + forms by washing with over 50 column volumes of 1.0 M lithium or cesium acetate, followed by at least 50 column volumes of water before bringing the column, in gradient fashion, to about 80/20 C ⁇ CN/water. All HPLC runs were performed isocratically. Concentration of samples from solvents was by rotary evaporation at 35 °C.
  • the Dowex AG50W used in all experiments refers to X8, 100-200 mesh, and Dowex AG1 refers to X8, 100-200 mesh (Bio-Rad). Dowex columns were washed with 20-30 column volumes of water immediately before use.
  • the ratios of CH3CN/H2O listed herein can be modified for any of the columns, depending on the degree of resolution required, but should not exceed 95/5, which will permanently modify column performance. Strict adherence to maximum flow rates specified by manufacturers and changing solvent ratios gradually is important.
  • the released oligosaccharide-alditols were initially separated into negatively charged and neutral fractions. The fraction containing the neutral oligosaccharide-alditols were taken up in 10 mL CH3CN/H2O, 74/26, injected in 0.5 mL batches onto the semipreparative column of Glycopak N (Bendiak, B. , et al. , Anal.
  • FIG. 1A Seven pools were collected (A-G). The first two pools (A and B) integrated for 76% of the total ultraviolet absorbance and contained the smallest structures (monosaccharide-alditols to fucosylated tetrasaccharide-alditols).
  • Fractions A and B were separately concentrated, taken up in 2 mL 80/20 CH3CN/H2O, and chromatographed on the analytical column of Glycopak N in batches. Fraction A was eluted isocratically with CH3CN/H2O, 85/15, at 1.0 mL/min; fraction B was chromatographed under similar conditions but eluted at a CH3CN/H2O ratio of 82/18. These conditions optimized the separation of the mono and disaccharide-alditols (FIG. IB) and trisaccharide-alditols (FIG. 2A).
  • the mono and disaccharide-alditols were resolved into at least 8 distinct fractions, labeled Al to A8, with fraction A3 itself containing at least 5 species, labeled A3(l) to A3(5) (see FIG. IB; the plot insert for fraction A3 is shown at 25-fold intensity).
  • the trisaccharide-alditols were resolved into 7 species, labeled Bl to B7 (see FIG. 2A).
  • fractions B2-B7 required additional purification.
  • the ratio of CH 3 CN/H 2 O during elution was 80/20 for fraction B2 (FIG. 2B), 78/22 for fraction B3 (FIG. 2C) and 76/24 for fractions B4, B5, B6 and B7 (FIGS. 2D-2G, respectively).
  • Fraction B3 separated into 7 species, B3a - B3g (FIG. 2C); fraction B5 resolved into 6 species, B5a- B5f (FIG. 2E). Fractions were again examined by NMR to determine purity.
  • fraction A6d3 is one of the oligosaccharide core structures (alditol form) of the current invention (GalNAc ⁇ l-3GalNAc-ol).
  • Fraction A3(3)e resolved into two fractions, A3(3)el and A3(3)e2 (FIG. 1J).
  • Fraction A3(3)el was subsequently identified as Fuc ⁇ l-4GalNAc-ol and fraction A3(3)e2 characterized as GalfNAc ⁇ l-6GalNAc-ol.
  • fraction B3g was resolved into 3 fractions, B3gl-B3g3 (FIG. 2K).
  • the major species was fraction B3g3, which is one of the oligosaccharide core structures (alditol form) of the present invention (GalNAc ⁇ l-3[GlcNAc ⁇ l- 6]GalNAc-ol).
  • fraction B5e separated into 3 fractions, B5el-B5e3 (FIG. 2L).
  • the predominant fraction, B5e3 is another one of the oligosaccharide core structures (alditol form) of the present invention (GlcNAc ⁇ l-3GalNAc ⁇ l -6GalNAc- ol). NMR was again used to determine the purity of the resulting fractions.
  • A3(3)g was resolved into 3 fractions, A3(3)gl-A3(3)g3 (FIG. II).
  • the A3(3)g3 fraction was the predominant species and is another of the novel oligosaccharide core structures (alditol form) provided by the current invention (Fuc ⁇ l-6GalNAc-ol).
  • Fraction A3(3)gl was shown by NM R to be identical to fraction A3(3)f2 (i.e. , Fuc ⁇ l-6GalNAc-ol).
  • NMR spectra were acquired either on a Bruker AM-500 or Varian Inova 500 or 600 MHz spectrometers. Some of the spectra were recorded at 600 MHz using a Nalorac Z- Spec MIDTG-600 3 mm probe. Samples were deuterium exchanged by concentrating from D2O prior to dissolving in this solvent. For the Nalorac 3 mm probe, samples were taken up in 120 ⁇ L and transferred to a Shigemi 3 mm microsample tube (matched to the magnetic susceptibility of D2O) using a flexible
  • Teflon tubing (Wilmad) attached to a Hamilton gas-tight Luer-lock syringe.
  • Time domain spectra were acquired with a spectral width of 2200 Hz collected over 4 K data points in t2 and 1 K experiments in t ⁇ . Thirty-two transients were accumulated per ti with a preparatory delay of 1.5s, with suppression of residual HOD by a presaturation pulse during the preparatory delay.
  • TOCSY spectra (Braunschweiler, L. and Ernst, R.R. , J. Magn. Reson., 53: 521-528, 1983) were recorded using the mLEV-17 pulse scheme (Bax,
  • TPPI rotating frame Overhauser effect spectroscopy experiments (ROESY) (see Bax, A. and Davis, D.G., J. Magn. Reson, . 63: 207-213, 1985) were performed with the spin- lock applied for 200 ⁇ s. Data processing was performed essentially as described for other TPPI experiments.
  • the heteronuclear multiple quantum coherence (HMQC) experiment (Bax, A. and Subramanian, S., J. Magn. Reson., 67: 565-567, 1986) was acquired with spectral widths of 2000 and 14000 Hz in E2 an d El, respectively, as 1024 x (512 x 2) hypercomplex files using
  • TPPI-States phase cycling (Marion, D. , et al. , J. Magn. Reson., 85: 393-399, 1989). Sixteen transients were accumulated per t
  • Oxidations with Pb(OAc)4 were carried out in a mixture of acetic acid/acetone/water, 3/2/2 at -30°C (Bendiak, B., et al., J. Org. Chem., 60: 8245- 8256, 1995).
  • a sample containing up to 10 ⁇ mol oligosaccharide-alditol in 0.6 mL water was transferred to a 20 mL screw-cap tube.
  • Acetone (0.6 mL) and glacial acetic acid (0.9 mL) were added, the sample was capped and taken to -30 °C in a methanol bath.
  • Lead tetraacetate (44.3 mg, 0.1 mmol, Fluka, free of brown lead oxide, weighed and transferred as quickly as possible), was added to the sample at - 30 °C, which was capped, and kept at -30 °C for 90 min with agitation by swirling the tube in the bath at about 5 min intervals . The tube was kept in the bath at all times.
  • the eluate was concentrated to a volume of about 200-300 ⁇ L by rotary evaporation. Care was taken not to evaporate exhaustively to a stiff syrup, or by freeze drying, because it was observed that acetaldehyde, present in the solution after stopping the reaction, may react with vicinal hydroxyls of sugars to give minor (about 10-15%) acetal products, but only when exhaustive removal of water occurs. Presumably, residual acetic acid present in these stiff syrups catalyzes this side- reaction, which can be completely avoided by not concentrating to dryness. For reduction, the sample was taken up in another 5.0 mL water, and again concentrated to about 200-300 ⁇ L to remove acetic acid virtually completely. It was taken up in 1.0 mL water and reduced with 1.0 mmole NaBH4 for 24 h.
  • the core structures of the present invention are not described in either the Carbbank database (http://www.ccrc.uga.edu) or the Sugabase database (the online database of NMR data of oligosaccharides, http://www.boc.chem.ruu.nl/). Structures were established by 1- and 2-D NMR experiments, ESMS and MSMS, permethylation analyses, highly regioselective oxidation of GalNAc-ol with Pb(OAc) 4 at -30 °C (Bendiak, B., J Org.
  • the H-NMR spectrum of this fraction (600 MHZ, 300 °K) is shown in FIG. 3A, and complete assignments are listed in Table I.
  • Electrospray MS in the presence of 75 ⁇ M ⁇ aCl gave a sodium adduct of m/z 392 (M + ⁇ a) + , which gave a fragmentation pattern (MSMS) characteristic of a deoxyHex-Hex ⁇ Ac-ol (FIG. 4A, Table II).
  • Electrospray MS gave a sodium adduct of m/z 231 (M + ⁇ a) + , (Table II), consistent with the structure, but attempting to fragment by MSMS resulted in stripping the sodium ion from the molecule, rather than fragmentation.
  • the second product isolated was 2-acetamido-
  • D-galactitol which corresponds to the core structure Fuc ⁇ l ⁇ 6Gal ⁇ Ac. As connected to the mucin protein moiety, the structure is Fuc ⁇ l ⁇ 6GalNAc ⁇ l ⁇ O-Ser or Thr.
  • the isolated oligosaccharide-alditol has the structure ⁇ -D-Gal ⁇ Ac-(l-3)-D-Gal ⁇ Ac-ol, which corresponds to a new core mucin structure of GalNAc ⁇ l ⁇ 3GalNAc.
  • the structure is GalNAc ⁇ l ⁇ 3GalNAc ⁇ l ⁇ O-Ser or Thr.
  • a 1-D 'H-NMR spectrum (600 MHZ, 300 °K, FIG. 6A) revealed 3 N-acetyl methyl signals (Table V, second column; not shown in FIG. 6A) and other characteristic signals indicative of two ⁇ -Hex ⁇ Ac sugars and Gal ⁇ Ac-ol.
  • the Gal ⁇ Ac-ol H-2 and H-5 signals nearly overlapped, the H-5 being slightly upfield.
  • GalNAc-ol H-5 observed by H-NMR, it was suspected that a GalNAc-ol substituted at the 3 and 6 positions was possible.
  • GalNAc-ol C-l, 63.2; C-2, 54.1; C-3, 79.5; C-4, 71.8; C-5, 70.2; C-6, 73.4.
  • a 2-D HMBC experiment showed interglycosidic through-bond 3 J CI! connectivities. These included ⁇ -GalNAc H-l to GalNAc-ol C-3, ⁇ -GalNAc C-l to GalNAc-ol H-3, ⁇ -GlcNAc H-l to GalNAc-ol C-6, and ⁇ -GlcNAc C-l to GalNAc-ol H-6 and H-6' . All these data are concordant with the structure GalNAc ⁇ 1 ⁇ 3 [GlcNAc ⁇ l ⁇ 6]GalNAc-ol. The corresponding core structure is
  • GalNAc ⁇ 1 ⁇ 3 [GlcNAc ⁇ l ⁇ 6]GalNAc which is yet another novel oligosaccharide core structure. As connected to the protein moiety, the structure is GalNAc ⁇ l ⁇ 3[GlcNAc ⁇ l ⁇ 6]GalNAc ⁇ l ⁇ O-Ser or Thr. D. Fraction B5e3 (GlcNAc ⁇ 1 ⁇ 3 GalN Ac ⁇ l ⁇ 6GalNAc-ol
  • a 2-D ROESY spectrum showed a strong interresidue dipolar correlation between the ⁇ -Glc ⁇ Ac H-l and ⁇ -Gal ⁇ Ac H-3, consistent with a 1-3 linkage between these residues. Also seen were interresidue dipolar correlations between ⁇ -Gal ⁇ Ac H-l and Gal ⁇ Ac-ol H-6 (weak) and H-6' (strong), consistent with a 1-6 linkage between these sugars.
  • FIG. 4D (FIG. 4D)) indicative of a C-C splitting of the Gal ⁇ Ac-ol between C-4 and C-5, where the Gal ⁇ Ac-ol is 6-substituted.
  • PMAA's that were generated (Table VI) demonstrated a 6-substituted Gal ⁇ Ac- ol, a 3-substituted GalNAc, and a terminal GlcNAc.
  • the data support the structure ⁇ -D-Glc ⁇ Ac-(l-3)- ⁇ -D-Gal ⁇ Ac-(l-6)-
  • D-GalNAc-ol corresponds to the novel oligosaccharide structure GlcNAc ⁇ l ⁇ 3GalNAc ⁇ l ⁇ 6GalNAc, that has the structure
  • This structure is particularly interesting as it indicates that elongation of a GalNAc ⁇ l- ⁇ GalNAc branch is possible.
  • the data indicate that the isolated oligosaccharide alditol is 2-acetamido-2-deoxy-4-0-( ⁇ -L-fucopyranosyl)-D-galactitol, that corresponds to the novel oligosaccharide core structure Fuc ⁇ l ⁇ 4GalNAc.
  • the structure is Fuc ⁇ l ⁇ 4GalNAc ⁇ l ⁇ O-Ser or Thr.
  • H-NMR spectra of this fraction (600 MHZ, 300 °K) are shown in Figure 7B, and complete assignments in Table I.
  • the molecule contained two N-acetyl groups, and electrospray MS in the presence of 50 ⁇ M ⁇ aCl gave a sodium adduct of m/z 449 (M + ⁇ a) + , that gave a fragmentation pattern (MSMS, m/z 449 selected; see FIG. 4G) indicative of a HexNAc-HexNAc-ol (Table II). From the 1-D and 2-D gradient COSY experiments, all J-couplings were determined and indicated a structure that did not have the usual pyranosyl ring form.
  • the coupling patterns were consistent with a 2-acetamido-2-deoxy- ⁇ -D-galactofuranosyl residue linked to a GalNAc-ol.
  • the data demonstrate that the isolated alditol is 2-acetamido-2-deoxy-6-0-(2-acetamido-2-deoxy- ⁇ -D-galactofuranosyl)-D- galactitol.
  • This alditol corresponds to the oligosaccharide core structure, Gal/NAc ⁇ l ⁇ 6 GalNAc.
  • the structure of this core structure as attached to the mucin protein backbone is Gal/NAc ⁇ l ⁇ 6GalNAc ⁇ l ⁇ O-Ser or Thr.
  • the data show that the isolated alditol is 2-acetamido-2-deoxy-4-O-( ⁇ -L- fucopyranosyl)-D-galactitol; this alditol corresponds to the oligosaccharide core structure of Fuc ⁇ l ⁇ 4GalNAc. As attached to the mucin protein backbone the structure is Fuc ⁇ l ⁇ 4GalNAc ⁇ l - ⁇ O-Ser or Thr.
  • Electrospray MS in the presence of 50 ⁇ M ⁇ aCl gave a sodium adduct of m/z 392 (M + ⁇ a) + that gave a fragmentation pattern (MSMS, m/z 392 selected for; see FIG. 4H) characteristic of a deoxyHex-HexNAc-ol (Table II).
  • the oligosaccharide core structure corresponding to this alditol is Fuc ⁇ l ⁇ GalNAc; when this core structure is connected to the mucin protein backbone, the structure is Fuc ⁇ l ⁇ GalNAc ⁇ l ⁇ O- Ser or Thr.
  • the method of preparing the carbohydrate antigens of the present invention typically involves: 1) attaching an optional linker such as serine or threonine to the anomeric carbon of the core ⁇ GalNAc residue, 2) linking additional sugar residues to the core ⁇ GalNAc to form an oligosaccharide of the present invention, 3) attaching an additional optional linker to act as a spacer and 4) conjugating the oligosaccharide, with or without linkers, to a carrier molecule.
  • the carrier molecule, R is chosen for its ability to elicit an immunogenic response when the antigen is injected into a host.
  • oligosaccharide can be attached to certain matrix molecules to prepare multivalent complexes that can then be attached to the carrier molecule.
  • the oligosaccharide can be reacted with a matrix molecule to form an antigenic cluster which in turn can be used to construct multivalent systems that are conjugated to a carrier molecule that can assist in eliciting an immune response.
  • the serine or threonine as attached to the GalNAc can be appropriately blocked at the amino group using temporary blocking groups such as 9-fluorenylmethylcarbonyl (Fmoc), t-butoxycarbonyl (Boc), allyloxycarbonyl (Aloe) or benzyloxycarbonyl (Cbz) groups.
  • temporary blocking groups such as 9-fluorenylmethylcarbonyl (Fmoc), t-butoxycarbonyl (Boc), allyloxycarbonyl (Aloe) or benzyloxycarbonyl (Cbz) groups.
  • Activation of the carboxyl group of the serine or threonine, to prepare a protein or peptide linkage can be performed using agents such as dicyclohexylcarbodiimide (DCC), benzotriazole-1 -yl-oxy-tris-(dimethylamino)- phosphonium hexafiuorophosphate (BOP), benzotriazole-1 -yl-oxy-tris-pyrrolidino- phosphonium hexafiuorophosphate (ByBOP), l-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) or 2-ethoxy-l-ethyoxycarbonyl-l,2-dihydroquinoline (EEDQ) (Le Nguyen, D., et al.
  • DCC dicyclohexylcarbodiimide
  • BOP benzotriazole-1 -yl-oxy-tris-(
  • Substitution of the core ⁇ -GalNAc residue at hydroxyl groups at both the 3 or 6 positions, or at the 3 or 6 positions independently, can be carried out using appropriate blocking strategies to permit glycosidation reactions to specifically occur independently at each hydroxyl group.
  • the 4 and 6 positions of an ⁇ -GalNAc residue can be simultaneously blocked as benzylidene or p-methoxybenzylidene acetals, FIG. 10, structure 2 (Abbas, S.A. and Matta, K.L., Carbohydr. Res., 132:137-141, 1984; Piskorz, C.F., et al., Carbohydr. Res., 126:115-124, 1984; Jain, R.K., et al, Carbohydr. Res., 275:231-243, 1995;
  • This reaction is well-known in the art to preferentially give a ⁇ -linked product, 3, both with 2-amino-2-deoxygalactose and 2-amino-2-deoxy-glucose having appropriate hydroxyl protection as glycosyl donors (Hindsgaul, V., et al., Can. J. Chem., 65:1645- 1652, 1987; Abbas, S.A., et al., Carbohydr. Res. 112:201-211, 1983; Abbas, S.A., et al., Carbohydr. Res., 1 13:63-70, 1983).
  • the product, 3 can then be deblocked using standard deblocking methods such as those described below to yield GalNAc ⁇ l-3GalNAc ⁇ -O-R 3 (where R 3 is an aglycon such as amino-blocked serine or threonine, benzyl, p-nitrophenyl or allyl group).
  • FIG. 1 Compounds having fucose linked at the 6 position of the core ⁇ - GalNAc residue can be synthesized as illustrated in FIG. 1 1.
  • the procedure typically involves a condensation reaction in which 2,3,4-tri-O-benzyl- ⁇ -L-fucopyranosyl bromide or 2,3,4-tri-O-acetyl- ⁇ -L-fucopyranosyl bromide, 4, is condensed with the 3- protected ⁇ -GalNAc derivative 2-acetamido-3-O-acetyl-2-deoxy- ⁇ -D- galactopyranoside, 7, (having a variable R 2 group, where R 2 is an aglycon such as an amino-blocked serine or threonine, benzyl, p-nitrophenyl or allyl group) (Abbas, S.A., et al., Carbohydr.
  • Substitution of the core ⁇ -GalNAc residue at the 4 position can be carried out using appropriate blocking strategies to permit glycosidation reactions to occur specifically at the 4 hydroxyl position.
  • FIGS. 13A and 13B and as described above in reference to FIG. 10 it is well known in the art that the 4 and 6 positions of an ⁇ -GalNAc residue can be simultaneously blocked by reacting the Cl -blocked core ⁇ -GalNAc residue, 13, with benzaldehyde or p-methoxy benzaldehyde to form the benzylidene or p-methoxy benzylidene acetals, 2 (Abbas, S.A., and Matta, K.L., Carbohydr. Res., 126:115-124, 1984; Janin, R.K., et al,
  • the 6-position of compound 15 can be blocked with a suitable blocking agent such as triphenylmethyl chloride to form the blocked compound, 16, in which the 4-position remains free for glycosidation.
  • a suitable blocking agent such as triphenylmethyl chloride
  • Triphenylmethyl (trityl, O-Tr) ethers can be introduced onto primary (6-hydroxyl) sites highly selectively (Barker, R.G., Methods Carbohydr. Chem., II, 168-171, 1963).
  • Fuc ⁇ l- 4GalNAc ⁇ -O-R 1 and Fuc ⁇ l-4GalNAc ⁇ -O-R' can be obtained (where R 1 is an aglycon such as amino-blocked serine or threonine, benzyl, p-nitrophenyl or allyl group).
  • the products having a 3-linked ⁇ -GalNAc and a 6-linked ⁇ -GlcNAc attached to a core ⁇ -GalNAc residue can be prepared by a method similar to that used in preparing the structures Gal ⁇ 1-3 [GlcNAc ⁇ 1 -6] GalNAc ⁇ -benzyl (Abbas, S.A., et al., Carbohydr. Res., 113:63-70, 1983), or GlcNAc ⁇ l-3[GlcNAc ⁇ l-6]GalNAc ⁇ - benzyl (Piskorz, C.F., et al., Carbohydr.
  • FIG. 14A and 14B One method for synthesizing this structure is set forth in FIG. 14A and 14B.
  • synthesis is initiated by specifically blocking the 6 position (a primary hydroxyl group) of the Cl -blocked core GalNAc residue, 13, with the trityl function to form blocked compound 20.
  • the 3 and 4 positions of the blocked core GalNAc residue, 20, are then blocked using benzyl chloride or benzyl bromide as described above such that the 3 and 4 positions are blocked with benzyl ethers, thus forming intermediate 21.
  • the trityl group can then be selectively removed using hydrogen bromide in acetic acid (Barker, R.G. Methods Carbohydr. Chem.
  • cleavage of benzylidene acetal groups can be accomplished by stirring in 60%o aqueous acetic acid at 70 °C for 4 h. Trityl functions can be deprotected highly selectively at room temperature using hydrogen bromide in acetic acid (Barker, R.G., Methods Carbohydr. Chem., II., 168-171, 1963). Benzyl groups can be removed by reduction with 10% Pd-C in glacial acetic acid, shaken under H 2 gas. Acetyl groups can be removed with KCN in methanol (Herzig, J., et al, J. Org. Chem., 51 : 727-730, 1986).
  • the N-phthalimido function (such as in compound 27) can be removed with hydrazine acetate in refluxing methanol (Kamath, V.P. and Hindsgaul, O., Carbohydr. Res., 280:323-330, 1996). Under these conditions, O-acetyl and some of the N-acetyl groups are removed.
  • the N-acetyl groups can be specifically reintroduced after deblocking the other functions using acetic anhydride with aqueous sodium bicarbonate.
  • the oligosaccharides of the present invention can be directly attached to a molecule that enhances the immunogenicity of the oligosaccharide.
  • the oligosaccharide can be attached to a matrix molecule (e.g., lysyllysine) to form a multivalent conjugate (i.e., a relatively small matrix molecule to which multiple oligosaccharides antigens are attached) which in turn is attached to a molecule that increases immunogenicity.
  • the oligosaccharide can also be used to synthesize antigen clusters. These antigen clusters can then be further attached to a matrix molecule such as lysyllysine to yield a multivalent system.
  • oligosaccharides can be attached to a variety of molecules that increase immunogenicity. Frequently, such molecules are polypeptides. However, as noted above, the molecule can also be a lipid, hpopeptides, liposaccharides, or other soluble polymers. For instance, U.S. Patent 5,660,834 to Kjeldsen et al.
  • the fucose residue existed as a pyranoside.
  • Assignments in column may need to be interchanged.
  • Electrospray MSMS data (or MS. where only the molecular ion is reported) for di- and t ⁇ saccha ⁇ de-alditols desc ⁇ bed herein, and products de ⁇ ved from their PMOAc)4 oxidation at -30°C ABH4 reduction.
  • TheNAc-ol The NAc-ol GalNAc ⁇ l-3TheNAc-ol GlcNAc ⁇ 1 -ethanediol Fuc ⁇ 1 -ethanediol

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Abstract

La présente invention concerne de nouvelles structures d'oligosaccharides ayant les structures suivantes: (a) Fucβ1→6GalNAc, (b) GalNAcβ1→3GalNAc, (c) GalNAcβ1→3[GlcNAcβ1-→6]GalNAc et (d) GlcNAcβ1⊃3GalNAcα1→6GalNAc, (e) Fucα1→4GalNAc, (f) GalfNAcα1→6GalNAc, (g) Fucβ1→4GalNAc et (h) Fucα1→6GalNAc et leurs dérivés, dans lesquelles Fuc représente glucose (sous sa forme pyranose), GalNAc représente N-acétyl-D-galactosamine (sous sa forme pyranose), GalfNAc représente N-acétyl-D-galactosamine (sous sa forme furanose) et GlcNAc N-acétyl-D-glucosamine (sous sa forme pyranose). Les structures d'oligosaccharides peuvent servir comme haptènes d'antigènes. L'invention concerne plus particulièrement des antigènes possédant les structures suivantes: Fucβ1⊃6GalNAcα1→X→R; GalNAcβ1→3GalNAcα1→X→R; GalNAcβ1→3[GlcNAcβ1→6]GalNAcα1→X→R; GalNAcβ1→3GalNAcα1→6GalNAcα1→X→R; Fucα1→4GalNAcα1→X→R; GalfNAcα1-⊃6GalNAcα1→X→R; Fucβ1→4GalNAcα1→X→R; Fucα1→6GalNAcα1→X→R, et leurs dérivés. Dans ces formules, X est un éventuel liant et R est un stimulateur d'antigénicité tel qu'un polypeptide, un lipide un polysaccharide ou un polymère soluble. La présente invention concerne aussi des anticorps possédant une spécificité de liaison vis-à-vis de ces antigènes, de même que des compositions pharmaceutiques contenant des antigènes ou des anticorps de l'invention.
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WO2003016915A1 (fr) * 2001-08-20 2003-02-27 Biotie Therapies Corp. Sequences d'un oligosaccharide specifiques de tumeurs et leur utilisation
WO2004017810A2 (fr) * 2002-08-20 2004-03-04 Biotie Therapies Corp. Epitopes d'oligosaccharide specifiques de tumeur et leur utilisation

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016915A1 (fr) * 2001-08-20 2003-02-27 Biotie Therapies Corp. Sequences d'un oligosaccharide specifiques de tumeurs et leur utilisation
JP2005500535A (ja) * 2001-08-20 2005-01-06 バイオティ セラピィーズ コープ 腫瘍特異的オリゴ糖配列およびその用途
US8236487B2 (en) 2001-08-20 2012-08-07 Glykos Finland Oy Tumor specific oligosaccharide sequences and use thereof
US8697061B2 (en) 2001-08-20 2014-04-15 Glykos Finland Oy Tumor specific oligosaccharide epitopes and use thereof
WO2004017810A2 (fr) * 2002-08-20 2004-03-04 Biotie Therapies Corp. Epitopes d'oligosaccharide specifiques de tumeur et leur utilisation
WO2004017810A3 (fr) * 2002-08-20 2004-06-10 Biotie Therapies Corp Epitopes d'oligosaccharide specifiques de tumeur et leur utilisation
JP2005535723A (ja) * 2002-08-20 2005-11-24 バイオティ セラピィーズ コープ 腫瘍特異的オリゴ糖エピトープおよびその用途
EP2322209A2 (fr) 2002-08-20 2011-05-18 Glykos Finland Oy Épitopes d'oligosaccharides spécifiques des tumeurs et leur utilisation.
EP2322209A3 (fr) * 2002-08-20 2012-02-01 Glykos Finland Oy Épitopes d'oligosaccharides spécifiques des tumeurs et leur utilisation.

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