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WO2009035494A2 - Methods for producing anti-glycan antibodies, vaccines and methods for treating cancer or infectious disease - Google Patents

Methods for producing anti-glycan antibodies, vaccines and methods for treating cancer or infectious disease Download PDF

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Publication number
WO2009035494A2
WO2009035494A2 PCT/US2008/009045 US2008009045W WO2009035494A2 WO 2009035494 A2 WO2009035494 A2 WO 2009035494A2 US 2008009045 W US2008009045 W US 2008009045W WO 2009035494 A2 WO2009035494 A2 WO 2009035494A2
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Prior art keywords
glycan
protein
containing molecules
antibodies
4glcnacβ
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PCT/US2008/009045
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French (fr)
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WO2009035494A3 (en
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Ola Blixt
M.G. Finn
Eiton Kaltgrad
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The Scripps Research Institute
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Publication of WO2009035494A2 publication Critical patent/WO2009035494A2/en
Publication of WO2009035494A3 publication Critical patent/WO2009035494A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the invention generally relates to a vaccine comprising protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules.
  • Methods for producing anti-glycan antibodies in a vertebrate subject are provided.
  • Methods for treatment or diagnosis of cancer or infectious disease in a vertebrate subject by administering the vaccine or the anti-glycan antibody to the subject are provided.
  • Viruses are generally highly antigenic, and Seeberger and coworkers have taken advantage of influenza virosomes to elicit a strong and isotype-crossed immune response against a tetrasaccharide incorporated into the lipid membrane of the particle.
  • the presentation of peptide and other small-molecule antigens on viral protein scaffolds is known to generate strong immune responses, and the structures of many such scaffolds are known to atomic resolution.
  • the invention generally relates to a protein nanoparticle display platform for glycan-containing molecules wherein the protein nanoparticles display enhanced antigenicity for the glycan-containing moleucules.
  • Vaccines are provided comprising protein nanoparticles, wherein at least some of the nanoparticles are covalently bound to glycan-containing molecules.
  • Methods for producing anti-glycan antibodies in a vertebrate subject are provided.
  • Methods for treatment or diagnosis of cancer or infectious disease are provided wherein the protein nanoparticle vaccine or anti-glycan antibody is administered to a vertebrate subject in need of such treatment for the disease.
  • Methods for producing anti-glycan antibodies in a vertebrate subject comprise administering a protein nanoparticle to the vertebrate subject, wherein at least some of the protein nanoparticles covalently bound to glycan-containing molecules.
  • These methods provide cost efficient production of specific anti-glycan antibodies in high yields for diagnostic and therapeutic purposes.
  • the glycan array technology allows for the efficient screening of a polyclonal set of antibodies for specific recognition of glycan motifs. These studies set the stage for similar efforts using the controlled structures, natural multivalency, and attachment chemistries available to virus particles as carrier proteins.
  • the protein nanoparticles comprising a plurality of glycan-containing molecules can also be used for the development of a vaccine for treatment of cancer or infectious disease.
  • a vaccine which comprises protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent.
  • at least some of the protein nanoparticles are bound to a plurality of different glycan-containing molecules.
  • the glycan- containing molecule can include, but are not limited to, a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide.
  • the protein nanoparticle can include, but are not limited to, a virus, bacteriophage, nucleoprotein nanoparticle, viral nanoparticle, viral capsid particle, vault protein, dendrimer or chaperonin.
  • the protein nanoparticle is a plant viral particle.
  • the plant viral particle can be a Comovirus, Tombusvirus, Sobemovirus, or,Nepovirus.
  • the plant viral particle can be a cowpea mosaic virus.
  • the protein nanoparticles are covalently bound to glycan- containing molecules through alkyne azide linkage. In a further aspect, the protein nanoparticles are covalently bound to glycan-containing molecules through a plurality of amino acid residues on the protein nanoparticle.
  • the protein nanoparticles can be covalently bound to glycan- containing molecules through N-hydroxysuccinimide ester linkage.
  • the vaccine is provided which comprises at least 100 glycan-containing molecules per protein nanoparticle, or at least 150 glycan-containing molecules per protein nanoparticle, or at least 200 glycan-containing molecules per protein nanoparticle.
  • a method for producing anti-glycan antibodies in a vertebrate subject comprises administering to the vertebrate subject a vaccine comprising protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent, and isolating the anti-glycan antibodies from a biological sample of the vertebrate subject.
  • a vaccine comprising protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent, and isolating the anti-glycan antibodies from a biological sample of the vertebrate subject.
  • at least some of the protein nanoparticles are bound to a plurality of different glycan-containing molecules.
  • the protein nanoparticles are covalently bound to glycan-containing molecules through alkyne azide linkage.
  • the protein nanoparticles are covalently bound to glycan-containing molecules through a plurality of amino acid residues on the protein nanoparticle.
  • the protein nanoparticles can be covalently bound to glycan-containing molecules through N-hydroxysuccinimide ester linkage.
  • the vertebrate subject is a mammalian subject or an avian subject.
  • the biological sample can be one or more eggs of the avian subject.
  • the biological sample can be serum of the mammalian subject or the avian subject.
  • the vaccine is provided which comprises at least 100 glycan-containing molecules per protein nanoparticle, or at least 150 glycan-containing molecules per protein nanoparticle, or at least 200 glycan-containing molecules per protein nanoparticle.
  • An anti-glycan antibody is provided which can be isolated by the method for producing antibodies.
  • a method for treating cancer in a vertebrate subject comprising, administering to the vertebrate subject a vaccine is provided which comprises protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent, wherein the vaccine elicits an immune response to the plurality of glycan molecules to reduce or eliminate cancer in the vertebrate subject.
  • a vaccine which comprises protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent, wherein the vaccine elicits an immune response to the plurality of glycan molecules to reduce or eliminate cancer in the vertebrate subject.
  • at least some of the protein nanoparticles are bound to a plurality of different glycan- containing molecules.
  • the glycan-containing molecule can include, but are not limited to, a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide.
  • the protein nanoparticles are covalently bound to glycan-containing molecules through alkyne azide linkage.
  • the protein nanoparticles are covalently bound to glycan-containing molecules through a plurality of amino acid residues on the protein nanoparticle.
  • the protein nanoparticles can be covalently bound to glycan-containing molecules through N-hydroxysuccinimide ester linkage.
  • the vaccine is provided which comprises at least 100 glycan-containing molecules per protein nanoparticle, or at least 150 glycan-containing molecules per protein nanoparticle, or at least 200 glycan-containing molecules per protein nanoparticle.
  • a method for preventing or treating infectious disease in a vertebrate subject comprising, administering to the vertebrate subject the anti-glycan antibody, in an amount effective to reduce or eliminate infectious disease in the vertebrate subject.
  • the anti-glycan antibody can be administered orally to the vertebrate subject.
  • the anti-glycan antibody can be administered to the vertebrate subject via an oral, pulmonary, oropharyngeal, nasopharyngeal, topical, intravenous, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular route.
  • at least some of the protein nanoparticles are bound to a plurality of different glycan-containing molecules.
  • the glycan-containing molecule can include, but are not limited to, a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide.
  • the vertebrate subject can bead mammalian subject or an avian subject.
  • the infectious disease can bead bacterial infectious disease, viral infectious disease, fungal infectious disease, or infectious parasitic disease.
  • the infectious disease can bean antibiotic-resistant bacterial infectious disease.
  • a method for diagnosing disease in a vertebrate subject comprises contacting a tissue sample from the vertebrate subject with the anti-glycan antibody, and detecting binding of the antibody to the tissue sample indicating presence of glycan- containing molecules relating to the disease in the vertebrate subject.
  • the disease can be inflammatory disease, infectious disease, cancer, or metabolic disease.
  • at least some of the protein nanoparticles are bound to a plurality of different glycan-containing molecules.
  • the glycan-containing molecule can include, but are not limited to, a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide.
  • Figures IA, IB, and 1C show glycan array binding analysis of crude IgY isolated after (a) no immunization (array vl.O); (b) immunization with 1 1 (array v2.0); and (c) immunization with 13 (array vl .O).
  • Figures 2A, 2B, 2C, and 2D show glycan array binding analysis using array version vl.O of anti-2 antibodies purified by affinity filtration through agarose-18.
  • Figures 3A and 3B show glycan array binding analysis using array version v2.0 of (a) anti-14 IgY (0.15 mg/mL), and (b) commercially available monoclonal IgG against globo- H (0.035 mg/mL, Axorra, Inc.).
  • Figure 4 shows 1 H NMR spectrum of 1.
  • Figure 5 shows MALDI-TOF mass spectra of BSA conjugates.
  • Figure 6 shows plots of ratios of significant signals (>20% of the maximum) from glycan array (v2.0) analyses of total IgY generated by different chickens (designated A and B) against ⁇ left) CPMV conjugate 15 and ⁇ right) KLH conjugate 16.
  • Figure 7 shows independent repeat of the experiment described in Figure 3, showing glycan array (v2.0) binding analysis of anti-14 IgY.
  • Figure 8 shows plots of ratios of averaged signals from glycan array (v2.0) analyses of total IgY generated by two different chickens against CPMV conjugate 15 vs. KLH conjugate 16; all IgY samples were used at 0.1 mg/mL.
  • Figure 9 shows plots of ratios of low-intensity signals ( ⁇ 20% of the maximum) from glycan array (v2.0) analyses of total IgY generated by two different chickens against CPMV conjugate 15 and KLH conjugate 16.
  • the invention generally relates to a protein nanoparticle display platform for glycan-containing molecules wherein the nanoparticles display enhanced antigenicity for the glycan-containing moleucules.
  • Vaccines are provided comprising protein nanoparticles, wherein at least some of the nanoparticles are covalently bound to glycan-containing molecules.
  • Methods for producing anti-glycan antibodies in a vertebrate subject are provided.
  • Methods for treatment or diagnosis of cancer or infectious disease are provided wherein the protein nanoparticle vaccine or anti-glycan antibody is administered to a vertebrate subject in need of treatment for the disease.
  • Tetra- and hexasaccharides were arrayed on the exterior surface of a protein nanoparticle, e.g., cowpea mosaic virus, using the copper-catalyzed azide-alkyne cycloaddition reaction.
  • Methods for producing anti-glycan antibodies in a vertebrate subject comprise the step of administering a nanoparticle-glycan conjugate to a mammalian subject to produce polyclonal anti-glycan IgG antibodies or administering a nanoparticle-glycan conjugate to an avian subject to produce polyclonal anti-glycan IgY antibodies.
  • avian IgY antibodies are produced in significantly higher yield than is possible for mouse or rabbit IgG, and exhibit reduced cross reactivity with native mammalian proteins.
  • affinity purification against immobilized mono-LacNAc was necessary to provide a set of antibodies with specific binding properties. Comparable performance was observed for the nanoparticle-glycan conjugate-based polyclonal vs. a commercial monoclonal antibody raised against the globo-H tetrasaccharide, highlighting the utility of the glycan microarray for both quality control and rapid in-depth analysis.
  • Virus-carbohydrate conjugates are promising candidates for development in diagnostic and immunotherapeutic applications.
  • Nanoparticle refers to protein nanoparticles or nucleoprotein nanoparticles having a regular or order structure and capable of covalently attaching a glycan-containing molecule.
  • the nanoparticles can include, but are not limited to, viruses, viral nanoparticles, vault proteins, dendrimers, chaperonins, or other large assemblies.
  • the protein nanoparticle can be an icosahedral virus, rod-shaped virus, animal virus, or plant virus.
  • the virus or viral nanoparticle can be a plant viral nanoparticle, for example, a Comovirus
  • the protein nanoparticle can be a virus, bacteriophage, virus-like particle, or viral capsid particle.
  • the protein nanoparticle can be derived, for example, from a plant viral particle, e.g., Comovirus.
  • the protein nanoparticle can be derived, for example, from R otavirus, Norwalkvirus, Alphavirus, Foot and Mouth Disease virus, Retrovirus, Hepatitis B virus, Tobacco mosaic virus, Flock House Virus, or papillomavirus. See, for example PCT Application WO 2000/032227.
  • Plant viral particle refers to any plant virus which is a small plant icosahedral virus composed of 60 identical copies of an asymmetric protein subunit assembled around a bipartite single strand (+) RNA genome.
  • Plant viral particles are plant viruses that include, but are not limited to, Comovirus, Tombusvirus, Sobemovirus, or Nepovirus.
  • the comovirus is cowpea mosaic virus. See U.S. Application No. 2006/0216238; Sen Gupta et al., RSC Chem. Commun. 2005, 34, 4315-4317.
  • Glycan-containing molecule refers to a carbohydrate or sugar-containing molecule including, but not limited to, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide.
  • Anti-glycan antibodies refer to antibodies isolated from a vertebrate subject, including, but not limited to, IgG antibodies from a mammalian subject or IgY antibodies from an avian subject following inoculation with a vaccine comprising protein nanoparticles covalently bound to glycan-containing molecules.
  • the antibodies produced in the vertebrate subject are typically polyclonal and can be identified as capable of binding to glycan-containing molecules or a plurality of different glycan containing molecules.
  • the antibodies can also be identified as capable of binding to the protein nanoparticle of the vaccine used to inoculate the vertebrate subject.
  • the anti-glycan antibodies can be isolated and purified from other antibodies, e.g., antibodies to the protein nanoparticle, by affinity purification.
  • "Patient”, “subject”, “vertebrate” or “mammal” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals.
  • Animals include all vertebrates, e.g., mammals and non-mammals, such as sheep, dogs, cows, pigs, chickens, amphibians, and reptiles.
  • Treating” or “treatment” includes the administration of the antibody compositions, or vaccine compositions, compounds or agents of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder (e.g., cancer, or infectious disease).
  • Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
  • Covalent attachment of the glycan-containing molecule to the viral particle can occur through alkyne-azide linkage chemistry as described herein. Covalent attachment can also occur through a variety of linkage chemistry to any of the other residues on the surface of the viral subunit, usually lysine, cysteine, tyrosine, aspartic acid, or glutamic acid, but also including chemically modified side chains and unnatural amino acids.
  • Each viral particle can have a number of identical viral subunits. For example, CPMV has 60 identical subunits, and other viral particles can have a multiple of 60 subunits. Each viral subunit has a multiple of available residues for linkage to the glycan-containing molecule. For example, CPMV has viral subunits with five available lysine residues per subunit.
  • Suitable linkage chemistries include maleimidyl linkers and alkyl halide linkers (which react with a sulfhydryl on the antibody moiety) and succinimidyl linkers (which react with a primary amine on the antibody moiety).
  • Several primary amine and sulfhydryl groups are present on immunoglobulins, and additional groups can be designed into recombinant immunoglobulin molecules. It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, 111.), can be employed as a linker group.
  • Coupling can be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues (see, e.g., U.S. Pat. No. 4,671,958).
  • glycan-containing molecules in the presence or absence of a therapeutic agent can be conjugated to the nanoparticles of the invention, for example, by any suitable technique, with appropriate consideration of the need for pharmacokinetic stability and reduced overall toxicity to the patient.
  • a glycan-containing molecule can be coupled to a suitable nanoparticle either directly or indirectly (e.g. via a linker group).
  • compositions and methods are provided for covalent linkage of glycan-containing molecules to nanoparticles providing methods for catalyzing a reaction between at least one terminal alkyne moieties, and at least one azide moieties, wherein one moiety is attached to the glycan-containing molecules and the other moiety is attached to the nanoparticle.
  • a nucleophilic group such as an amino or sulfhydryl group
  • a carbonyl-containing group such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide).
  • a suitable chemical linker group can be used.
  • a linker group can function as a spacer to distance a glycan-containing molecule from a nanoparticle in order to avoid interference with binding capabilities.
  • a linker group can also serve to increase the chemical reactivity of a substituent on a nanoparticle or glycan-containing molecule, and thus increase the coupling efficiency.
  • An increase in chemical reactivity can also facilitate the use of moieties, or functional groups on moieties, which otherwise would not be possible.
  • the glycan-containing molecules or the protein nanoparticles of the invention can be coupled or conjugated to one or more therapeutic or cytotoxic moieties.
  • Cytotoxic moiety refers to a moiety that inhibits cell growth or promotes cell death when proximate to or absorbed by a cell. Suitable cytotoxic moieties in this regard include radioactive agents or isotopes (radionuclides), chemotoxic agents such as differentiation inducers, inhibitors and small chemotoxic drugs, toxin proteins and derivatives thereof, as well as nucleotide sequences (or their antisense sequence). Therefore, the cytotoxic moiety can be, by way of non-limiting example, a chemotherapeutic agent, a photoactivated toxin or a radioactive agent.
  • cytotoxic agents can be coupled to the glycan-containing molecules of the invention, for example, through an oxidized carbohydrate group at a glycosylation site, as described in U.S. Pat. Nos. 5,057,313 and 5,156,840.
  • Yet another alternative method of coupling the glycan-containing molecules, e.g., glycoproteins, to the cytotoxic moiety is by the use of a non-covalent binding pair, such as streptavidin/biotin, or avidin/biotin.
  • one member of the pair is covalently coupled to the glycan- containing molecules and the other member of the binding pair is covalently coupled to the cytotoxic moiety.
  • a cytotoxic moiety is more potent when free from the glycan-containing molecules or the nanoparticles of the present invention
  • a number of different cleavable linker groups have been described.
  • the mechanisms for the intracellular release of a cytotoxic moiety agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710); by irradiation of a photolabile bond (e.g., U.S. Pat. No.
  • a glycan-containing molecule can be made useful as a contrasting agent for several visualization techniques, or a therapeutic antibody can be labeled for tracking by a visualization technique.
  • multiple molecules of a cytotoxic moiety are coupled to one glycan-containing molecule.
  • more than one type of moiety can be coupled to one glycan-containing molecule.
  • a therapeutic moiety such as an polynucleotide or antisense sequence
  • glycan-containing molecules in conjunction with a chemotoxic or radiotoxic moiety, to increase the effectiveness of the chemo- or radiotoxic therapy, as well as lowering the required dosage necessary to obtain the desired therapeutic effect.
  • immunoconjugates with more than one moiety can be prepared in a variety of ways. For example, more than one moiety can be coupled directly to CPMV viral nanoparticles, such that multiple sites for attachment (e.g., dendrimers) can be used. Alternatively, a carrier with the capacity to hold more than one cytotoxic moiety can be used.
  • a carrier can bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations.
  • Suitable covalent-bond carriers include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234), peptides, and polysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784), each of which has multiple sites for the attachment of moieties.
  • a carrier can also bear an agent by non- covalent associations, such as non-covalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088).
  • Encapsulation carriers are especially useful in chemotoxic therapeutic embodiments, as they can allow the therapeutic compositions to gradually release a chemotoxic moiety over time while concentrating it in the vicinity of the target cells.
  • the water soluble sulfonated bathophenanthroline ligand 2 can be used to promote a highly efficient Cu(I)-mediated azide- alkyne cycloaddition (CuAAC; "Click chemistry”) reaction for the chemoselective attachment of biologically relevant molecules to protein nanoparticles, e.g., cowpea mosaic virus (CPMV) nanoparticles.
  • CuAAC copper-mediated azide- alkyne cycloaddition
  • the ligated substrates included complex sugars, peptides, poly(ethylene oxide) polymers, and the iron carrier protein transferring (Tfn), with successful ligation even for cases that were previously resistant to azide-alkyne coupling using the conventional ligand tris(triazolyl)amine (1).
  • the use of 4-6 equiv of substrate was sufficient to achieve loadings of 60-1 15 molecules/virion in yields of 60-85%. Although it is sensitive to oxygen, the reliably efficient performance of the Cu-ligand»2 system makes it a useful tool for demanding bioconjugation applications.
  • compositions and methods are provided for covalent linkage of glycan- containing molecules to nanoparticles comprising catalyzing a reaction between at least one terminal alkyne moieties, and at least one azide moieties, wherein one moiety is attached to the glycan-containing molecules and the other moiety is attached to the nanoparticle, forming at least one triazole thereby.
  • a method for coupling a glycan-containing molecules to nanoparticles comprising catalyzing a reaction between at least one terminal alkyne moieties attached to the glycan-containing molecules, and at least one azide moieties attached to the nanoparticles, forming at least one triazole thereby, effecting catalysis by addition of a metal ion in the presence of a ligand, and providing a plurality of sites on the nanoparticles having azide moieties, such that a plurality of glycan-containing molecules can be coupled with the nanoparticles.
  • a further embodiment provides a method for coupling glycan-containing molecules to nanoparticles comprising catalyzing a ligation reaction between at least one terminal alkyne moieties attached to the nanoparticles, and at least one azide moieties attached to the glycan-containing molecules, forming at least one triazole thereby, effecting catalysis by addition of a metal ion in the presence of a ligand, and providing a plurality of sites on the nanoparticles having terminal alkyne moieties, such that a plurality of glycan-containing molecules can be coupled with the scaffold. See PCT Application WO 2007/01 1696.
  • “Plurality of sites” refers to two or more sites on a nanoparticle capable of binding two or more compounds per nanoparticle molecule. Depending upon the nature of the scaffold and the compounds, 100 or more, 200 or more, or 300 or more compound molecules can be bound per scaffold molecule.
  • the scaffold molecule is a protein of a viral nanoparticle, e.g., a CPMV nanoparticle.
  • Terminal alkyne moiety refers to an acetylenic bond (carbon-carbon triple bond) having a hydrogen attached to one carbon, e.g., R-C ⁇ C-H, wherein R is a compound including, but not limited to, polynucleotide, polypeptide, glycopolymer, chromophoric dye, glycan, or lipid.
  • Azide moiety refers to a moiety, N ⁇ N ⁇ -N ⁇ -.
  • An azide moiety can be attached to a compound having a general structure, N ⁇ N e -N ⁇ -R, wherein R is a compound including, but not limited to, polynucleotide, polypeptide, glycopolymer, chromophoric dye, glycan, or lipid.
  • the present invention provides an efficient strategy for end-functionalization of a compound, e.g., glycan-containing molecule, glycopolymer, carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide, polyethylene glycol, chromophoric dye, folic acid, lipid, polynucleotide, polypeptide, protein, or transferrin, using an azide-containing initiator for a living polymerization process followed by click chemistry elaboration of the unique azide end group.
  • a compound e.g., glycan-containing molecule, glycopolymer, carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidyl
  • the copper- catalyzed cycloaddition reaction provides very efficient coupling of such polymers to a functionalized viral coat protein with efficient use of coupling reagents, compound molecules, and scaffold molecules.
  • a well-defined side chain neoglycopolymer possessing a single activated chain end can be chemically conjugated efficiently to a protein or bionanoparticle in a "bioorthogonal" fashion.
  • the bioorthogonal labeling of biomolecules provides a unique, in vivo label that is an important tool for the study of biomolecule function and cellular fate. Attention is increasingly focused on labeling of biomolecules in living cells, since cell lysis introduces many artefacts.
  • the method further provides high diversity in the nature of the label used in the ligation reaction.
  • the method for coupling a compound to a scaffold comprises catalyzing a reaction between a first reactant having a terminal alkyne moiety and second reactant having an azide moiety for forming a product having a triazole moiety by addition of a metal ion in the presence of a ligand.
  • the metal ion includes, but is not limited to, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, Hf, Ta, W, Re, Os, Ir, Pt, Au, or Hg.
  • the metal includes, but is not limited to, Mn, Fe, Co, Cu, Mo, Tc, Ru, Rh, Pd, W, Re, Os, Ir, Pt, or Au. See for example, PCT International Application WO 2003/101972.
  • the metal is heterogeneous copper, metallic copper, copper oxide, or copper salts.
  • Copper(I) salts for example, Cu(I), CuOTf ⁇ C 6 H 6 and [Cu(NCCH 3 ) 4 ]PF 6 , can also be used directly in the absence of a reducing agent. These reactions usually require acetonitrile as co-solvent and one equivalent of a nitrogen base (e.g., 2,6-lutidine, triethylamine, diisopropylethylamine, or pyridine). However, formation of undesired byproducts, primarily diacetylenes, bis-triazoles, and 5-hydroxytriazoles, was often observed. For a recent summary of the reactions of Cu(I) complexes with dioxygen, see Schindler, Eur. J. Inorg. Chem.
  • the ligation reaction can be catalyzed by addition of Cu(I). IfCu(I) salt is used directly, no reducing agent is necessary, but acetonitrile or one of the other ligands indicate above can be used as a solvent (to prevent rapid oxidation of Cu(I) to Cu(II) and one equivalent of an amine can be added to accelerate the reaction. In this case, for better yields and product purity, oxygen should be excluded. Therefore, the ascorbate or any other reducing procedure is often preferred over the unreduced procedure.
  • the use of a reducing agent is procedurally simple, and furnishes triazole products in excellent yields and of high purity. Addition of an amine, such as triethylamine or 2,6-lutidine to the acetonitrile system, solves the problem of reactivity - the product is formed in quantitative yield after approximately 8 hours.
  • the ligation reaction can be catalyzed by addition of Cu(II) in the presence of a reducing agent for reducing the Cu(II) to Cu(I), in situ.
  • a reducing agent for reducing the Cu(II) to Cu(I) in situ.
  • Cu(II) salts e.g., CuSO 4 ⁇ SH 2 O
  • Reducing agents useful in this reaction include, but are not limited to ascorbic acid, sodium ascorbate, quinone, hydroquinone, vitamin Kl, glutathione, cysteine, Fe 2+ , Co 2+ , and an applied electric potential. See, for example, Davies, Polyhedron 11: 285-321 1992, and Creutz, Inorg. Chem.
  • metals can be employed as reducing agents to maintain the oxidation state of the Cu (I) catalyst or of other metal catalysts.
  • Metallic reducing agents include, but are not limited to, Cu, Al, Be, Co, Cr, Fe, Mg, Mn, Ni, and Zn.
  • an applied electric potential can be employed to maintain the oxidation state of the catalyst.
  • the ligation reaction can be catalyzed by addition of Cu(O) in the presence of an oxidizing agent for oxidizing the Cu(O) to Cu(I), in situ.
  • Metallic containers can also be used as a source of the catalytic species to catalyze the ligation reaction.
  • a copper container, Cu(O) can be employed to catalyzed the ligation reaction.
  • the reaction solution In order to supply the necessary ions, the reaction solution must make physical contact with the a copper surface of the container.
  • the reaction can be run in a non-metallic container, and the catalytic metal ions supplied by contacting the reaction solution with a copper wire, copper shavings, or other structures. Although these reactions may take longer to proceed to completion, the experimental procedure reduces the number of intervening steps.
  • the method for coupling a compound to a scaffold comprises catalyzing a reaction between a first reactant having a terminal alkyne moiety and second reactant having an azide moiety for forming a product having a triazole moiety by addition of a metal ion in the presence of a ligand for the metal ion.
  • the metal ion is coordinated to a ligand for solubilizing such metal ion within the solvent, for inhibiting oxidation of such metal ion, and for dissociating, in whole or in part, from such metal ion during the catalysis of the reaction.
  • Ligands can be, for example, monodentate ligands,bidentate (chelating) ligands, or multidentate ligands.
  • Monodentate ligands refers to Lewis bases that donate a single pair ("mono") of electrons to a metal atom.
  • Monodentate ligands can be either ions (usually anions) or neutral molecules.
  • Monodentate ligands include, but are not limited to, fluoride ion (F “ ), chloride ion (Cl “ ), bromide ion, (Br “ ), iodide ion (I “ ), water (H 2 O), ammonia (NH 3 ), hydroxide ion (OH “ ), carbon monoxide (CO), cyanide (CN “ ), or thiocyanate ion (CN-S “ ).
  • Bidentate ligands or chelating ligands refers to Lewis bases that donate two pairs of electrons to a metal atom.
  • Bidentate ligands include, but are not limited to, ethylenediamine, acetylacetonate ion, phenanthroline, sulfonated bathophenanthroline or oxalate ion.
  • Ligands include, but are not limited to, acetonitrile, cyanide, nitrile, isonitrile, water, primary, secondary or tertiary amine, a nitrogen bearing heterocycle carboxylate, halide, alcohol, and thiol sulfide, phosphine, and phosphite.
  • the halide is chloride and can be used at a concentration of 1-5 M.
  • Polyvalent ligands that include one or more functional groups selected from nitrile, isonitrile, primary, secondary, or tertiary amine, a nitrogen bearing heterocycle, carboxylate, halide, alcohol, thiol, sulfide, phosphine, and phosphite can also be employed.
  • the ligation reactions as provided herein are useful for in a method for coupling a compound to a scaffold.
  • the method provides catalyzing a ligation reaction between one or more terminal alkyne moieties and one or more azide moieties, for forming a product having a triazole moiety, the ligation reaction being catalyzed by addition of a metal ion in the presence of a ligand, and the scaffold having polyvalent sites for coupling to one or more compounds.
  • the one or more terminal alkyne moieties are attached to the compound, and the one or more azide moieties are attached to the scaffold.
  • the one or more terminal alkyne moieties are attached to the scaffold, and the one or more azide moieties are attached to the compound.
  • the scaffold can be a protein on a viral nanoparticle, for example, a cow pea mosaic viral nanoparticle.
  • the optimal design and composition of the vaccines can include glycan-containing molecules including, but not limited to, blood group A antigen, tri-LacNAc, sialyl Lewis", sialyl Lewis y , or globoH.
  • glycan-containing molecules including, but not limited to, blood group A antigen, tri-LacNAc, sialyl Lewis", sialyl Lewis y , or globoH.
  • candidate antigens being examined is Lewis y (Le y ), a blood group-related antigen that is overexpressed on the majority of human carcinomas.
  • Le y As a model for specificity, studies have examined the role of epitope clustering, carrier structure, and adjuvant on the immunogenicity of Le y conjugates in mice.
  • glycolipopeptide with clustered Le y epitopes are more effective than related structures with single Le y epitopes in producing antitumor cell antibodies; (ii) antibody responses to the clustered Le y -structure conjugated to KLH were skewed toward the immunizing carbohydrate structure; and (iii) totally synthetic constructs can be effective immunogens in conjunction with a suitable adjuvant, the effect of which is to bypass the need for specific T-cell help to stimulate IgG as well as IgM antibodies.
  • Vaccines comprising plant viral particles displaying a plurality of glycan- containing molecules or antibodies to glycan-containing molecules can be used.
  • the types of glycans bound by these antibodies is indicative of the type, extent and/or prognosis of the disease.
  • low-risk types of breast cancer as well as more aggressive types of breast cancer can be detected or treated using the present methods.
  • Patients with breast cancer have circulating antibodies that react with glycans such as ceruloplasmin glycans, Neu5Accx2- 6GalNAca, certain T-antigens carrying various substituents and other modifications, LNT-2 (a known ligand for tumor-promoting Galectin-4; see Huflejt & Leffler (2004).
  • GMl is a glycan that includes the following carbohydrate structure: Gal-beta3-GalNAc-beta4-[Neu5Ac-alpha3]-Gal-beta4-Glc-beta.
  • Sulfo-T is a T-antigen with sulfate residues, for example, Sulfo-T can include a carbohydrate of the following structure: Gal3GalNAc.
  • Globo-H is a glycan that includes the following carbohydrate structure: Fucose-alpha2-Gal-beta3-GalNAc-beta3-Gal-alpha4-Gal-beta4-Glc.
  • LNT-2 is a glycan that includes the following carbohydrate structure: GlcNAc-beta3-Gal-beta4-Glc-beta.
  • Sialylated Tn is a glycan with the following structure: Neu5Ac-alpha6-GalNAc-alpha.
  • Circulating antibodies from breast cancer patients can also react with the following glycans: Tri- LacNAc, LacNAc-LeX-LeX, LacNAc-LacNAc, H-type-2-LacNAc, H-type2-LacNAc-LacNAc, GlcNAcI33LacNAc, SLeXLacNAc, 3'SialylDiLacNAc, 3'Sialyl-tri-LacNAc, 6Sia-LacNAc- LeX-LeX, 6SiaLacNAc-LacNAc.
  • Methods for producing anti-glycan antibodies can be used for the diagnosis or treatment of cancer, e.g., breast cancer, with anti-glycan antibodies.
  • the nanoparticles, e.g., plant viral particles, comprising glycan-containing molecules can be used for detecting or treating breast cancer.
  • the plant viral particles comprising glycan-containing molecules include glycans that react with antibodies associated neoplasia in sera of mammals with benign or pre- malignant tumors. Such glycans have two or more sugar units.
  • the glycan-containing molecules include straight chain and branched oligosaccharides as well as naturally occurring and synthetic glycans.
  • Any type of sugar unit can be present in the glycans of the invention, including allose, altrose, arabinose, glucose, galactose, gulose, fucose, fructose, idose, lyxose, mannose, ribose, talose, xylose, neuraminic acid or other sugar units.
  • Such sugar units can have a variety of substituents.
  • Fatty acids, lipids, amino acids, peptides and proteins can also be attached to the glycan-containing molecules. See, for example, WO 2006/068758.
  • neutralizing antibodies known to be specific for HIV have been found to be reactive with mannose-containing glycans, in particular Man8 glycans.
  • HIV infection can be detected by detecting whether a patient has circulating antibodies that bind to Man8 glycans.
  • HIV infection can be treated or inhibited by administering a vaccine comprising a plant viral particle displaying a plurality of glycan-containing molecules, e.g., Man8 glycans, or administering anti-glycan antibodies produced by the methods of the present invention to a subject.
  • a method of detecting transplant tissue rejection or xenotransplant tissue rejection are provided.
  • Glycans specific for the transplanted or xenotranplanted tissue are used in glycan arrays to observe whether one or more glycan-containing molecules are bound by antibodies in the test sample.
  • Examples of glycan-containing molecules that can be used for diagnosis or treatment of transplant reject include any one of Gal-alpha3-Gal-beta, Gal-alpha3- Gal-beta4-GlcNAc[alpha3-Fucose]-beta, Gal-alpha3-Gal-beta4-Glc-beta, Gal-alpha3- Gal[alpha2-Fucose]-beta4-GlcNAc-beta, Gal-alpha3-Gal-beta4-GalAc-beta, GaI-alpha3-GalAc- alpha, Gal-alpha3 -Gal-beta, or Gal-beta4-GlcNAc[alpha3-Fucose]-beta, or a combination thereof.
  • the glycan-containing molecules can therefore include glycans that react with antibodies associated with particular disease or condition.
  • glycan-containing molecules or antibodies to glycan-containing molecules that are produced in response to cancer, bacterial infection, viral infection, inflammation, transplant rejection, autoimmune diseases and the like can be detected. See, for example, WO 2007/0059769.
  • CD Crohn's disease
  • IBD inflammatory bowel disease
  • Antibodies against a mannan epitope of Saccharomyces cerevisiae (gASCA), laminaribioside (ALCA), chitobioside (ACCA), mannobioside (AMCA), outer membrane porins (Omp) and the atypical perinuclear antibody pANCA were tested in serum samples of 1225 IBD patients, 200 healthy controls (HC) and 1 13 patients with non-IBD gastrointestinal inflammation (non-IBD GI). Antibody responses were correlated with type of disease and clinical characteristics. 76% of CD patients had at least one of the tested antibodies. For differentiation between CD and ulcerative colitis (UC), the combination of gASCA and pANCA was most accurate.
  • O-glycans Many secreted and cell surface glycoproteins are O-glycosylated.
  • the structures of these Ser/Thr-bound O-glycans change during differentiation and activation of cells, during apoptosis, and are often altered in disease.
  • O-glycans have been shown to be important for the functions of glycoproteins, for example, the expression of carbohydrate and peptide epitopes, the expression and stability of cell surface receptors, and cell adhesion.
  • the altered structures of O- glycans therefore may be in part responsible for the pathological properties of diseased cells.
  • the present study is aimed at defining the mechanisms by which O-glycans change in cystic fibrosis, cancer and inflammatory disease, and at studying the functional implications of these alterations.
  • breast cancer or leukaemia cells have increased sialyltransferase activities and produce hypersialylated O-glycans, compared to normal counterparts.
  • Sialylated O-glycans often cannot elongated further, and therefore remain truncated.
  • most breast cancer cell lines also show a lack of O-glycan branching by core 2 beta ⁇ -GlcNAc-transferase, and this leads to short and sialylated O-glycan structures.
  • Apoptosis is a process which may cause cells to undergo glycosylation changes. Apoptosis may also be influenced by specific glycan structures.
  • Apoptosis may be an important mechanism of xenograft rejection.
  • the enzymes synthesizing short sialylated structures are highly active, but the branching enzyme core 2 beta ⁇ -GlcNAc-transferase is less active in the apoptotic cell populations. This indicates that these enzymes are regulated differently during the process of apoptosis.
  • Increased angiogenesis is another feature of cancer and rheumatoid arthritis.
  • O-glycans and possibly other types of glycans, on cell surface glycoproteins of endothelial cells can be involved in the control of growth related processes.
  • Knowledge of the specific roles of O- glycans and the regulation of their synthesis in the endothelium can be useful in the control of transplant rejection, wound healing, cancer angiogenesis, inflammation and other clinical conditions involving cell growth and cell death.
  • Vaccines comprising a plant viral particle displaying a plurality of glycan- containing molecules or anti-glycan antibodies produced by the methods of the present invention are useful for treating disease related to transplant rejection, wound healing, cancer angiogenesis, cystic fibrosis, inflammation, and other clinical conditions involving cell growth and cell death.
  • the structures of Ser/Thr-bound O-glycans change during differentiation and activation of cells, during apoptosis, and are often altered in disease. Cancer, cystic fibrosis and inflammatory diseases are associated with alterations of O-glycans that are in part responsible for the pathology observed.
  • glycans described herein are referenced in abbreviated form. Many of the abbreviations used are provided in the following table. Moreover the glycans of the invention can have any of the sugar units, monosaccharides or core structures provided in this table.
  • D-GaIpNAc N-Acetylgalactop yranose AN
  • D-GaIpN D-Galacosamine AQ
  • D-ManpNAc D-NJ-Acetylmannopyranose MN
  • D-Neup5Ac N-Acetylneuraminic acid NN
  • D-Neu5G D-N-Glycolylneuraminic acid NJ
  • KDN* 2 Keto-3-deoxynananic acid
  • D-Ribp D-Ribopyranose B
  • D-Apip D-Apiopyranose P
  • KDN 3-deoxy-D-glycero-K-galacto-nonulosonic acid.
  • sugar units or other saccharide structures present in the glycans of the invention can be chemically modified in a variety of ways.
  • compositions and methods of the invention provide vaccines comprising a protein nanoparticle displaying a plurality of glycan-containing molecules or provide anti-glycan antibodies produced by the methods of the present invention that are useful for detecting and preventing cancer or infectious disease.
  • the glycans or glycan-containing molecules include numerous different types of carbohydrates and oligosaccharides. In general, the major structural attributes and composition of the separate glycans within the libraries have been identified. In some embodiments, the libraries consist of separate, substantially pure pools of glycans, carbohydrates and/or oligosaccharides. Further description of the types of glycans useful in the practice of the invention is provided in U.S. Provisional Ser. No.
  • the glycan-containing molecules of embodiments of the invention include straight chain and branched oligosaccharides as well as naturally occurring and synthetic glycans.
  • the glycan can be a glycoaminoacid, a glycopeptide, a glycolipid, a glycoaminoglycan (GAG), a glycoprotein, a whole cell, a cellular component, a glycoconjugate, a glycomimetic, a glycophospholipid anchor (GPI), glycosyl phosphatidylinositol (GPI)-linked glycoconjugates, bacterial lipopolysaccharides and endotoxins.
  • the glycans can also include N- glycans, O-glycans, glycolipids and glycoproteins.
  • the glycan-containing molecules of embodiments of the invention include 2 or more sugar units. Any type of sugar unit can be present in the glycans of the invention, including, for example, allose, altrose, arabinose, glucose, galactose, gulose, fucose, fructose, idose, lyxose, mannose, ribose, talose, xylose, or other sugar units.
  • sugar units can have a variety of modifications and substituents. Some examples of the types of modifications and substituents contemplated are provided in the tables herein.
  • sugar units can have a variety of substituents in place of the hydroxy (-OH), carboxylate (-COO " ), and methylenehydroxy (-CH 2 -OH) substituents.
  • lower alkyl moieties can replace any of the hydrogen atoms from the hydroxy (-OH), carboxylic acid (-COOH) and methylenehydroxy (- CH 2 -OH) substituents of the sugar units in the glycans of the invention.
  • amino acetyl can replace any of the hydrogen atoms from the hydroxy (-OH) , carboxylic acid (-C00H) and methylenehydroxy (-CH 2 -OH) substituents of the sugar units in the glycans of the invention.
  • N-acetylneuraminic acid can replace any of the hydrogen atoms from the hydroxy (-OH), carboxylic acid (-COOH) and methylenehydroxy (-CH 2 -OH) substituents of the sugar units in the glycans of the invention.
  • Sialic acid can replace any of the hydrogen atoms from the hydroxy (-OH), carboxylic acid (-C00H) and methylenehydroxy (- CH 2 -OH) substituents of the sugar units in the glycans of the invention.
  • Amino or lower alkyl amino groups can replace any of the OH groups on the hydroxy (-OH), carboxylic acid (-COOH) and methylenehydroxy (-CH 2 -OH) substituents of the sugar units in the glycans of the invention.
  • Sulfate (-SO 4 ) or phosphate (-PO 4 ) can replace any of the OH groups on the hydroxy (-OH), carboxylic acid (-COOH) and methylenehydroxy (-CH 2 -OH) substituents of the sugar units in the glycans of the invention.
  • Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl” embraces only the straight chain radical, when a branched chain isomer such as “isopropyl” has been specifically referred to.
  • Halo is fluoro, chloro, bromo, or iodo.
  • lower alkyl refers to (C
  • (C 3 -Ce)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;
  • C 3 -C 6 )cycloalkyl(Ci-Ce)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2- cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl;
  • (Ci-C 6 ) alkoxy can be methoxy, ethoxy, propoxy,
  • the glycans of the invention having one or more chiral centers may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a glycan of the invention, it being well known in the art how to prepare optically active forms, for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.
  • the libraries of the invention are particularly useful because diverse glycan structures are difficult to make and substantially pure solutions of a single glycan type are hard to generate.
  • the sugar units typically present in glycans have several hydroxyl (-OH) groups and each of those hydroxyl groups is substantially of equal chemical reactivity, manipulation of a single selected hydroxyl group is difficult. Blocking one hydroxyl group and leaving one free is not trivial and requires a carefully designed series of reactions to obtain the desired regioselectivity and stereoselectivity. Moreover, the number of manipulations required increases with the size of the oligosaccharide.
  • glycans of the invention have been obtained by a variety of procedures. For example, some of the chemical approaches developed to prepare N-acetyllactosamines by glycosylation between derivatives of galactose and N-acetyiglucosamine are described in AIy, M. R. E.jlbrahim, E.-S.
  • Synthesis of glycan-containing molecules can occur by synthesizing oligosaccharides using regiospecific and stereospecific enzymes, called glycosyltransferases, for coupling reactions between the monosaccharides.
  • glycosyltransferases catalyze the transfer of a monosaccharide from a glycosyl donor (usually a sugar nucleotide) to a glycosyl acceptor with high efficiency.
  • Most enzymes operate at room temperature in aqueous solutions (pH 6-8), which makes it possible to combine several enzymes in one pot for multi-step reactions.
  • the high regioselectivity, stereoselectivity and catalytic efficiency make enzymes especially useful for practical synthesis of oligosaccharides and glycoconjugates.
  • Bacterial expression systems lack the post-translational modification machinery that is required for correct folding and activity of the mammalian enzymes whereas the enzymes from the bacterial sources are compatible with this system.
  • bacterial enzymes are used as synthetic tools for generating glycans, rather than enzymes from the mammalian sources.
  • the repeating Gal ⁇ (l-4)GlcNAc-unit can be enzymatically synthesized by the concerted action of ⁇ 4-galactosyltransferase ( ⁇ 4GalT) and ⁇ 3-N- acetyllactosamninyltransferase ( ⁇ 3G IcNAcT). Fukuda, M., Biochim. Biophys. Acta.1984, 780:2, 119-150; Van den Eijnden, D. H.;Koenderman, A. H. L. and Schiphorst, W. E. C. M., J. Biol. Chem. 1988, 263, 12461-1247 1.
  • ⁇ 4GalT-GalE is a fusion protein constructed from ⁇ 4GalT and the uridine-5-diphospho-galactose-4'-epimerase (GaIE) for in situ conversion of inexpensive UDP-glucose to UDP-galactose providing a cost efficient strategy.
  • GaIE uridine-5-diphospho-galactose-4'-epimerase
  • glycans used in the compositions, libraries and arrays of the invention are described herein. However, in some cases a source of the glycan, rather than the precise structure of the glycan is given. Hence, a glycan from any available natural source can be used in the arrays and libraries of the invention. For example, known glycoproteins are a useful source of glycans.
  • the glycans from such glycoproteins can be isolated using available procedures or, for example, procedures provided herein. Such glycan preparations can then be used in the compositions, libraries and arrays of the invention.
  • the glycan arrays employ a library of characterized and well-defined glycan structures.
  • the array has been validated with a diverse set of carbohydrate binding proteins such as plant lectins and C-type lectins, Siglecs, Galectins, Influenza Hemaglutinins and anti- carbohydrate antibodies (both from crude sera and from purified serum fractions). Further description on how to make glycan arrays useful in the practice of the invention is provided in U.S. Provisional Ser. No. 60/550, 667, filed March 5, 2004, and U.S. Provisional Ser. No. 60/558,598, filed March 31, 2004, the contents of which are incorporated herein by reference.
  • the libraries, arrays and methods have several advantages.
  • One particular advantage is that the arrays and methods provide highly reproducible results.
  • the libraries and arrays permit screening of multiple glycans in one reaction.
  • the libraries and arrays provide large numbers and varieties of glycans.
  • the libraries and arrays have at least two, at least three, at least ten, or at least glycans.
  • the libraries and arrays have about 2 to about 100,000, or about 2 to about 10, 000, or about 2 to about 1,000, different glycans per array.
  • Such large numbers of glycans permit simultaneous assay of a multitude of glycan types.
  • the present arrays have been used for successfully screening a variety of glycan binding proteins.
  • the arrays can be used for more than one assay.
  • the arrays and methods provide high signal to noise ratios.
  • the screening methods provided are fast and easy because they involve only one or a few steps. No surface modifications or blocking procedures are typically required during the assay procedures.
  • composition of glycans on the arrays can be varied as needed by one of skill in the art.
  • Many different glycoconjugates can be incorporated into the arrays including, for example, naturally occurring or synthetic glycans, glycoproteins, glycopeptides, glycolipids, bacterial and plant cell wall glycans and the like. Immobilization procedures for attaching different glycans to the arrays are readily controlled to easily permit array construction.
  • Spacer molecules or groups can be used to link the glycans to the arrays.
  • spacer molecules or groups include fairly stable (e.g. substantially chemically inert) chains or polymers.
  • the spacer molecules or groups can be alkylene groups.
  • One example of an alkylene group is-(CH2) n-, where n is an integer of from 1 to 10.
  • the arrays are made by obtaining a library of glycan molecules, attaching linking moieties to the glycans in the library, obtaining a solid support that has a surface derivatized to react with the specific linking moieties present on the glycans of the library and attaching the glycan molecules to the solid support by forming a covalent linkage between the linking moieties and the derivatized surface of the solid support.
  • the derivatization reagent can be attached to the solid substrate via carbon- carbon bonds using, or example, substrates having (poly) trifluorochloroethylene surfaces, or more preferably, by siloxane bonds (using, for example, glass or silicon oxide as the solid substrate). Siloxane bonds with the surface of the substrate are formed in one embodiment via reactions of derivatization reagents bearing trichiorosilyl or trialkoxysilyl groups.
  • a glycan library can be employed that has been modified to contain primary amino groups.
  • the glycans can have amino moieties provided by attached alkylamine groups, amino acids, peptides, or proteins.
  • the glycans can have alkylamine groups such as the-OCH 2 C H 2 NH 2 (called Spi) or- OCH 2 CH 2 CH 2 NH 2 (called Sp2 or Sp3) groups attached that provide the primary amino group.
  • the primary amino groups on the glycans can react with an N-hydroxy succinimide (NHS)- derivatized surface of the solid support.
  • NHS-derivatized solid supports are commercially available.
  • NHS-activated glass slides are available from Accelr8 Technology Corporation, Denver, CO. After attachment of all the desired glycans, slides can further be incubated with ethanolamine buffer to deactivate remaining NHS functional groups on the solid support.
  • the array can be used without any further modification of the surface. No blocking procedures to prevent unspecific binding are typically needed.
  • Each type of glycan is contacted or printed onto to the solid support at a defined glycan probe location.
  • a microarray gene printer can be used for applying the various glycans to defined glycan probe locations. For example, about 0.1 nL to about 10 nL, or about 0.5 nL of glycan solution can be applied per defined glycan probe location.
  • concentrations of the glycan solutions can be contacted or printed onto the solid support. For example, a glycan solution of about 0.1 to about 1000 micromolar glycan or about 1.0 to about 500 micromolar glycan or about 10 to about 100 micromolar glycan can be employed.
  • each concentration may be advisable to apply to a replicate of several (for example, three to six) defined glycan probe locations.
  • Such replicates provide internal controls that confirm whether or not a binding reaction between a glycan and a test molecule is a real binding interaction.
  • glycans that bind to antibodies in test samples from cancer patients include ceruloplasmin, Neu5Gc(2-6) GaINAc, GMl, Sulfo-T, Globo-H, sialylated Tn (Neu5Ac-alpha6-GalNAc-alpha) and LNT-2.
  • Additional glycans to which antibodies from breast cancer patients bind include circulating antibodies from breast cancer patients can also react with the following glycans: Tri-LacNAc, LacNAc-LeX-LeX, LacNAc-LacNAc (glycan 76), H-type-2-LacNAc, H-type2-LacNAc-LacNAc, GlcNAc3LacNAc, SLeXLacNAc, 3' SialylDiLacNAc, 3' Sialyl-tri-LacNAc, 6Sia-LacNAc-LeX-LeX, 6SiaLacNAc- LacNAc. Because cancer patients have antibodies that can these glycans and the presence of such antibodies is indicative of breast cancer, many of these glycans should be present on glycan arrays used for detecting breast cancer.
  • Ceruloplasmin is human glycoprotein detectable in serum. Ceruloplasmin is mainly expressed and secreted by hepatocytes and is involved in copper metabolism and/or storage. See, e.g., Aouffen et a! 2001, Biochem Cell Biol, 79(4), 489-97; Wang et al, Oncogene, 2002, 21, 7598-7604; Chakravarty et al., Evaluation of Ceruloplasmine concentration in prognosis of human cancer, 1986, Acta, Med, Okayama 40 (2) 103-5; Senra et a!, Serum ceruloplasmine as a diagnoistic marker of cancer 1997, 121, 139-45.
  • Human ceruloplasmin (CAS Number 903 1-37-2) can be obtained from the Sigma-Aldrich Co., St. Louis, MO (catalog no. C4519). The entire ceruloplasmin glycoprotein can be printed or otherwise attached to a solid support during formation of a glycan array useful for detecting breast cancer.
  • glycans to which antibodies from metastatic breast cancer patients bind include Neu5Gc(2-6)GalNAc, GMl, Sulfo-T, Globo-H, Sialylated Tn and LNT-2.
  • GM 1 has the following structure: Gal-beta3-GalNAc-beta4-[Neu5Ac- alpha3]-Gal-b eta4-Glc-b eta.
  • the Sulfo-T antigens are T-antigens with sulfate residues.
  • T antigens have the structure Gal33GalNAc and the galactose sugar moieties of this glycan can have sulfate groups or other substituents.
  • Globo-H includes glycans with fucose-alpha2-Gal-beta3-GalNAc-beta3-Gal- alpha4-Gal-beta4-Glc.
  • the sialylated Tn glycan has the following structure: Neu5Ac-alpha6-GalNAc- alpha.
  • LNT-2 is a ligand for tumor-promoting Galectin-4. See Huflejt & Leffler (2004) Glycoconjugate J, 20: 247-255.
  • the structure of LNT-2 includes the following glycan: GIcNAc- beta3 -Gal-beta4-Glc-beta.
  • Anti-glycan antibodies produced by methods provided herein or a nanoparticle vaccine covalently linked to a plurality of glycan-containing molecules are effective when following a vaccination protocol and can enhance the memory or secondary immune response to cancerous cells in the patient.
  • Anti-glycan antibodies can be combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines and cell surface antigens, or used alone, to stimulate immunity.
  • an immunogenic agent such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines and cell surface antigens, or used alone, to stimulate immunity.
  • Methods provided herein to produce anti-glycan antibodies can boost GM-CSF- modified tumor cell vaccines to improve efficacy of vaccines in a number of experimental tumor models such as mammary carcinoma (Hurwitz et al, 1998, supra), primary prostate cancer (Hurwitz et al., Cancer Research, 60: 2444-8, 2000) and melanoma (van Elsas et al, J. Exp. Med., 190: 355-66, 1999).
  • non-immunogenic tumors such as the B 16 melanoma, have been rendered susceptible to destruction by the immune system.
  • the tumor cell vaccine can also be modified to express other immune activators such as IL2, and costimulatory molecules, among others.
  • Antineoplastic agent is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis is frequently a property of antineoplastic agents.
  • Chemotherapeutic agents can be used in combination with polyclonal anti- glycan antibodies in methods for treatment of neoplastic disease.
  • An antibody-cytotoxin conjugate comprising anti-glycan antibodies can also be used to boost immunity induced through standard cancer treatments. In these instances, it can be possible to reduce the dose of chemotherapeutic reagent administered (Mokyr et al, Cancer Research 58: 5301-5304, 1998).
  • the scientific rationale behind the combined use of anti-glycan antibodies is that cell death, that is a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway.
  • anti-glycan antibodies can boost an immune response primed to chemotherapy release of tumor cells.
  • a "solid tumor” includes, but is not limited to, sarcoma, melanoma, carcinoma, or other solid tumor cancer.
  • Sarcoma refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcom
  • Melanoma refers to a tumor arising from the melanocyte system of the skin and other organs.
  • Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.
  • Carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas further include, for example, epithelial cancer, colorectal carcinoma, gastric carcinoma, oral carcinoma, pancreatic carcinoma, ovarian carcinoma, or renal cell carcinoma.
  • Exemplary carcinomas further include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matr
  • Leukemia refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood—leukemic or aleukemic (subleukemic).
  • Leukemia includes, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocyte leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leuk
  • Additional cancers include, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer.
  • a method for producing anti-glycan antibodies in a vertebrate subject comprises administering to the vertebrate subject a vaccine comprising protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent, and isolating the anti-glycan antibodies from a biological sample of the vertebrate subject.
  • the antibodies can be produced in a vertebrate subject, e.g., immunoglobulin G (IgG) antibodies in a mammalian subject or immunoglobulin Y (IgY) antibodies in an avian subject.
  • Immunoglobulin Y is the major antibody found in eggs of birds, reptiles and amphibia, including chicken, Gallus domesticus. IgY can be used as an alternative to mammalian antibodies normally used in research, and its use in immunotherapy has recently been proposed. IgY is the functional equivalent of IgG in mammals. From an evolutionary perspective, IgY antibodies are considered to be the ancestor of mammalian IgG and IgE antibodies. Compared to mammalian antibodies, IgY possesses several biochemical advantages, including ease of purification from eggs.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Human light chains are classified as kappa and lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)) (incorporated by reference in its entirety for all purposes).
  • the variable regions of each light/heavy chain pair form the antibody binding site.
  • an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.
  • the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • both light and heavy chains comprise the domains FRl , CDRl, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. MoI. Biol. 196: 901-917, 1987; Chothia et ⁇ /., Nature 342: 878-883, 1989.
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79: 315-321, 1990, Kostelny et al., J. Immunol. 148: 1547-1553, 1992.
  • bispecific antibodies may be formed as "diabodies” (Holliger et al, PNAS USA 90: 6444-6448, 1993 or "Janusins" (Traunecker et al., EMBO J.
  • Bispecific antibodies do not exist in the form of fragments having a single binding site ⁇ e.g., Fab, Fab', and Fv).
  • Immunoglobulin Y is the major antibody found in eggs from chicken, Gallus domesticus. IgY can be used as an alternative to mammalian antibodies normally used in research, and its use in immunotherapy has recently been proposed. Compared to mammalian antibodies, IgY possesses several biochemical advantages and its simple purification from egg yolk prevents a stressful moment in animal handling, as no bleeding is necessary.
  • IgY Specific IgY against Pseudomonas aeruginosa given orally prolongs the time of intermittent colonization by six months, decrease the number of positive colonizations and might be a useful complement to antibiotic treatment. Immunoglobulin therapy may diminish the development of antibiotic resistant microorganisms.
  • IgY antibodies are the predominant serum immunoglobulin in birds, reptiles and amphibia, and are transferred in the female from serum to egg yolk to confer passive immunity to embryos and neonates. This process corresponds to placental IgG transfer in mammals, which confers passive immunity to the fetus. IgY is the functional equivalent of IgG. From an evolutionary perspective, IgY antibodies are considered to be the ancestor of mammalian IgG and IgE antibodies.
  • IgY immunogenicity against conserved mammalian proteins, due to the phylogenetic distance between donor and recipient organisms. This makes production of antibodies against conserved mammalian proteins generally more successful in chickens than in other mammals. In addition, IgY antibodies tend to recognize the same protein in a number of mammalian species, making them more widely applicable.
  • IgY antibodies are concentrated in egg yolks.
  • the isolation process involves separation of yolks from egg whites, followed by the purification of antibodies in yolks from lipids and other materials. Different materials have been used and various methods were developed, including polyethylene glycol (PEG) precipitation, DEAE fractionation, chloroform extraction, water dilution, precipitation with dextran sulphate or dextran blue or xanthan gums, separation in a two-phase system (phosphate and Triton X-100), a freeze-thaw cycle coupled with gel filtration on Biogel P-150.
  • PEG polyethylene glycol
  • DEAE fractionation DEAE fractionation
  • chloroform extraction water dilution
  • precipitation with dextran sulphate or dextran blue or xanthan gums separation in a two-phase system (phosphate and Triton X-100)
  • phosphate and Triton X-100 phosphate and Triton X-100
  • freeze-thaw cycle coupled with gel
  • a chicken usually lays about 280 eggs in a year and an egg yolk contains 100- 150 mg of IgY antibodies. This can result in 28 to 42 grams of IgY per year from each chicken through eggs. It was shown that antigen-specific IgY antibodies were between 2% and 10% of the total IgY harvested. As the industrialized scale of hosting and caring for millions of chickens has been well developed in industry, the production of IgY antibodies can be readily scalable [0132] Despite the similarities between IgY and IgG antibodies, there are some profound differences in their chemical structures. The IgY heavy chain is 65-70 kDa, whereas the molecular mass of the mammalian IgG heavy chain is approximately 50 kDa.
  • the IgY light chain is 19-21 kDa; the IgG is 22-23 kDa.
  • the greater molecular mass of IgY is due to an increased number of heavy-chain constant domains and an extra pair of carbohydrate chains.
  • the hinge region of IgY is shorter and less flexible compared to that of mammalian IgG.
  • IgY is a more hydrophobic molecule than IgG, which matches the lipid-rich environment of the egg yolk.
  • the structural and the amino acid sequence differences determine the differences between the two types of antibodies in their biochemical features and immunological functions.
  • IgY antibodies of avian species do not bind to bacterial Fc receptors such as staphylococcal protein A or streptococcal protein G, indicating the immunological difference of the Fc region from that of IgG, although there has been a study showing that the protein A- reactive site was generated after IgY bound antigen.
  • Chicken egg-yolk immunoglobulins do not react with mammalian IgG nor IgM, neither with human anti-mouse IgG antibodies (HAMA), nor binding to the rheumatoid factor (RF), which is an antiimmunoglobulin autoantibody found in many different diseases.
  • IgY antibodies were shown to have high avidity against bacterial or human proteins (4) The immunoprecipitation characteristics of IgY are different from that of IgG, presumably due to the different structure of their hinge regions. (5) IgY is stable at pH 4-9 and temperature up to 65°C in aqueous condition, which is different from the stability of IgG at pH 3-10 and temperature up to 70 0 C. However, the resistance of IgY to the more extreme pH ranges increases if high salt conditions or stabilizing reagents such as sorbitol are present. IgY was reported to be stable at 40°C for an extended period.
  • Polypeptide fragment refers to a polypeptide that has an amino- terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acids long, more preferably at least 20 amino acids long, usually at least 50 amino acids long, and even more preferably at least 70 amino acids long.
  • analog refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and which has at least one of the following properties: (1) specific binding to glycan-containing molecules, under suitable binding conditions, (2) ability to infection or replication of infectious bacteria, viruses, fungi, or parasites in vitro or in vivo; or (3) ability to inhibit tumor cell growth in vitro or in vivo.
  • polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence.
  • Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.
  • Peptide analogs are commonly used in the pharmaceutical industry as non- peptide drugs with properties analogous to those of the template peptide. These types of non- peptide compound are termed "peptide mimetics" or "peptidomimetics". Fauchere, J. Adv. Drug Res. 15: 29, 1986; Veber and Freidinger TINS p. 392 (1985); and Evans et al. J. Med. Chem. 30: 1229, 1987, which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • a paradigm polypeptide ⁇ i.e., a polypeptide that has a biochemical property or pharmacological activity
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type ⁇ e.g., D-lysine in place of L-lysine may be used to generate more stable peptides.
  • constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch ⁇ r ⁇ j. Rev. Biochem. 61: 387, 1992, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • the term "substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.
  • amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% homology.
  • conservative amino acid replacements are contemplated.
  • Conservative amino acid replacement does not against the overall homology which can be maintained at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% homology.
  • Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • More preferred families are: serine and threonine are aliphatic- hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family.
  • Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases.
  • computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253: 164, 1991.
  • sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.
  • Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs.
  • Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally- occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts.
  • a conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N. Y. (1991)); and Thornton et at. Nature 354: 105, 1991, which are each incorporated herein by reference.
  • Antibody or "antibody peptide(s)” refer to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab') 2 , Fv, and single-chain antibodies.
  • An intact “antibody” comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • HCVR heavy chain variable region
  • the heavy chain constant region is comprised of three domains, CHi, CH 2 and CH 3 .
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.
  • the term antibody includes antigen-binding portions of an intact antibody that retain capacity to bind glycan-containing molecules.
  • binding examples include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and CHl domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, Nature 341: 544-546, 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the V L , V H , C L and CHl domains
  • F(ab') 2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • An antibody other than a "bispecific” or “bifunctional” antibody is understood to have each of its binding sites identical.
  • An antibody substantially inhibits adhesion of a receptor to a counterreceptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay).
  • Fab antibodies or "Fab fragments” refers to antibody fragments lacking all or part of an immunoglobulin constant region, and containing the Fab regions of the antibodies. Fab antibodies are prepared as described herein.
  • Single chain antibodies or “single chain Fv (scFv)” refers to an antibody fusion molecule of the two domains of the Fv fragment, V L and V H - Although the two domains of the Fv fragment, V L and V H , are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al, Science 242: 423-426, 1988; and Huston et al, Proc. Natl. Acad. Sci. USA, 85: 5879-5883, 1988).
  • Such single chain antibodies are included by reference to the term “antibody” fragments can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.
  • Human sequence antibody includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences.
  • the human sequence antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences ⁇ e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • Such antibodies can be generated in non- human transgenic animals, e.g., as described in PCT Publication Nos. WO 01/14424 and WO 00/37504.
  • human sequence antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., humanized antibodies).
  • recombinant immunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567, incorporated herein by reference in its entirety and for all purposes; and Queen et al., Proc. Natl Acad. Sci. USA 86: 10029-10033, 1989.
  • Monoclonal antibody refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • the term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions (if present) derived from human germline immunoglobulin sequences.
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • Polyclonal antibody refers to a preparation of more than 1 (two or more) different antibodies to glycan-containing molecules. Similarly antibodies to glycan-containing molecules can act as peptidomimetics that bind to cell surface glycans and thus inhibit interaction with cells presenting glycans on a cell surface. These and other antibodies suitable for use in the present invention can be prepared according to methods that are well known in the art and/or are described in the references cited here.
  • anti-glycan antibodies used in the invention are mammalian antibodies or avian antibodies— e.g., antibodies isolated from a mammalian species, e.g., human, sheep, dogs, cows, pigs, or an avian species, e.g., chicken.
  • mammalian antibodies or avian antibodies e.g., antibodies isolated from a mammalian species, e.g., human, sheep, dogs, cows, pigs, or an avian species, e.g., chicken.
  • Immuno cell response refers to the response of immune system cells to external or internal stimuli ⁇ e.g., antigen, glycan-containing molecules, cell surface receptors, cytokines, chemokines, and other cells) producing biochemical changes in the immune cells that result in immune cell migration, killing of target cells, phagocytosis, production of antibodies, other soluble effectors of the immune response, and the like.
  • external or internal stimuli e.g., antigen, glycan-containing molecules, cell surface receptors, cytokines, chemokines, and other cells
  • Immuno response refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, metastatic epithelial cancer, colorectal carcinoma, gastric carcinoma, oral carcinoma, pancreatic carcinoma, ovarian carcinoma, or renal cell carcinoma, or infectious disease from invading pathogens, cells or tissues infected with pathogens, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • T lymphocyte response and “T lymphocyte activity” are used here interchangeably to refer to the component of immune response dependent on T lymphocytes (e.g., the proliferation and/or differentiation of T lymphocytes into helper, cytotoxic killer, or suppressor T lymphocytes, the provision of signals by helper T lymphocytes to B lymphocytes that cause or prevent antibody production, the killing of specific target cells by cytotoxic T lymphocytes, and the release of soluble factors such as cytokines that modulate the function of other immune cells).
  • T lymphocyte response e.g., the proliferation and/or differentiation of T lymphocytes into helper, cytotoxic killer, or suppressor T lymphocytes, the provision of signals by helper T lymphocytes to B lymphocytes that cause or prevent antibody production, the killing of specific target cells by cytotoxic T lymphocytes, and the release of soluble factors such as cytokines that modulate the function of other immune cells).
  • compositions comprising one or a combination of antibodies, e.g., anti-glycan antibodies, or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan-containing molecules, formulated together with a pharmaceutically acceptable carrier.
  • Some compositions include a combination of multiple (e.g., two or more) polyclonal antibodies or antigen-binding portions thereof of the invention.
  • each of the antibodies or antigen-binding portions thereof of the composition is a polyclonal antibody or a human sequence antibody that binds to a distinct, pre-selected epitope of an antigen.
  • compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition (i.e., a neoplastic disease or infectious disease) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • a disease or condition i.e., a neoplastic disease or infectious disease
  • compositions or medicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease.
  • An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose.
  • agents are usually administered in several dosages until a sufficient immune response has been achieved. Typically, the immune response is monitored and repeated dosages are given if the immune response starts to wane.
  • Effective doses of the antibody compositions of the present invention e.g., anti- glycan antibodies produced by the methods of the present invention, or vaccines comprising a nanoparticle, at least some of the nanoparticles covalently bound to glycan-containing molecules, for the treatment of cancer-related conditions, e.g., metastic cancer, or for treatment of infectious disease as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the vertebrate subject, whether the patient is a vertebrate, e,g., mammalian or avian species, human, primate, rat, mouse, dog, cat, rabbit, cow, horse, or goat, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the vertebrate subject is a human but nonhuman mammals, including transgenic mammals, and avian species can also be treated. Treatment dosages need to be titrated to optimize safety and efficacy.
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1- 10 mg/kg.
  • An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months.
  • two or more antibodies, or two or more plant viral particles with different binding specificities are administered simultaneously, in which case the dosage of each antibody or nanoparticle vaccine administered falls within the ranges indicated.
  • Antibody or nanoparticle vaccine is usually administered on multiple occasions.
  • Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 ⁇ g/ml and in some methods 25-300 ⁇ g/ml.
  • antibody or nanoparticle vaccine can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody or nanoparticle vaccine in the patient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.
  • Doses for antibody or nanoparticle vaccine range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ⁇ g to 10 mg, or 30-300 ⁇ g antibody per patient. Doses for nanoparticle vaccine vary from 10-100, or more, particles per dose.
  • Antibody compositions of the present invention e.g., anti-glycan antibodies produced by the methods of the present invention, or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan-containing molecules, for the treatment of cancer- related conditions, e.g., metastic cancer, or for treatment of infectious disease as described herein can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic as inhalants for antibody preparations targeting brain lesions, and/or therapeutic treatment.
  • the most typical route of administration of an immunogenic agent or nanoparticle vaccine is subcutaneous although other routes can be equally effective.
  • intramuscular injection This type of injection is most typically performed in the arm or leg muscles.
  • agents are injected directly into a particular tissue where deposits have accumulated, for example intracranial injection.
  • Intramuscular injection on intravenous infusion are preferred for administration of antibody.
  • particular therapeutic antibodies are injected directly into the cranium.
  • antibodies are administered as a sustained release composition or device, such as a MedipadTM device.
  • Antibodies or nanoparticle vaccines of the invention can optionally be administered in combination with other agents that are at least partly effective in treating various diseases including various cancer-related diseases.
  • antibodies or nanoparticle vaccines can also be administered in conjunction with other agents that increase passage of the agents of the invention across the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • Antibody compositions of the present invention e.g., anti-glycan antibodies produced by the methods of the present invention, or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan-containing molecules, for the treatment of cancer- related conditions, e.g., metastatic cancer, or for treatment of infectious disease as described herein are often administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components.
  • an active therapeutic agent i.e., and a variety of other pharmaceutically acceptable components.
  • compositions can also include, depending on the formulation desired, pharmaceutically- acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functional ized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).
  • compositions of antibodies or nanoparticle vaccine of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol.
  • a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol.
  • auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions.
  • Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil.
  • glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • Antibodies can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient.
  • An exemplary composition comprises monoclonal antibody at 5 mg/mL, formulated in aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-1 19, 1997.
  • the agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.
  • binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
  • Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%- 95% of active ingredient, preferably 25%-70%.
  • Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins. Glenn et al., Nature 391: 851, 1998. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.
  • transdermal delivery can be achieved using a skin patch or using transferosomes. Paul et al., Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.
  • compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • antibodies and antibody compositions of the invention of any epitope binding specificity and very high binding affinity to any desired antigen.
  • the higher the binding affinity of an antibody for its target the more stringent wash conditions can be performed in an immunoassay to remove nonspecifically bound material without removing target antigen.
  • antibodies and antibody compositions of the invention used in the above assays usually have binding affinities of at least 10 8 , 10 9 , 10 10 , 10 11 or 10 12 M " '.
  • it is desirable that antibodies used as diagnostic reagents have a sufficient on-rate to reach equilibrium under standard conditions in at least 12 hours, preferably at least five hours and more preferably at least one hour.
  • Antibodies and antibody compositions of the invention used in the claimed methods preferably have a high immunoreactivity, that is, percentages of antibodies molecules that are correctly folded so that they can specifically bind their target antigen. Such can be achieved by expression of sequences encoding the antibodies in E. coli as described above. Such expression usually results in immunoreactivity of at least 80%, 90%, 95% or 99%.
  • Some methods of the invention employ polyclonal preparations of antibodies and antibody compositions of the invention as diagnostic reagents.
  • the use of polyclonal mixtures has a number of advantages with respect to compositions made of one monoclonal antibody.
  • polyclonal antibodies or other polypeptides can generate a stronger signal (for diagnostics) than a monoclonal that binds to a single site.
  • a polyclonal preparation can bind to numerous variants of a prototypical target sequence ⁇ e.g., allelic variants, species variants, strain variants, drug-induced escape variants) whereas a monoclonal antibody may bind only to the prototypical sequence or a narrower range of variants thereto.
  • the preparation typically contains an assortment of antibodies with different epitope specificities to the intended target antigen.
  • a difference in epitope binding specificities can be determined by a competition assay.
  • Samples and Target Although human or avian antibodies can be used as diagnostic reagents for any kind of sample, and are useful as diagnostic reagents for human samples. Samples can be obtained from any tissue or body fluid of a patient. Preferred sources of samples include, whole blood, plasma, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. Samples can also be obtained from biopsies of internal organs or from cancers. Samples can be obtained from clinical patients for diagnosis or research or can be obtained from undiseased individuals, as controls or for basic research.
  • target antigens including tumor antigens, for example, tumor antigens for metastatic epithelial cancer, colorectal carcinoma, gastric carcinoma, oral carcinoma, pancreatic carcinoma, ovarian carcinoma, or renal cell carcinoma, or target antigens or infectious bacteria, virus, fungi or parasites.
  • target antigens are human proteins whose expression levels or compositions have been correlated with human disease or other phenotype. Examples of such antigens include adhesion proteins, hormones, growth factors, cellular receptors, autoantigens, autoantibodies, and amyloid deposits.
  • targets of interest include tumor cell antigens, such as carcinoembryonic antigen.
  • Other antigens of interest are class I and class II MHC antigens.
  • Immunometric or sandwich assays are a preferred format. See U.S. Pat. Nos. 4,376,110; 4,486,530; 5,914,241 ; and 5,965,375, each incorporated herein by reference in their entirety and for all purposes.
  • Such assays use one antibody or population of antibodies immobilized to a solid phase, and another antibody or population of antibodies in solution. Typically, the solution antibody or population of antibodies is labelled. If an antibody population is used, the population typically contains antibodies binding to different epitope specificities within the target antigen. Accordingly, the same population can be used for both solid phase and solution antibody. Solid phase and solution antibodies can be contacted with target antigen in either order or simultaneously.
  • the assay is referred to as being a forward assay. Conversely, if the solution antibody is contacted first, the assay is referred to as being a reverse assay. If target is contacted with both antibodies simultaneously, the assay is referred to as a simultaneous assay. After contacting the target with antibody, a sample is incubated for a period that usually varies from about 10 min to about 24 hr and is usually about 1 hr. A wash step is then performed to remove components of the sample not specifically bound to the antibody being used as a diagnostic reagent. When solid phase and solution antibodies are bound in separate steps, a wash can be performed after either or both binding steps.
  • binding is quantified, typically by detecting label linked to the solid phase through binding of labelled solution antibody.
  • a calibration curve is prepared from samples containing known concentrations of target antigen. Concentrations of antigen in samples being tested are then read by interpolation from the calibration curve. Analyte can be measured either from the amount of labelled solution antibody bound at equilibrium or by kinetic measurements of bound labelled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of target in a sample.
  • Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, and also particles, such as agarose, a dextran-based gel, dipsticks, particulates, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEXTM. (Amersham Pharmacia Biotech, Piscataway N.J.) Immobilization can be by absorption or by covalent attachment.
  • antibodies can be joined to a linker molecule, such as biotin for attachment to a surface bound linker, such as avidin.
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H 5 14 C, 35 S, 125 I, 121 I, 112 In, "mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18 F, 11 C, 15 O, (for Positron emission tomography), 99m TC, 111 In (for Single photon emission tomography), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g.
  • magnetic beads e.g., DynabeadsTM
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti- ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • an anti- ligand e.g., streptavidin
  • a number of ligands and anti-ligands can be used.
  • a ligand has a natural anti- ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands.
  • any haptenic or antigenic compound can be used in combination with an antibody.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbel liferone, and the like
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple calorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
  • the antibody compositions of the present invention e.g., anti-glycan antibodies produced by the methods of the present invention, or vaccines comprising a plant viral particle displaying a plurality of glycan-containing molecules, for the treatment of cancer-related conditions, e.g., metastic cancer, or for treatment of infectious disease as described herein, will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • a therapeutically effective dose of the antibody compositions of the present invention e.g., anti-glycan antibodies produced by the methods of the present invention, or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan- containing molecules, for the treatment of cancer-related conditions, e.g., metastic cancer, or for treatment of infectious disease as described herein will provide therapeutic benefit without causing substantial toxicity.
  • cancer-related conditions e.g., metastic cancer
  • infectious disease as described herein will provide therapeutic benefit without causing substantial toxicity.
  • Toxicity of the antibodies or nanoparticle vaccine described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 (the dose lethal to 50% of the population) or the LDioo (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of the antibodies or plant viral particles described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et ai, 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1,
  • kits comprising the compositions ⁇ e.g., polyclonal anti-glycan antibodies or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan-containing molecules) of the invention and instructions for use.
  • the kit can further contain a least one additional reagent, or one or more additional human antibodies of the invention ⁇ e.g., a human antibody having a complementary activity which binds to an epitope in the antigen distinct from the first human antibody).
  • Kits typically include a label indicating the intended use of the contents of the kit.
  • the term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • Carbohydrates 1-4 (Scheme 1) - each significant in biology and human pathology (see Supporting Information) - were prepared as 2-azidoethanol adducts by enzymatic and chemical methods as previously described.
  • the 30-nm wild-type cowpea mosaic virus (CPMV) particle was acylated on exterior lysine side chains with N- hydroxysuccinimide linkers 5 or 6, containing azide or alkyne groups, respectively (Scheme 1, bottom).
  • CPMV cowpea mosaic virus
  • the CuAAC method allows for the convenient covalent deposition of minimal quantities of these highly valuable compounds on proteins, allowing us to achieve high loadings on the viral surface with the use of relatively modest amounts of material.
  • the virus-carbohydrate conjugates were used to inoculate chickens by intramuscular injection, in order to evaluate their capacities for inducing an IgY immune response.
  • the avian IgY isotype is the functional equivalent of mammalian IgG and is considered to be its evolutionary precursor.
  • IgY's have several practical advantages over IgG's for use as polyclonal reagents, including large isolated yields from the eggs of immunized hens and reduced cross-reactivity with native mammalian proteins.
  • Total IgY was isolated by poly(ethyleneglycol) precipitation from eggs collected from the immunized birds, providing approximately 1 gram of polyclonal antibody per experiment from a total of 12 eggs per bird. Unless otherwise indicated, the antibodies obtained in this manner were used without further purification. Each inoculation was performed on two chickens in parallel and the results analyzed independently.
  • oligosaccharides In addition to chicken, the attachment of oligosaccharides to the exterior surfaces of capsids derived from bacteriophage Q-beta and cowpea mosaic virus has also resulted in effective anti-glycan immune response in mice.
  • the ganglioside GM3 carbohydrate (Neu5Ac ⁇ 2-3Gal ⁇ l-4Glc ⁇ -l inker) was conjugated to Q-beta and administered in the standard manner.
  • Binding specificities were determined by analysis of the total serum antibody on an extensive glycan array, covering all major glycan types. Similar experiments were also performed with CPMV and the Tn antigen, which produced similar strong immune response (see, Miermont et al., Chem. Eur. J. 2008, 14, 4939).
  • Antibody specificities are assayed on a glycan microarray
  • Figure 1 shows glycan array binding analysis of crude IgY isolated after (a) no immunization (array vl .O); (b) immunization with 11 (array v2.0); and (c) immunization with 13 (array vl .O).
  • the dominant motif recognized by these antibodies is shown in the box; a key to the symbols used is boxed in panel b.
  • a few strong peaks are provided by non- sialyl carbohydrates that are likely to be present in serum due to bacterial exposure, such as 127 (a peptidoglycan from gram negative bacterial cell wall) and 200 (rhamnose, a common bacterial sugar).
  • Polyclonal IgY antibodies can be enriched through affinity purification to increase specificity
  • Figure 2 shows glycan array binding analysis using array version vl .O of anti-2 antibodies purified by affinity filtration through agarose-18: (a) total IgY from immunization with 12; in the box is the cartoon representation of the structure of the virus-displayed carbohydrate; (b) flow-through after washing; (c) binding profile of antibodies eluted with pH 2 buffer followed by neutralization to pH 7. (d) ELISA assay for anti-CPMV antibodies of the material obtained at each step of affinity purification of polyclonal anti-12, at identical protein concentrations. These data show that CPMV-binding antibodies are removed in the flow-through step, and are therefore not present in the glycan-binding "elution" fraction. To the right of (d) is the LacNAc structure attached to agarose-alkyne to create the affinity purification column.
  • Globo-H decorated CPMV elicits polyclonal antibodies with comparable specificity to a monoclonal antibody
  • Danishefsky and coworkers have administered a KLH-globo-H conjugate to cancer patients and characterized the polyclonal immune response in comparison to bacterial polysaccharide vaccines (Wang et al., Proc. Natl. Acad. Sci. USA 2000, 97, 2719-2724; and Slovin et al., Proc. Natl. Acad. Sci. USA 1999, 96, 5710-5715).
  • the avian IgY response to this antigen was highly potent and selective, comparing favorably with, if not quite matching, the specificity of a commercially available monoclonal IgM antibody against globo-H ( Figure 3a).
  • Figure 3b displayed strongest binding to globo-H itself (glycan 56 on the array) and the H-type-3 trisaccharide Fuc((l-2)Gal((l-3)GalNAc(- (58), but also recognized the related ganglioside fucosyl-GMl (59, 60) and GlcNAc((l-2)Gal((l-3)GalNAc(- (158).
  • the avian IgY showed the strongest affinity for the same globo-H related antigens (56, 58), but also recognized a somewhat wider range of Fuc((l-2)Gal((l-3) structures including the H-type-1 (Fuc((l-2)Gal((l-3)GlcNAc sugars 63 and 64, and, more weakly, H-type-2 (Fuc((l-2)Gal((l-4)GlcNAc) and 2'-fucosyllactose (Fuc((l- 2)Gal((l-4)Glc) structures (69,72-74).
  • Compound 55 (Fuc((l-2)Gal((l-3)GalNAc((l-3)Gal(-, a terminal fragment of globo-H, was recognized much less well by both monoclonal IgM and polyclonal IgY antibodies than the full structure 56. Both antibodies distinguish between globo- H related antigens and Le b and Le y sugars that bear additional fucosylation adjacent to the terminal fucose unit (57, 65-68).
  • Figure 3 shows glycan array binding analysis using array version v2.0 of (a) anti-14 IgY (0.15 mg/mL), and (b) commercially available monoclonal IgG against globo-H (0.035 mg/mL, Axorra, Inc.).
  • array version v2.0 of (a) anti-14 IgY (0.15 mg/mL), and (b) commercially available monoclonal IgG against globo-H (0.035 mg/mL, Axorra, Inc.).
  • symbolic representations of the structures of the glycans most strongly bound are shown; a key to the symbols used is boxed in panel b.
  • the identification number and average signal intensities are listed for each glycan structurel.
  • the glycan array technology used here is a convenient and effective way to define anti-carbohydrate specificities, and will have two important uses in our future studies.
  • the binding profiles provided by the arrays represent a wealth of information that we hope will allow us to correlate antibody properties with the structures and conditions of immunization.
  • glycan array profiling is a highly useful method of quality control for the development of carbohydrate-targeted reagents and immunotherapies.
  • the trisaccharide (150 mg, 0.25 mmol) and UDP-GIcNAc (350mg, 0.57 mmol) were dissolved in aqueous buffer (25 mL) containing MnCl 2 (40 mM) and NAD + , (0.3 mM).
  • UDPGal-4'-epimerase (350 U) and GTA (10 U) were added and the pH was adjusted to 7.5. The reaction was stirred at 37°C for 24 hours to give 85-90% product formation as judged by thin-layer chromatography.
  • Virus-carbohydrate conjugates 11, 12, and 14. CPMV-azide (0.5 mL of 2 mg/mL solution) was incubated with complementary alkyne (8, 5 mM) in Tris buffer (0.1 M, pH 8, 0.5 mL) containing 9 (3 mM), CuSO 4 (2 mM) and tris(carboxyethyl)phosphine (4 mM), for 18 h at room temperature.
  • the resulting virus conjugates were purified by ultracentrifugation over a 10-40% sucrose gradient, pelleting of the recovered virus, and dissolution of the resulting material in potassium phosphate buffer (0.1 M, pH 7).
  • Mass recoveries of derivatized viruses were typically 60-80%; all such samples were composed of >95% intact particles as determined by analytical size-exclusion FPLC.
  • the carbohydrate loading on these conjugates was determined by quantitative measurement of fluorescein absorbance against calibrated protein-dye standards.
  • CPMV at 0.10 mg/mL gives a standard absorbance of 0.80, and fluorescein exhibits a molar absorbtivity of 70,000 M "1 cm "1 at 495 nm.
  • the average molecular weight of the CPMV virion is 5.6 x 10 6 g/mol.
  • Carbohydrate syntheses were prepared as 2-azidoethanol ⁇ - anomeric adducts by enzymatic and chemical synthesis as previously described (Vasiliu et al., Carb. Res. 2006, 341, 1447-1457; and Huang et al., Proc. Natl. Acad. Sci. USA 2006, 103, 15- 20).
  • a representative example (compound 1) of the last step and purification is given in the Experimental section.
  • the 1 H NMR (500 MHz, D 2 O) spectrum of 1 is shown in Figure 4.
  • the resulting suspension was bubbled with a gentle flow of nitrogen for 1 min, capped, and rotated at room temperature for 12-18 hours.
  • the reaction mixture was drained and washed sequentially with approximately 5 column volumes each of DMF, H 2 O, MeOH, 0.1 M aq EDTA, H 2 O and DMF to obtain carbohydrate-agarose beads.
  • a parallel experiment with a dye-azide under otherwise identical conditions provided highly colored beads confirming a positive reaction.
  • Bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) at 4 mg/mL in 0.1 M phosphate buffer (pH 7.0) were treated with 335 equiv. of NHS-alkyne linker 6.
  • the reactions were agitated by gentle rocking overnight at room temperature and the products purified by two rounds of dialysis in 1 liter of distilled H 2 O.
  • the resulting conjugates were degassed by gentle N 2 sparging before storing in under nitrogen atmosphere.
  • Fluorescein-azide and tri-LacNAc-azide (2) were joined with these alkyne- derivatized proteins under the same conditions as used for 12 and 13 (2 mg/mL protein, 0.3 mM azide, 1 mM CuOTf , 2 mM ligand 10, in 0.1 M Tris buffer, pH 8). Each conjugate was allowed to proceed under nitrogen for 18 hours at room temperature and purified by dialysis as described above.
  • the column was then washed with approximately 100 mL of standard buffer (PBS, pH 7.4) and then with 0.1 M glycine buffer (pH 2, 5 mL, "elution" fraction) which was immediately neutralized in 1.0 M Tris-HCl buffer (pH 8.0).
  • the flowthrough fraction was re-incubated in the mono-LacNAc agarose column for 1 hour and eluted in the same fashion twice more to ensure maximum yield of tri-LacNAc specific antibodies. After three such rounds, the elution fractions were pooled and concentrated to 1 mg/mL for analysis on the glycan array.
  • the concentrated "wash" fractions contained no protein as determined by uv-vis spectroscopy.
  • Figure 6 shows plots of ratios of significant signals (>20% of the maximum) from glycan array (v2.0) analyses of total IgY generated by different chickens (designated A and B) against (left) CPMV conjugate 15 and (right) KLH conjugate 16. Numerical data for these plots can be found in Supporting Information.
  • Figure 7 shows a repeat of the generation and analysis of anti-14 (globo-H on CPMV). Very good reproducibility was observed.
  • Figure 7 shows independent repeat of the experiment described in Figure 3, showing glycan array (v2.0) binding analysis of anti-14 IgY. The only significant difference between these data and the analysis shown in Figure 3 is the strong response to globoside Galqtl-4)Gal ⁇ l-4)Glc (glycan 1 1 1).
  • FIG. 8 shows plots of ratios of averaged signals from glycan array (v2.0) analyses of total IgY generated by two different chickens against CPMV conjugate 15 vs. KLH conjugate 16; all IgY samples were used at 0.1 mg/mL. To the right are the structures of the glycans exhibiting a greater than 100-fold difference in absolute signal intensity comparing anti- 15 to anti-16 or vice versa. For clarity, not all of the glycan numbers are shown on the x-axis.
  • Figure 9 shows plots of ratios of low-intensity signals ( ⁇ 20% of the maximum) from glycan array (v2.0) analyses of total IgY generated by two different chickens against CPMV conjugate 15 and KLH conjugate 16. Numerical data for this plot can be found in Table 4. To the right are the structures of the glycans exhibiting a greater than 50-fold difference in absolute signal intensity comparing anti-15 to anti-16 or vice versa.
  • Table 1 Test of reproducibility in IgY generation and glycan array analysis (v2.0) for total IgY derived from immunization with 15 (CPMV display; Figure 6). Samples derived from two different chickens are designated “exp. A” and “exp. B.”
  • Glycans giving average signals >20% of maximum intensity, ordered by glycan number.
  • Table 2 Test of reproducibility in IgY generation and glycan array analysis (v2.0) for total IgY derived from immunization with 16 (KLH display; Figure 6). Samples derived from two different chickens are designated “exp. A” and “exp. B.”
  • Glycans giving average signals >20% of maximum intensity, ordered by glycan number
  • Table 3 (data for Figure 8; comparison of CPMV and KLH platforms). Glycan array analysis (v2.0) for total IgY derived from immunization with 15 vs. 16. Average signals are derived from the independent experiments listed in Tables Sl and S2.
  • Glycans giving average signals >20% of maximum intensity for either ant ⁇ -15 or ant ⁇ -16, ordered by glycan number
  • G ra r g. signal for g. signal for Glycan structure lycan # tio 15/16 atio 16/15 anti-15 anti-16
  • G ra r g. signal for g. signal for Glycan structure lycan U tio 15/16 atio 16/15 anti-15 anti-16
  • GalNAc ⁇ l-3 (Fuc ⁇ l- 106 Gal ⁇ l-3Gal ⁇ l-4Glc ⁇ - Gal ⁇ l-3GlcNAc ⁇ l- 80 2)Gal ⁇ l-4(Fuc ⁇ l- SpO 3Gal ⁇ l -4GlcNAc ⁇ -SpO
  • GalNAc ⁇ l-4(Fuc ⁇ l- .. were Gal ⁇ l-3(Fuc ⁇ l- Gal ⁇ l-4GalNAc ⁇ l-

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Abstract

The invention provides vaccines that comprise protein nanoparticles at least some of which are covalently bound to glycan-containing molecules. The invention also provides methods for producing anti-glycan antibodies in a vertebrate subject. The invention further provides methods for treating cancer or infectious diseases.

Description

METHODS FOR PRODUCING ANTI-GLYCAN ANTIBODIES, VACCINES AND METHODS FOR TREATING CANCER OR INFECTIOUS DISEASE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Number 60/952,794 (filed July 30, 2007). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made by U.S. government support by Grant No. GM621 16 and CAl 12075 from National Institutes of Health. The Government has therefore certain rights in this invention.
FIELD OF INVENTION
[0003] The invention generally relates to a vaccine comprising protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules. Methods for producing anti-glycan antibodies in a vertebrate subject are provided. Methods for treatment or diagnosis of cancer or infectious disease in a vertebrate subject by administering the vaccine or the anti-glycan antibody to the subject are provided.
BACKGROUND
[0004] The development of anti-glycan antibodies has attracted considerable interest in recent years for diagnostic and therapeutic purposes, due to the aberrant expression of unique glycans on the proteins and lipids of specific cell types, but the breaking of immunological tolerance with carbohydrates is generally difficult. D. H. Dube et al. Nat. Rev. Drug Disc. 2005, 4, 477-488; S. Hakomori, Proc. Natl. Acad. Sci. USA 2002, 99, 225-232; B. E. Collins et al., Current Opinion in Chemical Biology 2004, 8, 1-9; S. F. Slovin et al. Immunology and Cell Biology 2005, 83, 418-428. Viruses are generally highly antigenic, and Seeberger and coworkers have taken advantage of influenza virosomes to elicit a strong and isotype-crossed immune response against a tetrasaccharide incorporated into the lipid membrane of the particle. X. Liuet al., ACS Chem. Biol. 2006, 1, 161-164. The presentation of peptide and other small-molecule antigens on viral protein scaffolds is known to generate strong immune responses, and the structures of many such scaffolds are known to atomic resolution. M. F. Bachmann et al., Science 1993, 262, 1448-1451 ; R. M. Zinkernagel, Science 1996, 271, 173-178; T. Fehr et al., Proc. Natl. Acad. Sci. USA 1998, 95, 9477-9481 ; C. Portaet al., Med. Virol. 1998, 8, 25-41.
[0005] Danishefsky and coworkers have displayed synthetic complex carbohydrates on keyhole limpet hemocyanin (KLH) as the carrier protein in the development of candidate vaccines. O. Ouerfelli et al., Expert Review of Vaccines 2005, 4, 677-685. In one example, a KLH conjugate displaying seven different carbohydrates was shown to elicit antibodies to all of the glycan immunogens. E. Kagan et al., Cancer Immunol. Immunother. 2005, 54, 524-430; G. Ragupathi et al., Cancer Immunol. Immunother. 2003, 52, 608-616; P. Livingston, Curr. Opin. Immunol. 1992, 4, 624-629. In these efforts, as in others, multivalency is important, but the structure of KLH is ill defined. In a remarkable example, a KLH conjugate displaying seven different carbohydrates was shown to elicit antibodies to all of the glycan immunogens. E. Kagan et al., Cancer Immunol. Immunother. 2005, 54, 524-430; G. Ragupathi et al., Cancer Immunol. Immunother. 2003, 52, 608-616; P. Livingston, Curr. Opin. Immunol. 1992, 4, 624- 629. Other laboratories have also turned towards multivalent display of different but related sugars such as gangliosides, which are commonly overexpressed in tumors of neuroectodermal origin. P. Livingston, Curr. Opin. Immunol. 1992, 4, 624-629. Several polyvalent scaffolds other than standard protein adjuvants have been used to display carbohydrates, including dendrimers, polymers made by ring-opening metathesis polymerization and other technologies, polyacrylamide chains, amino acids, liposomes, and small organic molecules fitted for a specific binding site. S. Andre et al., Glycobiology 1999, 9, 1253-1261 ; M. J. Cloninger, Curr. Opin. Chem. Biol. 2002, 6, 742-748; E. K. Woller et al., Org. Lett. 2002, 4, 7-10; E. K. Woller et al., J. Am. Chem. Soc. 2003, 125, 8820-8826; D. Page et al., Bioconj. Chem. 1997, 8, 714-723; D. Page et al., Bioorg. Med. Chem. 1996, 4, 1949-1961 ; J. E. Gestwicki et al., J. Am. Chem. Soc. 2002, 124, 14922-14933; L. Kiessling, Curr. Opin. Chem. Biol. 2000, 4, 696-703; K. H. Mortell et al., J. Am. Chem. Soc. 1994, 116, 12053-12054; K. H. Mortell et al., J. Am. Chem. Soc. 1996, 118, 2297-2298; W. J. Sanders et al., J. Biol. Chem. 1999, 274, 5271-5278; L. E. Strong et al., J. Am. Chem. Soc. 1999, 121, 6193-6196; M. Kanai et al., J. Am. Chem. Soc. 1997, 119, 9931- 9932; M. C. Schuster et al., J. MoI. Catal. A 1997, 116, 209-216; L. L. Kiessling et al., Chem. Biol. 1996, 3, 71-77; M. Kanai et al., J. Am. Chem. Soc. 1997, 119, 9931 -9932; M. C. Schuster et al., J. MoI. Catal. A 1997, 116, 209-216; R. Roy et al., Carb. Res. 1988, 777, C1-C4 ; W. Spevak et al., J. Am. Chem. Soc. 1993, 115, 1 146-1 147; S. A. DeFrees et al., J. Am. Chem. Soc. 1996, 118, 6101-6104; J. E. Kingery-Wood et al., J. Am. Chem. Soc. 1992, 114, 7303-7305; H. Kamitakahara et al., Angew. Chem. Int. Ed. 1998, 37, 1524-1528; M. Mammen et al., Chem. Biol. 1996, 3, 757-763; c) N. K. Sauter et al., Biochemistry 1989, 28, 8388-8396; G. B. Sigal et al., J. Am. Chem. Soc. 1996, 118, 3789-3800 ; J. Allen et al., Chem. Eur. J. 2000, 6, 1366-1375; J. Allen et al., J. Am. Chem. Soc. 2001, 123, 1890-1897 ; J. E. Kingery-Wood et al., J. Am. Chem. Soc. 1992, 114, 7303-7305; P. I. Kitov et al., Nature 2000, 4, 669-672.
[0006] A need exists in the art for a structurally more well defined platform for glycan display that induces strong antigenicity toward the attached glycan molecules. This will boost the efficiency of production of anti-glycan antibodies and also provide improved vaccines to treat cancer and infectious disease.
SUMMARY
[0007] The invention generally relates to a protein nanoparticle display platform for glycan-containing molecules wherein the protein nanoparticles display enhanced antigenicity for the glycan-containing moleucules. Vaccines are provided comprising protein nanoparticles, wherein at least some of the nanoparticles are covalently bound to glycan-containing molecules. Methods for producing anti-glycan antibodies in a vertebrate subject are provided. Methods for treatment or diagnosis of cancer or infectious disease are provided wherein the protein nanoparticle vaccine or anti-glycan antibody is administered to a vertebrate subject in need of such treatment for the disease.
[0008] Methods for producing anti-glycan antibodies in a vertebrate subject are provided which comprise administering a protein nanoparticle to the vertebrate subject, wherein at least some of the protein nanoparticles covalently bound to glycan-containing molecules. These methods provide cost efficient production of specific anti-glycan antibodies in high yields for diagnostic and therapeutic purposes. The glycan array technology allows for the efficient screening of a polyclonal set of antibodies for specific recognition of glycan motifs. These studies set the stage for similar efforts using the controlled structures, natural multivalency, and attachment chemistries available to virus particles as carrier proteins. The protein nanoparticles comprising a plurality of glycan-containing molecules can also be used for the development of a vaccine for treatment of cancer or infectious disease.
[0009] A vaccine is provided which comprises protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent. In one aspect, at least some of the protein nanoparticles are bound to a plurality of different glycan-containing molecules. The glycan- containing molecule can include, but are not limited to, a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide. The protein nanoparticle can include, but are not limited to, a virus, bacteriophage, nucleoprotein nanoparticle, viral nanoparticle, viral capsid particle, vault protein, dendrimer or chaperonin. In one aspect, the protein nanoparticle is a plant viral particle. The plant viral particle can be a Comovirus, Tombusvirus, Sobemovirus, or,Nepovirus. The plant viral particle can be a cowpea mosaic virus.
[0010] In one aspect, the protein nanoparticles are covalently bound to glycan- containing molecules through alkyne azide linkage. In a further aspect, the protein nanoparticles are covalently bound to glycan-containing molecules through a plurality of amino acid residues on the protein nanoparticle. The protein nanoparticles can be covalently bound to glycan- containing molecules through N-hydroxysuccinimide ester linkage. The vaccine is provided which comprises at least 100 glycan-containing molecules per protein nanoparticle, or at least 150 glycan-containing molecules per protein nanoparticle, or at least 200 glycan-containing molecules per protein nanoparticle.
[0011] A method for producing anti-glycan antibodies in a vertebrate subject is provided which comprises administering to the vertebrate subject a vaccine comprising protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent, and isolating the anti-glycan antibodies from a biological sample of the vertebrate subject. In one aspect, at least some of the protein nanoparticles are bound to a plurality of different glycan-containing molecules. In one aspect, the protein nanoparticles are covalently bound to glycan-containing molecules through alkyne azide linkage. In a further aspect, the protein nanoparticles are covalently bound to glycan-containing molecules through a plurality of amino acid residues on the protein nanoparticle. The protein nanoparticles can be covalently bound to glycan-containing molecules through N-hydroxysuccinimide ester linkage. In one aspect, the vertebrate subject is a mammalian subject or an avian subject. The biological sample can be one or more eggs of the avian subject. The biological sample can be serum of the mammalian subject or the avian subject. The vaccine is provided which comprises at least 100 glycan-containing molecules per protein nanoparticle, or at least 150 glycan-containing molecules per protein nanoparticle, or at least 200 glycan-containing molecules per protein nanoparticle.
[0012] An anti-glycan antibody is provided which can be isolated by the method for producing antibodies.
[0013] A method for treating cancer in a vertebrate subject comprising, administering to the vertebrate subject a vaccine is provided which comprises protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent, wherein the vaccine elicits an immune response to the plurality of glycan molecules to reduce or eliminate cancer in the vertebrate subject. In one aspect, at least some of the protein nanoparticles are bound to a plurality of different glycan- containing molecules. The glycan-containing molecule can include, but are not limited to, a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide. In one aspect, the protein nanoparticles are covalently bound to glycan-containing molecules through alkyne azide linkage. In a further aspect, the protein nanoparticles are covalently bound to glycan-containing molecules through a plurality of amino acid residues on the protein nanoparticle. The protein nanoparticles can be covalently bound to glycan-containing molecules through N-hydroxysuccinimide ester linkage. The vaccine is provided which comprises at least 100 glycan-containing molecules per protein nanoparticle, or at least 150 glycan-containing molecules per protein nanoparticle, or at least 200 glycan-containing molecules per protein nanoparticle.
[0014] A method for preventing or treating infectious disease in a vertebrate subject comprising, administering to the vertebrate subject the anti-glycan antibody, in an amount effective to reduce or eliminate infectious disease in the vertebrate subject. The anti-glycan antibody can be administered orally to the vertebrate subject. The anti-glycan antibody can be administered to the vertebrate subject via an oral, pulmonary, oropharyngeal, nasopharyngeal, topical, intravenous, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular route. In one aspect, at least some of the protein nanoparticles are bound to a plurality of different glycan-containing molecules. The glycan-containing molecule can include, but are not limited to, a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide. The vertebrate subject can bead mammalian subject or an avian subject. The infectious disease can bead bacterial infectious disease, viral infectious disease, fungal infectious disease, or infectious parasitic disease. The infectious disease can bean antibiotic-resistant bacterial infectious disease.
[0015] A method for diagnosing disease in a vertebrate subject is provided which comprises contacting a tissue sample from the vertebrate subject with the anti-glycan antibody, and detecting binding of the antibody to the tissue sample indicating presence of glycan- containing molecules relating to the disease in the vertebrate subject. The disease can be inflammatory disease, infectious disease, cancer, or metabolic disease. In one aspect, at least some of the protein nanoparticles are bound to a plurality of different glycan-containing molecules. The glycan-containing molecule can include, but are not limited to, a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figures IA, IB, and 1C show glycan array binding analysis of crude IgY isolated after (a) no immunization (array vl.O); (b) immunization with 1 1 (array v2.0); and (c) immunization with 13 (array vl .O).
[0017] Figures 2A, 2B, 2C, and 2D show glycan array binding analysis using array version vl.O of anti-2 antibodies purified by affinity filtration through agarose-18.
[0018] Figures 3A and 3B show glycan array binding analysis using array version v2.0 of (a) anti-14 IgY (0.15 mg/mL), and (b) commercially available monoclonal IgG against globo- H (0.035 mg/mL, Axorra, Inc.).
[0019] Figure 4 shows 1H NMR spectrum of 1.
[0020] Figure 5 shows MALDI-TOF mass spectra of BSA conjugates.]
[0021] Figure 6 shows plots of ratios of significant signals (>20% of the maximum) from glycan array (v2.0) analyses of total IgY generated by different chickens (designated A and B) against {left) CPMV conjugate 15 and {right) KLH conjugate 16.
[0022] Figure 7 shows independent repeat of the experiment described in Figure 3, showing glycan array (v2.0) binding analysis of anti-14 IgY.
[0023] Figure 8 shows plots of ratios of averaged signals from glycan array (v2.0) analyses of total IgY generated by two different chickens against CPMV conjugate 15 vs. KLH conjugate 16; all IgY samples were used at 0.1 mg/mL.
[0024] Figure 9 shows plots of ratios of low-intensity signals (<20% of the maximum) from glycan array (v2.0) analyses of total IgY generated by two different chickens against CPMV conjugate 15 and KLH conjugate 16.
DETAILED DESCRIPTION
[0025] The invention generally relates to a protein nanoparticle display platform for glycan-containing molecules wherein the nanoparticles display enhanced antigenicity for the glycan-containing moleucules. Vaccines are provided comprising protein nanoparticles, wherein at least some of the nanoparticles are covalently bound to glycan-containing molecules. Methods for producing anti-glycan antibodies in a vertebrate subject are provided. Methods for treatment or diagnosis of cancer or infectious disease are provided wherein the protein nanoparticle vaccine or anti-glycan antibody is administered to a vertebrate subject in need of treatment for the disease.
[0026] Tetra- and hexasaccharides were arrayed on the exterior surface of a protein nanoparticle, e.g., cowpea mosaic virus, using the copper-catalyzed azide-alkyne cycloaddition reaction. Methods for producing anti-glycan antibodies in a vertebrate subject comprise the step of administering a nanoparticle-glycan conjugate to a mammalian subject to produce polyclonal anti-glycan IgG antibodies or administering a nanoparticle-glycan conjugate to an avian subject to produce polyclonal anti-glycan IgY antibodies. As demonstrated in the Examples below, inoculation of chickens with the nanoparticle-glycan conjugates gave rise to large quantities of polyclonal anti-glycan IgY antibodies, which displayed excellent avidity and specificity on analysis using printed glycan microarrays. In addition to antibody responses in chicken, the present inventors performed similar studies by inoculating mice with the nanoparticle-glycan conjugates. The results indicate that high and selective anti-carbohydrate antibody responses were also produced in mice.
[0027] As exemplified in the Examples, avian IgY antibodies are produced in significantly higher yield than is possible for mouse or rabbit IgG, and exhibit reduced cross reactivity with native mammalian proteins. For a tri-LacNAc antigen, affinity purification against immobilized mono-LacNAc was necessary to provide a set of antibodies with specific binding properties. Comparable performance was observed for the nanoparticle-glycan conjugate-based polyclonal vs. a commercial monoclonal antibody raised against the globo-H tetrasaccharide, highlighting the utility of the glycan microarray for both quality control and rapid in-depth analysis. Virus-carbohydrate conjugates are promising candidates for development in diagnostic and immunotherapeutic applications.
[0028] It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.
[0029] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. [0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.
[0031] "Nanoparticle" refers to protein nanoparticles or nucleoprotein nanoparticles having a regular or order structure and capable of covalently attaching a glycan-containing molecule. The nanoparticles can include, but are not limited to, viruses, viral nanoparticles, vault proteins, dendrimers, chaperonins, or other large assemblies. The protein nanoparticle can be an icosahedral virus, rod-shaped virus, animal virus, or plant virus. In a detailed aspect, the virus or viral nanoparticle can be a plant viral nanoparticle, for example, a Comovirus
[0032] The protein nanoparticle can be a virus, bacteriophage, virus-like particle, or viral capsid particle. The protein nanoparticle can be derived, for example, from a plant viral particle, e.g., Comovirus. The protein nanoparticle can be derived, for example, from R otavirus, Norwalkvirus, Alphavirus, Foot and Mouth Disease virus, Retrovirus, Hepatitis B virus, Tobacco mosaic virus, Flock House Virus, or papillomavirus. See, for example PCT Application WO 2000/032227.
[0033] "Plant viral particle" refers to any plant virus which is a small plant icosahedral virus composed of 60 identical copies of an asymmetric protein subunit assembled around a bipartite single strand (+) RNA genome. Plant viral particles are plant viruses that include, but are not limited to, Comovirus, Tombusvirus, Sobemovirus, or Nepovirus. In one embodiment, the comovirus is cowpea mosaic virus. See U.S. Application No. 2006/0216238; Sen Gupta et al., RSC Chem. Commun. 2005, 34, 4315-4317.
[0034] "Glycan-containing molecule" refers to a carbohydrate or sugar-containing molecule including, but not limited to, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide.
[0035] "Anti-glycan antibodies" refer to antibodies isolated from a vertebrate subject, including, but not limited to, IgG antibodies from a mammalian subject or IgY antibodies from an avian subject following inoculation with a vaccine comprising protein nanoparticles covalently bound to glycan-containing molecules. The antibodies produced in the vertebrate subject are typically polyclonal and can be identified as capable of binding to glycan-containing molecules or a plurality of different glycan containing molecules. The antibodies can also be identified as capable of binding to the protein nanoparticle of the vaccine used to inoculate the vertebrate subject. The anti-glycan antibodies can be isolated and purified from other antibodies, e.g., antibodies to the protein nanoparticle, by affinity purification.
[0036] "Patient", "subject", "vertebrate" or "mammal" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as sheep, dogs, cows, pigs, chickens, amphibians, and reptiles.
[0037] "Treating" or "treatment" includes the administration of the antibody compositions, or vaccine compositions, compounds or agents of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder (e.g., cancer, or infectious disease). Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
[0038] "Covalent attachment" of the glycan-containing molecule to the viral particle can occur through alkyne-azide linkage chemistry as described herein. Covalent attachment can also occur through a variety of linkage chemistry to any of the other residues on the surface of the viral subunit, usually lysine, cysteine, tyrosine, aspartic acid, or glutamic acid, but also including chemically modified side chains and unnatural amino acids. Each viral particle can have a number of identical viral subunits. For example, CPMV has 60 identical subunits, and other viral particles can have a multiple of 60 subunits. Each viral subunit has a multiple of available residues for linkage to the glycan-containing molecule. For example, CPMV has viral subunits with five available lysine residues per subunit.
[0039] Suitable linkage chemistries include maleimidyl linkers and alkyl halide linkers (which react with a sulfhydryl on the antibody moiety) and succinimidyl linkers (which react with a primary amine on the antibody moiety). Several primary amine and sulfhydryl groups are present on immunoglobulins, and additional groups can be designed into recombinant immunoglobulin molecules. It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, 111.), can be employed as a linker group. Coupling can be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues (see, e.g., U.S. Pat. No. 4,671,958). [0040] In general, glycan-containing molecules in the presence or absence of a therapeutic agent can be conjugated to the nanoparticles of the invention, for example, by any suitable technique, with appropriate consideration of the need for pharmacokinetic stability and reduced overall toxicity to the patient. A glycan-containing molecule can be coupled to a suitable nanoparticle either directly or indirectly (e.g. via a linker group). A direct reaction between an glycan-containing molecule and a nanoparticle is possible when each possesses a functional group capable of reacting with the other. Compositions and methods are provided for covalent linkage of glycan-containing molecules to nanoparticles providing methods for catalyzing a reaction between at least one terminal alkyne moieties, and at least one azide moieties, wherein one moiety is attached to the glycan-containing molecules and the other moiety is attached to the nanoparticle. As a further example, a nucleophilic group, such as an amino or sulfhydryl group, can be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide). Alternatively, a suitable chemical linker group can be used. A linker group can function as a spacer to distance a glycan-containing molecule from a nanoparticle in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on a nanoparticle or glycan-containing molecule, and thus increase the coupling efficiency. An increase in chemical reactivity can also facilitate the use of moieties, or functional groups on moieties, which otherwise would not be possible.
[0041] In certain embodiments of the invention, the glycan-containing molecules or the protein nanoparticles of the invention, for example, can be coupled or conjugated to one or more therapeutic or cytotoxic moieties. "Cytotoxic moiety" refers to a moiety that inhibits cell growth or promotes cell death when proximate to or absorbed by a cell. Suitable cytotoxic moieties in this regard include radioactive agents or isotopes (radionuclides), chemotoxic agents such as differentiation inducers, inhibitors and small chemotoxic drugs, toxin proteins and derivatives thereof, as well as nucleotide sequences (or their antisense sequence). Therefore, the cytotoxic moiety can be, by way of non-limiting example, a chemotherapeutic agent, a photoactivated toxin or a radioactive agent.
[0042] As an alternative coupling method, cytotoxic agents can be coupled to the glycan-containing molecules of the invention, for example, through an oxidized carbohydrate group at a glycosylation site, as described in U.S. Pat. Nos. 5,057,313 and 5,156,840. Yet another alternative method of coupling the glycan-containing molecules, e.g., glycoproteins, to the cytotoxic moiety is by the use of a non-covalent binding pair, such as streptavidin/biotin, or avidin/biotin. In these embodiments, one member of the pair is covalently coupled to the glycan- containing molecules and the other member of the binding pair is covalently coupled to the cytotoxic moiety.
[0043] Where a cytotoxic moiety is more potent when free from the glycan-containing molecules or the nanoparticles of the present invention, it can be desirable to use a linker group which is cleavable during or upon internalization into a cell, or which is gradually cleavable over time in the extracellular environment. A number of different cleavable linker groups have been described. The mechanisms for the intracellular release of a cytotoxic moiety agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710); by irradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014); by hydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045); by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958); and acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789).
[0044] It can be desirable to couple more than one therapeutic or cytotoxic moieties to a glycan-containing molecules or coupled to nanoparticles of the invention. By poly-derivatizing the CPMV viral nanoparticle, for example, several cytotoxic strategies can be simultaneously implemented, a glycan-containing molecule can be made useful as a contrasting agent for several visualization techniques, or a therapeutic antibody can be labeled for tracking by a visualization technique. In one embodiment, multiple molecules of a cytotoxic moiety are coupled to one glycan-containing molecule. In another embodiment, more than one type of moiety can be coupled to one glycan-containing molecule. For instance, a therapeutic moiety, such as an polynucleotide or antisense sequence, can be conjugated to glycan-containing molecules in conjunction with a chemotoxic or radiotoxic moiety, to increase the effectiveness of the chemo- or radiotoxic therapy, as well as lowering the required dosage necessary to obtain the desired therapeutic effect. Regardless of the particular embodiment, immunoconjugates with more than one moiety can be prepared in a variety of ways. For example, more than one moiety can be coupled directly to CPMV viral nanoparticles, such that multiple sites for attachment (e.g., dendrimers) can be used. Alternatively, a carrier with the capacity to hold more than one cytotoxic moiety can be used.
[0045] As explained above, a carrier can bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations. Suitable covalent-bond carriers include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234), peptides, and polysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784), each of which has multiple sites for the attachment of moieties. A carrier can also bear an agent by non- covalent associations, such as non-covalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Encapsulation carriers are especially useful in chemotoxic therapeutic embodiments, as they can allow the therapeutic compositions to gradually release a chemotoxic moiety over time while concentrating it in the vicinity of the target cells.
CovALENT LINKAGE OF GLYCAN-CONTAINING MOLECULES BY AZIDE ALKYNE CYCLOADDITION
[0046] Covalent bond formation to proteins is made difficult by their multiple unprotected functional groups and normally low concentrations. The water soluble sulfonated bathophenanthroline ligand 2 can be used to promote a highly efficient Cu(I)-mediated azide- alkyne cycloaddition (CuAAC; "Click chemistry") reaction for the chemoselective attachment of biologically relevant molecules to protein nanoparticles, e.g., cowpea mosaic virus (CPMV) nanoparticles. The ligated substrates included complex sugars, peptides, poly(ethylene oxide) polymers, and the iron carrier protein transferring (Tfn), with successful ligation even for cases that were previously resistant to azide-alkyne coupling using the conventional ligand tris(triazolyl)amine (1). The use of 4-6 equiv of substrate was sufficient to achieve loadings of 60-1 15 molecules/virion in yields of 60-85%. Although it is sensitive to oxygen, the reliably efficient performance of the Cu-ligand»2 system makes it a useful tool for demanding bioconjugation applications.
Figure imgf000013_0001
[0047] Compositions and methods are provided for covalent linkage of glycan- containing molecules to nanoparticles comprising catalyzing a reaction between at least one terminal alkyne moieties, and at least one azide moieties, wherein one moiety is attached to the glycan-containing molecules and the other moiety is attached to the nanoparticle, forming at least one triazole thereby. A method for coupling a glycan-containing molecules to nanoparticles is provided comprising catalyzing a reaction between at least one terminal alkyne moieties attached to the glycan-containing molecules, and at least one azide moieties attached to the nanoparticles, forming at least one triazole thereby, effecting catalysis by addition of a metal ion in the presence of a ligand, and providing a plurality of sites on the nanoparticles having azide moieties, such that a plurality of glycan-containing molecules can be coupled with the nanoparticles.. A further embodiment provides a method for coupling glycan-containing molecules to nanoparticles is provided comprising catalyzing a ligation reaction between at least one terminal alkyne moieties attached to the nanoparticles, and at least one azide moieties attached to the glycan-containing molecules, forming at least one triazole thereby, effecting catalysis by addition of a metal ion in the presence of a ligand, and providing a plurality of sites on the nanoparticles having terminal alkyne moieties, such that a plurality of glycan-containing molecules can be coupled with the scaffold. See PCT Application WO 2007/01 1696.
[0048] "Plurality of sites" refers to two or more sites on a nanoparticle capable of binding two or more compounds per nanoparticle molecule. Depending upon the nature of the scaffold and the compounds, 100 or more, 200 or more, or 300 or more compound molecules can be bound per scaffold molecule. In one aspect, the scaffold molecule is a protein of a viral nanoparticle, e.g., a CPMV nanoparticle.
[0049] "Terminal alkyne moiety" refers to an acetylenic bond (carbon-carbon triple bond) having a hydrogen attached to one carbon, e.g., R-C ≡ C-H, wherein R is a compound including, but not limited to, polynucleotide, polypeptide, glycopolymer, chromophoric dye, glycan, or lipid.
[0050] "Azide moiety" refers to a moiety, N≡NΦ-NΘ-. An azide moiety can be attached to a compound having a general structure, N≡Ne-Nθ-R, wherein R is a compound including, but not limited to, polynucleotide, polypeptide, glycopolymer, chromophoric dye, glycan, or lipid.
[0051] The present invention provides an efficient strategy for end-functionalization of a compound, e.g., glycan-containing molecule, glycopolymer, carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide, polyethylene glycol, chromophoric dye, folic acid, lipid, polynucleotide, polypeptide, protein, or transferrin, using an azide-containing initiator for a living polymerization process followed by click chemistry elaboration of the unique azide end group. The copper- catalyzed cycloaddition reaction provides very efficient coupling of such polymers to a functionalized viral coat protein with efficient use of coupling reagents, compound molecules, and scaffold molecules. In an embodiment of the invention, a well-defined side chain neoglycopolymer possessing a single activated chain end can be chemically conjugated efficiently to a protein or bionanoparticle in a "bioorthogonal" fashion. The bioorthogonal labeling of biomolecules provides a unique, in vivo label that is an important tool for the study of biomolecule function and cellular fate. Attention is increasingly focused on labeling of biomolecules in living cells, since cell lysis introduces many artefacts. The method further provides high diversity in the nature of the label used in the ligation reaction.
[0052] In one embodiment, the method for coupling a compound to a scaffold comprises catalyzing a reaction between a first reactant having a terminal alkyne moiety and second reactant having an azide moiety for forming a product having a triazole moiety by addition of a metal ion in the presence of a ligand. The metal ion includes, but is not limited to, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, Hf, Ta, W, Re, Os, Ir, Pt, Au, or Hg. In a detailed embodiment, the metal includes, but is not limited to, Mn, Fe, Co, Cu, Mo, Tc, Ru, Rh, Pd, W, Re, Os, Ir, Pt, or Au. See for example, PCT International Application WO 2003/101972.
[0053] In a further detailed embodiment, the metal is heterogeneous copper, metallic copper, copper oxide, or copper salts.
[0054] Copper(I) salts, for example, Cu(I), CuOTf^C6H6 and [Cu(NCCH3)4]PF6, can also be used directly in the absence of a reducing agent. These reactions usually require acetonitrile as co-solvent and one equivalent of a nitrogen base (e.g., 2,6-lutidine, triethylamine, diisopropylethylamine, or pyridine). However, formation of undesired byproducts, primarily diacetylenes, bis-triazoles, and 5-hydroxytriazoles, was often observed. For a recent summary of the reactions of Cu(I) complexes with dioxygen, see Schindler, Eur. J. Inorg. Chem. 2311-2326, 2000 and Blackman and Tolman in Structure and Bonding, B. Meunier, Ed., Springer- Verlag, Berlin, Heidelberg, 97: 179-211, 2000. This complication with direct use of Cu(I) species was minimized when 2,6-lutidine was used, and exclusion of oxygen further improved product purity and yield.
[0055] In one embodiment, the ligation reaction can be catalyzed by addition of Cu(I). IfCu(I) salt is used directly, no reducing agent is necessary, but acetonitrile or one of the other ligands indicate above can be used as a solvent (to prevent rapid oxidation of Cu(I) to Cu(II) and one equivalent of an amine can be added to accelerate the reaction. In this case, for better yields and product purity, oxygen should be excluded. Therefore, the ascorbate or any other reducing procedure is often preferred over the unreduced procedure. The use of a reducing agent is procedurally simple, and furnishes triazole products in excellent yields and of high purity. Addition of an amine, such as triethylamine or 2,6-lutidine to the acetonitrile system, solves the problem of reactivity - the product is formed in quantitative yield after approximately 8 hours.
[0056] In a further embodiment, the ligation reaction can be catalyzed by addition of Cu(II) in the presence of a reducing agent for reducing the Cu(II) to Cu(I), in situ. Cu(II) salts, e.g., CuSO4^SH2O, can be less costly and often purer than Cu(I) salts. Reducing agents useful in this reaction include, but are not limited to ascorbic acid, sodium ascorbate, quinone, hydroquinone, vitamin Kl, glutathione, cysteine, Fe2+, Co2+, and an applied electric potential. See, for example, Davies, Polyhedron 11: 285-321 1992, and Creutz, Inorg. Chem. 20: 4449, 1981. In a further example, metals can be employed as reducing agents to maintain the oxidation state of the Cu (I) catalyst or of other metal catalysts. Metallic reducing agents include, but are not limited to, Cu, Al, Be, Co, Cr, Fe, Mg, Mn, Ni, and Zn. Alternatively, an applied electric potential can be employed to maintain the oxidation state of the catalyst.
[0057] In a further embodiment, the ligation reaction can be catalyzed by addition of Cu(O) in the presence of an oxidizing agent for oxidizing the Cu(O) to Cu(I), in situ. Metallic containers can also be used as a source of the catalytic species to catalyze the ligation reaction. For example, a copper container, Cu(O), can be employed to catalyzed the ligation reaction. In order to supply the necessary ions, the reaction solution must make physical contact with the a copper surface of the container. Alternatively, the reaction can be run in a non-metallic container, and the catalytic metal ions supplied by contacting the reaction solution with a copper wire, copper shavings, or other structures. Although these reactions may take longer to proceed to completion, the experimental procedure reduces the number of intervening steps.
[0058] In one embodiment, the method for coupling a compound to a scaffold comprises catalyzing a reaction between a first reactant having a terminal alkyne moiety and second reactant having an azide moiety for forming a product having a triazole moiety by addition of a metal ion in the presence of a ligand for the metal ion. The metal ion is coordinated to a ligand for solubilizing such metal ion within the solvent, for inhibiting oxidation of such metal ion, and for dissociating, in whole or in part, from such metal ion during the catalysis of the reaction. Ligands can be, for example, monodentate ligands,bidentate (chelating) ligands, or multidentate ligands. Monodentate ligands refers to Lewis bases that donate a single pair ("mono") of electrons to a metal atom. Monodentate ligands can be either ions (usually anions) or neutral molecules. Monodentate ligands include, but are not limited to, fluoride ion (F"), chloride ion (Cl"), bromide ion, (Br"), iodide ion (I"), water (H2O), ammonia (NH3), hydroxide ion (OH"), carbon monoxide (CO), cyanide (CN"), or thiocyanate ion (CN-S").
[0059] Bidentate ligands or chelating ligands refers to Lewis bases that donate two pairs of electrons to a metal atom. Bidentate ligands include, but are not limited to, ethylenediamine, acetylacetonate ion, phenanthroline, sulfonated bathophenanthroline or oxalate ion.
[0060] Ligands include, but are not limited to, acetonitrile, cyanide, nitrile, isonitrile, water, primary, secondary or tertiary amine, a nitrogen bearing heterocycle carboxylate, halide, alcohol, and thiol sulfide, phosphine, and phosphite. In a detailed embodiment, the halide is chloride and can be used at a concentration of 1-5 M. Polyvalent ligands that include one or more functional groups selected from nitrile, isonitrile, primary, secondary, or tertiary amine, a nitrogen bearing heterocycle, carboxylate, halide, alcohol, thiol, sulfide, phosphine, and phosphite can also be employed.
[0061] The ligation reactions as provided herein are useful for in a method for coupling a compound to a scaffold. The method provides catalyzing a ligation reaction between one or more terminal alkyne moieties and one or more azide moieties, for forming a product having a triazole moiety, the ligation reaction being catalyzed by addition of a metal ion in the presence of a ligand, and the scaffold having polyvalent sites for coupling to one or more compounds. In one aspect, the one or more terminal alkyne moieties are attached to the compound, and the one or more azide moieties are attached to the scaffold. In a further aspect, the one or more terminal alkyne moieties are attached to the scaffold, and the one or more azide moieties are attached to the compound. In a detailed aspect, the scaffold can be a protein on a viral nanoparticle, for example, a cow pea mosaic viral nanoparticle.
TREATMENT OF CANCER OR INFECTIOUS DISEASE WITH VACCINES COMPRISING NANOPARTICLES COVALENTLY LINKED TO GLYCAN-CONT AINING MOLECULES
[0062] Studies have explored the feasibility of using vaccines comprising nanoparticles wherein at least some of the nanoparticles are covalently bound to a plurality of glycan- containing molecules for the immunotherapy of cancer. The optimal design and composition of the vaccines can include glycan-containing molecules including, but not limited to, blood group A antigen, tri-LacNAc, sialyl Lewis", sialyl Lewisy, or globoH. Among the candidate antigens being examined is Lewisy (Ley), a blood group-related antigen that is overexpressed on the majority of human carcinomas. Using Ley as a model for specificity, studies have examined the role of epitope clustering, carrier structure, and adjuvant on the immunogenicity of Ley conjugates in mice. A glycolipopeptide containing a cluster of three contiguous Ley-serine epitopes and the Pan^Cys immunostimulating moiety was found to be superior to a similar construct containing only one Ley-serine epitope in eliciting antitumor cell antibodies. Reexamination of the clustered Ley-serine Pan^Cys conjugate with the adjuvant QS-21 resulted in the identification of both IgG and IgM antibodies reacting with tumor cells, thus demonstrating the feasibility of an entirely synthetic carbohydrate-based anticancer vaccine in an animal model. In practical terms, one can conclude that (i) glycolipopeptide with clustered Ley epitopes are more effective than related structures with single Ley epitopes in producing antitumor cell antibodies; (ii) antibody responses to the clustered Ley-structure conjugated to KLH were skewed toward the immunizing carbohydrate structure; and (iii) totally synthetic constructs can be effective immunogens in conjunction with a suitable adjuvant, the effect of which is to bypass the need for specific T-cell help to stimulate IgG as well as IgM antibodies. Kudryashov et al., PNAS USA 98: 3264-3269, 2001.
[0063] Vaccines comprising plant viral particles displaying a plurality of glycan- containing molecules or antibodies to glycan-containing molecules can be used. The types of glycans bound by these antibodies is indicative of the type, extent and/or prognosis of the disease. Thus, low-risk types of breast cancer as well as more aggressive types of breast cancer can be detected or treated using the present methods. Patients with breast cancer have circulating antibodies that react with glycans such as ceruloplasmin glycans, Neu5Accx2- 6GalNAca, certain T-antigens carrying various substituents and other modifications, LNT-2 (a known ligand for tumor-promoting Galectin-4; see Huflejt & Leffler (2004). Glycoconjugate J, 20: 247-255), Globo-H-, and GM 1 -antigens. GMl is a glycan that includes the following carbohydrate structure: Gal-beta3-GalNAc-beta4-[Neu5Ac-alpha3]-Gal-beta4-Glc-beta. Sulfo-T is a T-antigen with sulfate residues, for example, Sulfo-T can include a carbohydrate of the following structure: Gal3GalNAc. Globo-H is a glycan that includes the following carbohydrate structure: Fucose-alpha2-Gal-beta3-GalNAc-beta3-Gal-alpha4-Gal-beta4-Glc. LNT-2 is a glycan that includes the following carbohydrate structure: GlcNAc-beta3-Gal-beta4-Glc-beta. Sialylated Tn is a glycan with the following structure: Neu5Ac-alpha6-GalNAc-alpha. Circulating antibodies from breast cancer patients can also react with the following glycans: Tri- LacNAc, LacNAc-LeX-LeX, LacNAc-LacNAc, H-type-2-LacNAc, H-type2-LacNAc-LacNAc, GlcNAcI33LacNAc, SLeXLacNAc, 3'SialylDiLacNAc, 3'Sialyl-tri-LacNAc, 6Sia-LacNAc- LeX-LeX, 6SiaLacNAc-LacNAc.
[0064] Methods for producing anti-glycan antibodies can be used for the diagnosis or treatment of cancer, e.g., breast cancer, with anti-glycan antibodies. The nanoparticles, e.g., plant viral particles, comprising glycan-containing molecules can be used for detecting or treating breast cancer. The plant viral particles comprising glycan-containing molecules include glycans that react with antibodies associated neoplasia in sera of mammals with benign or pre- malignant tumors. Such glycans have two or more sugar units. The glycan-containing molecules include straight chain and branched oligosaccharides as well as naturally occurring and synthetic glycans. Any type of sugar unit can be present in the glycans of the invention, including allose, altrose, arabinose, glucose, galactose, gulose, fucose, fructose, idose, lyxose, mannose, ribose, talose, xylose, neuraminic acid or other sugar units. Such sugar units can have a variety of substituents. For example, substituents that can be present instead of, or in addition to, the substituents typically present on the sugar units include amino, carboxy, thiol, azide, N-acetyl, N-acetylneuraminic acid, oxy (=0), sialic acid, sulfate (-SO4), phosphate (-PO4), lower alkoxy, lower alkanoyloxy, lower acyl, andlor lower alkanoylaminoalkyl. Fatty acids, lipids, amino acids, peptides and proteins can also be attached to the glycan-containing molecules. See, for example, WO 2006/068758.
[0065] In another example, neutralizing antibodies known to be specific for HIV have been found to be reactive with mannose-containing glycans, in particular Man8 glycans. Hence, HIV infection can be detected by detecting whether a patient has circulating antibodies that bind to Man8 glycans. Moreover, HIV infection can be treated or inhibited by administering a vaccine comprising a plant viral particle displaying a plurality of glycan-containing molecules, e.g., Man8 glycans, or administering anti-glycan antibodies produced by the methods of the present invention to a subject.
[0066] A method of detecting transplant tissue rejection or xenotransplant tissue rejection are provided. Glycans specific for the transplanted or xenotranplanted tissue are used in glycan arrays to observe whether one or more glycan-containing molecules are bound by antibodies in the test sample. Examples of glycan-containing molecules that can be used for diagnosis or treatment of transplant reject include any one of Gal-alpha3-Gal-beta, Gal-alpha3- Gal-beta4-GlcNAc[alpha3-Fucose]-beta, Gal-alpha3-Gal-beta4-Glc-beta, Gal-alpha3- Gal[alpha2-Fucose]-beta4-GlcNAc-beta, Gal-alpha3-Gal-beta4-GalAc-beta, GaI-alpha3-GalAc- alpha, Gal-alpha3 -Gal-beta, or Gal-beta4-GlcNAc[alpha3-Fucose]-beta, or a combination thereof.
[0067] The glycan-containing molecules can therefore include glycans that react with antibodies associated with particular disease or condition. For example, glycan-containing molecules or antibodies to glycan-containing molecules that are produced in response to cancer, bacterial infection, viral infection, inflammation, transplant rejection, autoimmune diseases and the like can be detected. See, for example, WO 2007/0059769.
[0068] In a further aspect, several antibodies have been associated with Crohn's disease (CD) and are associated with distinct clinical phenotypes. A panel of antibodies against bacterial peptides and glycans have been tested to differentiate inflammatory bowel disease (IBD), and to determine whether they were associated with particular clinical manifestations. Antibodies against a mannan epitope of Saccharomyces cerevisiae (gASCA), laminaribioside (ALCA), chitobioside (ACCA), mannobioside (AMCA), outer membrane porins (Omp) and the atypical perinuclear antibody pANCA were tested in serum samples of 1225 IBD patients, 200 healthy controls (HC) and 1 13 patients with non-IBD gastrointestinal inflammation (non-IBD GI). Antibody responses were correlated with type of disease and clinical characteristics. 76% of CD patients had at least one of the tested antibodies. For differentiation between CD and ulcerative colitis (UC), the combination of gASCA and pANCA was most accurate. For differentiation between IBD, HC and non-IBD GI, the combination of gASCA, pANCA and ALCA had the best accuracy. Increasing amount and level of antibody responses towards gASCA, ALCA, ACCA, AMCA and Omp was associated with more complicated disease behaviour (44.7% vs. 53.6% vs. 71.1% vs. 82.0%, p < .001), and higher frequency of CD-related abdominal surgery (38.5% vs. 48.8% vs. 60.7% vs. 75.4%, p < .001). Using this panel of serological markers, the study showed that number and magnitude of immune responses to different microbial antigens are associated with severity of the disease. Ferrante, et al., Gut, April 24, 2007; Henckaerts, et al., Gut, June 26, 2007.
[0069] Many secreted and cell surface glycoproteins are O-glycosylated. The structures of these Ser/Thr-bound O-glycans change during differentiation and activation of cells, during apoptosis, and are often altered in disease. O-glycans have been shown to be important for the functions of glycoproteins, for example, the expression of carbohydrate and peptide epitopes, the expression and stability of cell surface receptors, and cell adhesion. The altered structures of O- glycans therefore may be in part responsible for the pathological properties of diseased cells. The present study is aimed at defining the mechanisms by which O-glycans change in cystic fibrosis, cancer and inflammatory disease, and at studying the functional implications of these alterations. In cancer cells, drastic changes of O-glycans occur, seen in the expression of cancer-associated epitopes such as the Tn and sialyl-Tn (STn) antigens. STn, sialyl a2-6 GalNAc-Ser/Thr, is associated with a poor prognosis in colorectal and other cancer patients.
[0070] Several possible mechanisms for the expression of sialyl-Tn antigen in cancer have been proposed. There may be a block in the synthesis of common O-glycan core structures, there may be increased synthesis of sialyl-Tn, or increased exposure of the antigen to antibodies due to decreased modifications of sialic acids. There may also be altered synthesis of specific glycoproteins in cancer. This may increase STn expression since the peptide moieties of glycoproteins in part direct the synthesis of O-glycans. Human colon cancer cells LSC highly express STn and lack the ability to form the common O-glycan core structures. Thus only Tn and STn can be synthesized. However, different mechanisms regulate O-glycan synthesis in other cancer cells. For example, breast cancer or leukaemia cells have increased sialyltransferase activities and produce hypersialylated O-glycans, compared to normal counterparts. Sialylated O-glycans often cannot elongated further, and therefore remain truncated. In addition to increased sialylation most breast cancer cell lines also show a lack of O-glycan branching by core 2 betaό-GlcNAc-transferase, and this leads to short and sialylated O-glycan structures. Apoptosis is a process which may cause cells to undergo glycosylation changes. Apoptosis may also be influenced by specific glycan structures. Several diseases, including cancer and arthritis, are associated with abnormal apoptosis. Apoptosis may be an important mechanism of xenograft rejection. Studies showed that apoptotic pig aortic endothelial cells exhibit a shift of O- glycosylation biosynthetic pathways as well as in the exposure of cell surface glycans. The enzymes synthesizing short sialylated structures are highly active, but the branching enzyme core 2 betaό-GlcNAc-transferase is less active in the apoptotic cell populations. This indicates that these enzymes are regulated differently during the process of apoptosis. Increased angiogenesis is another feature of cancer and rheumatoid arthritis. Cyclic changes of endothelial glycosyltransferase activities were observed during angiogenesis in vivo and in vitro. O-glycans, and possibly other types of glycans, on cell surface glycoproteins of endothelial cells can be involved in the control of growth related processes. Knowledge of the specific roles of O- glycans and the regulation of their synthesis in the endothelium can be useful in the control of transplant rejection, wound healing, cancer angiogenesis, inflammation and other clinical conditions involving cell growth and cell death.
[0071] Vaccines comprising a plant viral particle displaying a plurality of glycan- containing molecules or anti-glycan antibodies produced by the methods of the present invention are useful for treating disease related to transplant rejection, wound healing, cancer angiogenesis, cystic fibrosis, inflammation, and other clinical conditions involving cell growth and cell death. The structures of Ser/Thr-bound O-glycans change during differentiation and activation of cells, during apoptosis, and are often altered in disease. Cancer, cystic fibrosis and inflammatory diseases are associated with alterations of O-glycans that are in part responsible for the pathology observed. Cancer and endothelial cells were chosen as model systems to show how the biosynthetic pathways of O-glycans are rearranged in disease and during apoptosis. Proceedings of the Royal Society of Medicine's 5th Jenner Symposium (Glycobiology and Medicine Conference), July 10-1 1, 2000.
[0072] Some of the structural elements of the glycans described herein are referenced in abbreviated form. Many of the abbreviations used are provided in the following table. Moreover the glycans of the invention can have any of the sugar units, monosaccharides or core structures provided in this table.
Trivial Name Monosaccharide / Core Code D-Glcp: D-Glucopyranose G
D-GaIp: D-Galactopyranose A
D-GlcpNAc: N-Acetylglucopyranose GN
D-GlcpN: D-Glucosamine GQ
D-GaIpNAc: N-Acetylgalactop yranose AN
D-GaIpN: D-Galacosamine AQ
D-Manp: D-Mannopyranose M
D-ManpNAc: D-NJ-Acetylmannopyranose MN
D-Neup5Ac: N-Acetylneuraminic acid NN
D-Neu5G: D-N-Glycolylneuraminic acid NJ
D-Neup: Neuraminic acid N
KDN* 2: Keto-3-deoxynananic acid K
Kdo: 3-deoxy-D-manno-2 octulopyranosylono W
D-GaIpA: D-Galactoronic acid L
D-Idop: D-Iodoronic acid I
L-Rhap: L-Rhamnopyranose H
L-Fucp: L-Fucopyranose F
D-XyIp: D-Xylopyranose X
D-Ribp: D-Ribopyranose B
L-Araf: L-Arabinofuranose R
D-GlcpA: D-Glucoronic acid U
D-AlIp: D-Allopyranose O
D-Apip: D-Apiopyranose P
D-Tagp: D-Tagopyranose T
D-Abep: D-Abequopyranose Q
D-XuIp: D-Xylulopyranose D
D-Fruf: D-Fructofuranose E *
[0073] Another description of KDN is: 3-deoxy-D-glycero-K-galacto-nonulosonic acid. The sugar units or other saccharide structures present in the glycans of the invention can be chemically modified in a variety of ways.
[0074] A listing of some of the types of modifications and substituents that the sugar units in the glycans of the invention can possess, along with the abbreviations for these modifications/substituents are listed below.
Modification type/ Symbol Modification type/ Symbol Acid A Acid A
N-Methylcarbamoyl ECO deacetylated N-Acetyl (amine) Q pentyl EE Deoxy Y octyl EH Ethyl ET ethyl ET Hydroxyl OH inositol IN Inositol IN
N-Glycolyl J Methyl ME methyl ME N-Acetyl N
N-Acetyl N N-Glycolyl J hydroxyl OH N-Methylcarbamoyl ECO phosphate P N-Sulfate QS phosphocholine PC O-Acetyl T
Phosphoethanolamine (2- aminoethylphosphate) PE Octyl EH
Pyrovat acetal PYR* Pentyl EE deacetylated N-Acetyl (amine) Q Phosphate P
N-Sulfate QS Phosphocholine PC sulfate S Phosphoethanolamine (2- aminoethyiphosphate) PE
O-Acetyl T Pyrovat acetal PYR* deoxy Y Sulfate S
*when written on position 3, it means 3,4, when to 4 it means 4,6.
GLYCAN-CONTAINING MOLECULES
[0075] The compositions and methods of the invention provide vaccines comprising a protein nanoparticle displaying a plurality of glycan-containing molecules or provide anti-glycan antibodies produced by the methods of the present invention that are useful for detecting and preventing cancer or infectious disease. The glycans or glycan-containing molecules include numerous different types of carbohydrates and oligosaccharides. In general, the major structural attributes and composition of the separate glycans within the libraries have been identified. In some embodiments, the libraries consist of separate, substantially pure pools of glycans, carbohydrates and/or oligosaccharides. Further description of the types of glycans useful in the practice of the invention is provided in U.S. Provisional Ser. No. 60/550,667, filed March 5, 2004, and U.S. Provisional Ser. No. 60/558, 598, filed March 31, 2004, the contents of which are incorporated herein by reference. [0076] The glycan-containing molecules of embodiments of the invention include straight chain and branched oligosaccharides as well as naturally occurring and synthetic glycans. For example, the glycan can be a glycoaminoacid, a glycopeptide, a glycolipid, a glycoaminoglycan (GAG), a glycoprotein, a whole cell, a cellular component, a glycoconjugate, a glycomimetic, a glycophospholipid anchor (GPI), glycosyl phosphatidylinositol (GPI)-linked glycoconjugates, bacterial lipopolysaccharides and endotoxins. The glycans can also include N- glycans, O-glycans, glycolipids and glycoproteins.
[0077] The glycan-containing molecules of embodiments of the invention include 2 or more sugar units. Any type of sugar unit can be present in the glycans of the invention, including, for example, allose, altrose, arabinose, glucose, galactose, gulose, fucose, fructose, idose, lyxose, mannose, ribose, talose, xylose, or other sugar units. The tables provided herein list other examples of sugar units that can be used in the glycans of the invention. Such sugar units can have a variety of modifications and substituents. Some examples of the types of modifications and substituents contemplated are provided in the tables herein. For example, sugar units can have a variety of substituents in place of the hydroxy (-OH), carboxylate (-COO" ), and methylenehydroxy (-CH2-OH) substituents. Thus, lower alkyl moieties can replace any of the hydrogen atoms from the hydroxy (-OH), carboxylic acid (-COOH) and methylenehydroxy (- CH2-OH) substituents of the sugar units in the glycans of the invention. For example, amino acetyl (-NH-CO-CH3) can replace any of the hydrogen atoms from the hydroxy (-OH) , carboxylic acid (-C00H) and methylenehydroxy (-CH2-OH) substituents of the sugar units in the glycans of the invention. N-acetylneuraminic acid can replace any of the hydrogen atoms from the hydroxy (-OH), carboxylic acid (-COOH) and methylenehydroxy (-CH2-OH) substituents of the sugar units in the glycans of the invention. Sialic acid can replace any of the hydrogen atoms from the hydroxy (-OH), carboxylic acid (-C00H) and methylenehydroxy (- CH2-OH) substituents of the sugar units in the glycans of the invention. Amino or lower alkyl amino groups can replace any of the OH groups on the hydroxy (-OH), carboxylic acid (-COOH) and methylenehydroxy (-CH2-OH) substituents of the sugar units in the glycans of the invention. Sulfate (-SO4) or phosphate (-PO4) can replace any of the OH groups on the hydroxy (-OH), carboxylic acid (-COOH) and methylenehydroxy (-CH2-OH) substituents of the sugar units in the glycans of the invention. Hence, substituents that can be present instead of, or in addition to, the substituents typically present on the sugar units include N-acetyl, N-acetylneuraminic acid, oxy (=0), sialic acid, sulfate (-SO4), phosphate (-PO4), lower alkoxy, lower alkanoyloxy, lower acyl, andlor lower alkanoylaminoalkyl. [0078] The following definitions are used, unless otherwise described: Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl" embraces only the straight chain radical, when a branched chain isomer such as "isopropyl" has been specifically referred to. Halo is fluoro, chloro, bromo, or iodo.
[0079] Specifically, lower alkyl refers to (C|-C6)alkyl, which can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C3-Ce)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C3-C6)cycloalkyl(Ci-Ce)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2- cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl; (Ci-C6) alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3- pentoxy, or hexyloxy.
[0080] It will be appreciated by those skilled in the art that the glycans of the invention having one or more chiral centers may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a glycan of the invention, it being well known in the art how to prepare optically active forms, for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.
[0081] Specific and preferred values listed below for substituents and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges or for the substituents.
[0082] The libraries of the invention are particularly useful because diverse glycan structures are difficult to make and substantially pure solutions of a single glycan type are hard to generate. For example, because the sugar units typically present in glycans have several hydroxyl (-OH) groups and each of those hydroxyl groups is substantially of equal chemical reactivity, manipulation of a single selected hydroxyl group is difficult. Blocking one hydroxyl group and leaving one free is not trivial and requires a carefully designed series of reactions to obtain the desired regioselectivity and stereoselectivity. Moreover, the number of manipulations required increases with the size of the oligosaccharide. Hence, while synthesis of a disaccharide may require 5 to 12 steps, as many as 40 chemical steps can be involved in synthesis of a typical tetrasaccharide. In the past, chemical synthesis of oligosaccharides was therefore fraught with purification problems, low yields and high costs. However the invention has solved these problems by providing libraries and arrays of numerous structurally distinct glycans. [0083] The glycans of the invention have been obtained by a variety of procedures. For example, some of the chemical approaches developed to prepare N-acetyllactosamines by glycosylation between derivatives of galactose and N-acetyiglucosamine are described in AIy, M. R. E.jlbrahim, E.-S. I.; El-Ashry, E.-S. H. E. and Schmidt, R. R., Carbohydr. Res. 1999, 316, 121-132; Ding, Y.; Fukuda, M. and Hindsgaul, 0., Bioorg. Med. Chem. Lett. 1998, 8, 1903-1908; Kretzschmar, G. and Stahl, W., Tetrahedr. 1998, 54, 634 1-6358. These procedures can be used to make the glycans of the present libraries, but because there are multiple tedious protection- deprotection steps involved in such chemical syntheses, the amounts of products obtained in these methods can be low, for example, in milligram quantities.
[0084] Synthesis of glycan-containing molecules can occur by synthesizing oligosaccharides using regiospecific and stereospecific enzymes, called glycosyltransferases, for coupling reactions between the monosaccharides. These enzymes catalyze the transfer of a monosaccharide from a glycosyl donor (usually a sugar nucleotide) to a glycosyl acceptor with high efficiency. Most enzymes operate at room temperature in aqueous solutions (pH 6-8), which makes it possible to combine several enzymes in one pot for multi-step reactions. The high regioselectivity, stereoselectivity and catalytic efficiency make enzymes especially useful for practical synthesis of oligosaccharides and glycoconjugates. See Koeller, K. M. and Wong, C- H., Nature 2001, 409, 232-240; Wymer, N. and Toone, E. J., Curr. Opin. Chem. Biol. 2000, 4, 1 10-119; Gijsen, H. J. M.; Qiao, L.;Fitz, W. and Wong, C-H., Chem. Rev. 1996, 96, 443-473.
[0085] Recent advances in isolating and cloning glycosyltransferases from mammalian and non-mammalian sources such as bacteria facilitate production of various oligosaccharides. DeAngelis, P. L., Glycobiol. 2002, 12, 9R-16R; Endo, T. and Koizumi, S., Curr. Opin. Struct. Biol. 2000, 10, 536-541; Johnson, K. F., Glycoconj. 1 1999, 16, 141-146. In general, bacterial glycosyltransferases are more relaxed regarding donor and acceptor specificities than mammalian glycosyltransferases. Moreover, bacterial enzymes are well expressed in bacterial expression systems such as E. coli that can easily be scaled up for over expression of the enzymes. Bacterial expression systems lack the post-translational modification machinery that is required for correct folding and activity of the mammalian enzymes whereas the enzymes from the bacterial sources are compatible with this system. Thus, in many embodiments, bacterial enzymes are used as synthetic tools for generating glycans, rather than enzymes from the mammalian sources.
[0086] For example, the repeating Galβ(l-4)GlcNAc-unit can be enzymatically synthesized by the concerted action of β4-galactosyltransferase (β4GalT) and β3-N- acetyllactosamninyltransferase (β3G IcNAcT). Fukuda, M., Biochim. Biophys. Acta.1984, 780:2, 119-150; Van den Eijnden, D. H.;Koenderman, A. H. L. and Schiphorst, W. E. C. M., J. Biol. Chem. 1988, 263, 12461-1247 1. Studies have previously cloned and characterized the bacterial N. meningitides enzymes β4GalT-GalE and β3G IcNAcT and demonstrated their utility in preparative synthesis of various galactosides. Blixt, O.;et al., J Org. Chem. 2001, 66, 2442-2448; Blixt, O.;et al., Glycobiol. 1999, 9, 1061-1071. β4GalT-GalE is a fusion protein constructed from β4GalT and the uridine-5-diphospho-galactose-4'-epimerase (GaIE) for in situ conversion of inexpensive UDP-glucose to UDP-galactose providing a cost efficient strategy. See WO 2006/068758 and WO 2007/0059769, the contents of which are incorporated herein by reference.
[0087] In most cases, the structures of the glycans used in the compositions, libraries and arrays of the invention are described herein. However, in some cases a source of the glycan, rather than the precise structure of the glycan is given. Hence, a glycan from any available natural source can be used in the arrays and libraries of the invention. For example, known glycoproteins are a useful source of glycans.
[0088] The glycans from such glycoproteins can be isolated using available procedures or, for example, procedures provided herein. Such glycan preparations can then be used in the compositions, libraries and arrays of the invention.
GLYCAN ARRAYS FOR DETECTING CANCER AND INFECTIOUS DISEASE
[0089] The glycan arrays employ a library of characterized and well-defined glycan structures. The array has been validated with a diverse set of carbohydrate binding proteins such as plant lectins and C-type lectins, Siglecs, Galectins, Influenza Hemaglutinins and anti- carbohydrate antibodies (both from crude sera and from purified serum fractions). Further description on how to make glycan arrays useful in the practice of the invention is provided in U.S. Provisional Ser. No. 60/550, 667, filed March 5, 2004, and U.S. Provisional Ser. No. 60/558,598, filed March 31, 2004, the contents of which are incorporated herein by reference.
[0090] The libraries, arrays and methods have several advantages. One particular advantage is that the arrays and methods provide highly reproducible results.
[0091] Another advantage is that the libraries and arrays permit screening of multiple glycans in one reaction. Thus, the libraries and arrays provide large numbers and varieties of glycans. For example, the libraries and arrays have at least two, at least three, at least ten, or at least glycans. In some embodiments, the libraries and arrays have about 2 to about 100,000, or about 2 to about 10, 000, or about 2 to about 1,000, different glycans per array. Such large numbers of glycans permit simultaneous assay of a multitude of glycan types. [0092] Moreover, as described herein, the present arrays have been used for successfully screening a variety of glycan binding proteins. Such experiments demonstrate that little degradation of the glycan occurs and only small amounts of glycan binding proteins are consumed during a screening assay. Hence, the arrays can be used for more than one assay. The arrays and methods provide high signal to noise ratios. The screening methods provided are fast and easy because they involve only one or a few steps. No surface modifications or blocking procedures are typically required during the assay procedures.
[0093] The composition of glycans on the arrays can be varied as needed by one of skill in the art. Many different glycoconjugates can be incorporated into the arrays including, for example, naturally occurring or synthetic glycans, glycoproteins, glycopeptides, glycolipids, bacterial and plant cell wall glycans and the like. Immobilization procedures for attaching different glycans to the arrays are readily controlled to easily permit array construction.
[0094] Spacer molecules or groups can be used to link the glycans to the arrays.
[0095] Such spacer molecules or groups include fairly stable (e.g. substantially chemically inert) chains or polymers. For example, the spacer molecules or groups can be alkylene groups. One example of an alkylene group is-(CH2) n-, where n is an integer of from 1 to 10.
[0096] Unique libraries of different glycans are attached to defined regions on the solid support of the array surface by any available procedure. In general, the arrays are made by obtaining a library of glycan molecules, attaching linking moieties to the glycans in the library, obtaining a solid support that has a surface derivatized to react with the specific linking moieties present on the glycans of the library and attaching the glycan molecules to the solid support by forming a covalent linkage between the linking moieties and the derivatized surface of the solid support.
[0097] The derivatization reagent can be attached to the solid substrate via carbon- carbon bonds using, or example, substrates having (poly) trifluorochloroethylene surfaces, or more preferably, by siloxane bonds (using, for example, glass or silicon oxide as the solid substrate). Siloxane bonds with the surface of the substrate are formed in one embodiment via reactions of derivatization reagents bearing trichiorosilyl or trialkoxysilyl groups.
[0098] For example, a glycan library can be employed that has been modified to contain primary amino groups. For example, the glycans can have amino moieties provided by attached alkylamine groups, amino acids, peptides, or proteins. In some embodiments the glycans can have alkylamine groups such as the-OCH2C H2NH2 (called Spi) or- OCH2CH2CH2NH2 (called Sp2 or Sp3) groups attached that provide the primary amino group. The primary amino groups on the glycans can react with an N-hydroxy succinimide (NHS)- derivatized surface of the solid support. Such NHS-derivatized solid supports are commercially available. For example, NHS-activated glass slides are available from Accelr8 Technology Corporation, Denver, CO. After attachment of all the desired glycans, slides can further be incubated with ethanolamine buffer to deactivate remaining NHS functional groups on the solid support. The array can be used without any further modification of the surface. No blocking procedures to prevent unspecific binding are typically needed.
[0099] Each type of glycan is contacted or printed onto to the solid support at a defined glycan probe location. A microarray gene printer can be used for applying the various glycans to defined glycan probe locations. For example, about 0.1 nL to about 10 nL, or about 0.5 nL of glycan solution can be applied per defined glycan probe location. Various concentrations of the glycan solutions can be contacted or printed onto the solid support. For example, a glycan solution of about 0.1 to about 1000 micromolar glycan or about 1.0 to about 500 micromolar glycan or about 10 to about 100 micromolar glycan can be employed. In general, it may be advisable to apply each concentration to a replicate of several (for example, three to six) defined glycan probe locations. Such replicates provide internal controls that confirm whether or not a binding reaction between a glycan and a test molecule is a real binding interaction.
[0100] As illustrated herein, glycans that bind to antibodies in test samples from cancer patients, e.g., breast cancer, include ceruloplasmin, Neu5Gc(2-6) GaINAc, GMl, Sulfo-T, Globo-H, sialylated Tn (Neu5Ac-alpha6-GalNAc-alpha) and LNT-2. Additional glycans to which antibodies from breast cancer patients bind include circulating antibodies from breast cancer patients can also react with the following glycans: Tri-LacNAc, LacNAc-LeX-LeX, LacNAc-LacNAc (glycan 76), H-type-2-LacNAc, H-type2-LacNAc-LacNAc, GlcNAc3LacNAc, SLeXLacNAc, 3' SialylDiLacNAc, 3' Sialyl-tri-LacNAc, 6Sia-LacNAc-LeX-LeX, 6SiaLacNAc- LacNAc. Because cancer patients have antibodies that can these glycans and the presence of such antibodies is indicative of breast cancer, many of these glycans should be present on glycan arrays used for detecting breast cancer.
[0101] Ceruloplasmin is human glycoprotein detectable in serum. Ceruloplasmin is mainly expressed and secreted by hepatocytes and is involved in copper metabolism and/or storage. See, e.g., Aouffen et a! 2001, Biochem Cell Biol, 79(4), 489-97; Wang et al, Oncogene, 2002, 21, 7598-7604; Chakravarty et al., Evaluation of Ceruloplasmine concentration in prognosis of human cancer, 1986, Acta, Med, Okayama 40 (2) 103-5; Senra et a!, Serum ceruloplasmine as a diagnoistic marker of cancer 1997, 121, 139-45. [0102] Human ceruloplasmin (CAS Number 903 1-37-2) can be obtained from the Sigma-Aldrich Co., St. Louis, MO (catalog no. C4519). The entire ceruloplasmin glycoprotein can be printed or otherwise attached to a solid support during formation of a glycan array useful for detecting breast cancer.
[0103] Other glycans to which antibodies from metastatic breast cancer patients bind include Neu5Gc(2-6)GalNAc, GMl, Sulfo-T, Globo-H, Sialylated Tn and LNT-2.
[0104] Thus, GM 1 has the following structure: Gal-beta3-GalNAc-beta4-[Neu5Ac- alpha3]-Gal-b eta4-Glc-b eta.
[0105] The Sulfo-T antigens are T-antigens with sulfate residues. In general, T antigens have the structure Gal33GalNAc and the galactose sugar moieties of this glycan can have sulfate groups or other substituents.
[0106] Globo-H includes glycans with fucose-alpha2-Gal-beta3-GalNAc-beta3-Gal- alpha4-Gal-beta4-Glc.
[0107] The sialylated Tn glycan has the following structure: Neu5Ac-alpha6-GalNAc- alpha.
[0108] LNT-2 is a ligand for tumor-promoting Galectin-4. See Huflejt & Leffler (2004) Glycoconjugate J, 20: 247-255. The structure of LNT-2 includes the following glycan: GIcNAc- beta3 -Gal-beta4-Glc-beta.
CANCER TREATMENT
[0109] Anti-glycan antibodies produced by methods provided herein or a nanoparticle vaccine covalently linked to a plurality of glycan-containing molecules are effective when following a vaccination protocol and can enhance the memory or secondary immune response to cancerous cells in the patient. Anti-glycan antibodies can be combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines and cell surface antigens, or used alone, to stimulate immunity. Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, ASCO Educational Book Spring: 60-62, 2000; Logothetis, ASCO Educational Book Spring: 300-302, 2000; Khayat, ASCO Educational Book Spring: 414-428, 2000; Foon, ASCO Educational Book Spring: 730-738, 2000; see also Restifo et ai, Cancer: Principles and Practice of Oncology, 61: 3023-3043, 1997. In one of these strategies, a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination. Dranoff et al, Proc. Natl. Acad. Sci U.S.A., 90: 3539-43, 1993.
[0110] Methods provided herein to produce anti-glycan antibodies can boost GM-CSF- modified tumor cell vaccines to improve efficacy of vaccines in a number of experimental tumor models such as mammary carcinoma (Hurwitz et al, 1998, supra), primary prostate cancer (Hurwitz et al., Cancer Research, 60: 2444-8, 2000) and melanoma (van Elsas et al, J. Exp. Med., 190: 355-66, 1999). In these instances, non-immunogenic tumors, such as the B 16 melanoma, have been rendered susceptible to destruction by the immune system. The tumor cell vaccine can also be modified to express other immune activators such as IL2, and costimulatory molecules, among others.
[0111] "Antineoplastic agent" is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis is frequently a property of antineoplastic agents.
[0112] Chemotherapeutic agents can be used in combination with polyclonal anti- glycan antibodies in methods for treatment of neoplastic disease. An antibody-cytotoxin conjugate comprising anti-glycan antibodies can also be used to boost immunity induced through standard cancer treatments. In these instances, it can be possible to reduce the dose of chemotherapeutic reagent administered (Mokyr et al, Cancer Research 58: 5301-5304, 1998). The scientific rationale behind the combined use of anti-glycan antibodies is that cell death, that is a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway. Thus, anti-glycan antibodies can boost an immune response primed to chemotherapy release of tumor cells.
[0113] A "solid tumor" includes, but is not limited to, sarcoma, melanoma, carcinoma, or other solid tumor cancer.
[0114] "Sarcoma" refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiecta ic sarcoma.
[0115] "Melanoma" refers to a tumor arising from the melanocyte system of the skin and other organs. Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.
[0116] "Carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas further include, for example, epithelial cancer, colorectal carcinoma, gastric carcinoma, oral carcinoma, pancreatic carcinoma, ovarian carcinoma, or renal cell carcinoma. Exemplary carcinomas further include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidernoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet- ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma viflosum.
[0117] "Leukemia" refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood—leukemic or aleukemic (subleukemic). Leukemia includes, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocyte leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocyte leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblasts leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocyte leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.
[0118] Additional cancers include, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer. ANTIBODY STRUCTURE
[0119] A method for producing anti-glycan antibodies in a vertebrate subject is provided which comprises administering to the vertebrate subject a vaccine comprising protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent, and isolating the anti-glycan antibodies from a biological sample of the vertebrate subject. The antibodies can be produced in a vertebrate subject, e.g., immunoglobulin G (IgG) antibodies in a mammalian subject or immunoglobulin Y (IgY) antibodies in an avian subject. Immunoglobulin Y (IgY) is the major antibody found in eggs of birds, reptiles and amphibia, including chicken, Gallus domesticus. IgY can be used as an alternative to mammalian antibodies normally used in research, and its use in immunotherapy has recently been proposed. IgY is the functional equivalent of IgG in mammals. From an evolutionary perspective, IgY antibodies are considered to be the ancestor of mammalian IgG and IgE antibodies. Compared to mammalian antibodies, IgY possesses several biochemical advantages, including ease of purification from eggs.
[0120] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site.
[0121] Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.
[0122] The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FRl , CDRl, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. MoI. Biol. 196: 901-917, 1987; Chothia et α/., Nature 342: 878-883, 1989.
[0123] A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79: 315-321, 1990, Kostelny et al., J. Immunol. 148: 1547-1553, 1992. In addition, bispecific antibodies may be formed as "diabodies" (Holliger et al, PNAS USA 90: 6444-6448, 1993 or "Janusins" (Traunecker et al., EMBO J. 10: 3655-3659, 1991 and Traunecker et al., Int J Cancer 7:51-52, 1992). Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies. Bispecific antibodies do not exist in the form of fragments having a single binding site {e.g., Fab, Fab', and Fv).
[0124] Immunoglobulin Y (IgY) is the major antibody found in eggs from chicken, Gallus domesticus. IgY can be used as an alternative to mammalian antibodies normally used in research, and its use in immunotherapy has recently been proposed. Compared to mammalian antibodies, IgY possesses several biochemical advantages and its simple purification from egg yolk prevents a stressful moment in animal handling, as no bleeding is necessary.
[0125] Small amount of antigen (1 mg) can be used to elicit an immune response in chickens and there are low intra-individual differences regarding antibody concentration found in yolk. By studying two chicken breeds and their cross, a genetic correlation was shown regarding the IgY concentration, which implies a possibility by breeding to increase IgY concentrations. By using IgY instead of goat antibody as capture antibody in ELISA, it is possible reduce interferences by complement activation. After oral administration of IgY to healthy volunteers, IgY activity was present in saliva 8 hours later, indicating a protective effect. This effect has been studied in an open clinical trial with cystic fibrosis patients. Specific IgY against Pseudomonas aeruginosa given orally prolongs the time of intermittent colonization by six months, decrease the number of positive colonizations and might be a useful complement to antibiotic treatment. Immunoglobulin therapy may diminish the development of antibiotic resistant microorganisms.
[0126] IgY antibodies are the predominant serum immunoglobulin in birds, reptiles and amphibia, and are transferred in the female from serum to egg yolk to confer passive immunity to embryos and neonates. This process corresponds to placental IgG transfer in mammals, which confers passive immunity to the fetus. IgY is the functional equivalent of IgG. From an evolutionary perspective, IgY antibodies are considered to be the ancestor of mammalian IgG and IgE antibodies.
[0127] One of the important advantages of using IgY is that there is an enhanced immunogenicity against conserved mammalian proteins, due to the phylogenetic distance between donor and recipient organisms. This makes production of antibodies against conserved mammalian proteins generally more successful in chickens than in other mammals. In addition, IgY antibodies tend to recognize the same protein in a number of mammalian species, making them more widely applicable.
[0128] Chicken IgY antibodies were found to have high affinity (avidity). IgY antibodies with high avidity against bacterial or human proteins have been developed. The avian immune response was also shown to be persistent: 20-30 μg of a highly conserved mammalian antigen induced high and long-lasting IgY titers in the yolk from immunized hens.
[0129] Distinct from bleeding immunized animals each time for production of antiserum, collecting eggs from immunized hens for isolation of IgY antibodies is a noninvasive, non-stressful process for the animal and for the human handler. This is not only a much easier and more reliable procedure, but also a method of biological production favorable to animal welfare.
[0130] IgY antibodies are concentrated in egg yolks. The isolation process involves separation of yolks from egg whites, followed by the purification of antibodies in yolks from lipids and other materials. Different materials have been used and various methods were developed, including polyethylene glycol (PEG) precipitation, DEAE fractionation, chloroform extraction, water dilution, precipitation with dextran sulphate or dextran blue or xanthan gums, separation in a two-phase system (phosphate and Triton X-100), a freeze-thaw cycle coupled with gel filtration on Biogel P-150. The isolation procedures are generally efficient and economical, although the various methods generate IgY antibodies with different yields, purity, stability and activity.
[0131] A chicken usually lays about 280 eggs in a year and an egg yolk contains 100- 150 mg of IgY antibodies. This can result in 28 to 42 grams of IgY per year from each chicken through eggs. It was shown that antigen-specific IgY antibodies were between 2% and 10% of the total IgY harvested. As the industrialized scale of hosting and caring for millions of chickens has been well developed in industry, the production of IgY antibodies can be readily scalable [0132] Despite the similarities between IgY and IgG antibodies, there are some profound differences in their chemical structures. The IgY heavy chain is 65-70 kDa, whereas the molecular mass of the mammalian IgG heavy chain is approximately 50 kDa. The IgY light chain is 19-21 kDa; the IgG is 22-23 kDa. The greater molecular mass of IgY is due to an increased number of heavy-chain constant domains and an extra pair of carbohydrate chains. In addition, the hinge region of IgY is shorter and less flexible compared to that of mammalian IgG. Recently, it has also been suggested that IgY is a more hydrophobic molecule than IgG, which matches the lipid-rich environment of the egg yolk. The structural and the amino acid sequence differences determine the differences between the two types of antibodies in their biochemical features and immunological functions.
[0133] Several major differences are observed between IgY antibodies of avian species and IgG antibodies of mammals: (1) IgY antibodies do not bind to bacterial Fc receptors such as staphylococcal protein A or streptococcal protein G, indicating the immunological difference of the Fc region from that of IgG, although there has been a study showing that the protein A- reactive site was generated after IgY bound antigen. (2) Chicken egg-yolk immunoglobulins do not react with mammalian IgG nor IgM, neither with human anti-mouse IgG antibodies (HAMA), nor binding to the rheumatoid factor (RF), which is an antiimmunoglobulin autoantibody found in many different diseases. (3) IgY antibodies were shown to have high avidity against bacterial or human proteins (4) The immunoprecipitation characteristics of IgY are different from that of IgG, presumably due to the different structure of their hinge regions. (5) IgY is stable at pH 4-9 and temperature up to 65°C in aqueous condition, which is different from the stability of IgG at pH 3-10 and temperature up to 700C. However, the resistance of IgY to the more extreme pH ranges increases if high salt conditions or stabilizing reagents such as sorbitol are present. IgY was reported to be stable at 40°C for an extended period.
[0134] "Polypeptide fragment" as used herein refers to a polypeptide that has an amino- terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acids long, more preferably at least 20 amino acids long, usually at least 50 amino acids long, and even more preferably at least 70 amino acids long. The term "analog" as used herein refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and which has at least one of the following properties: (1) specific binding to glycan-containing molecules, under suitable binding conditions, (2) ability to infection or replication of infectious bacteria, viruses, fungi, or parasites in vitro or in vivo; or (3) ability to inhibit tumor cell growth in vitro or in vivo. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.
[0135] Peptide analogs are commonly used in the pharmaceutical industry as non- peptide drugs with properties analogous to those of the template peptide. These types of non- peptide compound are termed "peptide mimetics" or "peptidomimetics". Fauchere, J. Adv. Drug Res. 15: 29, 1986; Veber and Freidinger TINS p. 392 (1985); and Evans et al. J. Med. Chem. 30: 1229, 1987, which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide {i.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: -CH2NH-, -CH2S-, -CH2-CH2-, ~CH=CH-(cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type {e.g., D-lysine in place of L-lysine) may be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Λrøj. Rev. Biochem. 61: 387, 1992, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
[0136] As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.
[0137] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% homology. In particular, conservative amino acid replacements are contemplated. Conservative amino acid replacement does not against the overall homology which can be maintained at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% homology. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are aliphatic- hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253: 164, 1991. Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.
[0138] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally- occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N. Y. (1991)); and Thornton et at. Nature 354: 105, 1991, which are each incorporated herein by reference.
[0139] "Antibody" or "antibody peptide(s)" refer to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies. An intact "antibody" comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHi, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system. The term antibody includes antigen-binding portions of an intact antibody that retain capacity to bind glycan-containing molecules. Examples of binding include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, Nature 341: 544-546, 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
[0140] An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. An antibody substantially inhibits adhesion of a receptor to a counterreceptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay).
[0141] "Fab antibodies" or "Fab fragments" refers to antibody fragments lacking all or part of an immunoglobulin constant region, and containing the Fab regions of the antibodies. Fab antibodies are prepared as described herein.
[0142] "Single chain antibodies" or "single chain Fv (scFv)" refers to an antibody fusion molecule of the two domains of the Fv fragment, VL and VH- Although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al, Science 242: 423-426, 1988; and Huston et al, Proc. Natl. Acad. Sci. USA, 85: 5879-5883, 1988). Such single chain antibodies are included by reference to the term "antibody" fragments can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.
[0143] "Human sequence antibody" includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. The human sequence antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences {e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Such antibodies can be generated in non- human transgenic animals, e.g., as described in PCT Publication Nos. WO 01/14424 and WO 00/37504. However, the term "human sequence antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., humanized antibodies).
[0144] Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567, incorporated herein by reference in its entirety and for all purposes; and Queen et al., Proc. Natl Acad. Sci. USA 86: 10029-10033, 1989.
[0145] "Monoclonal antibody" refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions (if present) derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
[0146] "Polyclonal antibody" refers to a preparation of more than 1 (two or more) different antibodies to glycan-containing molecules. Similarly antibodies to glycan-containing molecules can act as peptidomimetics that bind to cell surface glycans and thus inhibit interaction with cells presenting glycans on a cell surface. These and other antibodies suitable for use in the present invention can be prepared according to methods that are well known in the art and/or are described in the references cited here. In preferred embodiments, anti-glycan antibodies used in the invention are mammalian antibodies or avian antibodies— e.g., antibodies isolated from a mammalian species, e.g., human, sheep, dogs, cows, pigs, or an avian species, e.g., chicken.
[0147] "Immune cell response" refers to the response of immune system cells to external or internal stimuli {e.g., antigen, glycan-containing molecules, cell surface receptors, cytokines, chemokines, and other cells) producing biochemical changes in the immune cells that result in immune cell migration, killing of target cells, phagocytosis, production of antibodies, other soluble effectors of the immune response, and the like.
[0148] "Immune response" refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, metastatic epithelial cancer, colorectal carcinoma, gastric carcinoma, oral carcinoma, pancreatic carcinoma, ovarian carcinoma, or renal cell carcinoma, or infectious disease from invading pathogens, cells or tissues infected with pathogens, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
[0149] "T lymphocyte response" and "T lymphocyte activity" are used here interchangeably to refer to the component of immune response dependent on T lymphocytes (e.g., the proliferation and/or differentiation of T lymphocytes into helper, cytotoxic killer, or suppressor T lymphocytes, the provision of signals by helper T lymphocytes to B lymphocytes that cause or prevent antibody production, the killing of specific target cells by cytotoxic T lymphocytes, and the release of soluble factors such as cytokines that modulate the function of other immune cells).
TREATMENT REGIMES
[0150] The invention provides pharmaceutical compositions comprising one or a combination of antibodies, e.g., anti-glycan antibodies, or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan-containing molecules, formulated together with a pharmaceutically acceptable carrier. Some compositions include a combination of multiple (e.g., two or more) polyclonal antibodies or antigen-binding portions thereof of the invention. In some compositions, each of the antibodies or antigen-binding portions thereof of the composition is a polyclonal antibody or a human sequence antibody that binds to a distinct, pre-selected epitope of an antigen.
[0151] In prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition (i.e., a neoplastic disease or infectious disease) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. In therapeutic applications, compositions or medicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient immune response has been achieved. Typically, the immune response is monitored and repeated dosages are given if the immune response starts to wane. EFFECTIVE DOSAGES
[0152] Effective doses of the antibody compositions of the present invention, e.g., anti- glycan antibodies produced by the methods of the present invention, or vaccines comprising a nanoparticle, at least some of the nanoparticles covalently bound to glycan-containing molecules, for the treatment of cancer-related conditions, e.g., metastic cancer, or for treatment of infectious disease as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the vertebrate subject, whether the patient is a vertebrate, e,g., mammalian or avian species, human, primate, rat, mouse, dog, cat, rabbit, cow, horse, or goat, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the vertebrate subject is a human but nonhuman mammals, including transgenic mammals, and avian species can also be treated. Treatment dosages need to be titrated to optimize safety and efficacy.
[0153] For administration with an antibody or a nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan-containing molecules, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1- 10 mg/kg. An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months. In some methods, two or more antibodies, or two or more plant viral particles with different binding specificities are administered simultaneously, in which case the dosage of each antibody or nanoparticle vaccine administered falls within the ranges indicated. Antibody or nanoparticle vaccine is usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, antibody or nanoparticle vaccine can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody or nanoparticle vaccine in the patient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime. [0154] Doses for antibody or nanoparticle vaccine range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg antibody per patient. Doses for nanoparticle vaccine vary from 10-100, or more, particles per dose.
ROUTES OF ADMINISTRATION
[0155] Antibody compositions of the present invention, e.g., anti-glycan antibodies produced by the methods of the present invention, or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan-containing molecules, for the treatment of cancer- related conditions, e.g., metastic cancer, or for treatment of infectious disease as described herein can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic as inhalants for antibody preparations targeting brain lesions, and/or therapeutic treatment. The most typical route of administration of an immunogenic agent or nanoparticle vaccine is subcutaneous although other routes can be equally effective. The next most common route is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles. In some methods, agents are injected directly into a particular tissue where deposits have accumulated, for example intracranial injection. Intramuscular injection on intravenous infusion are preferred for administration of antibody. In some methods, particular therapeutic antibodies are injected directly into the cranium. In some methods, antibodies are administered as a sustained release composition or device, such as a Medipad™ device.
[0156] Antibodies or nanoparticle vaccines of the invention can optionally be administered in combination with other agents that are at least partly effective in treating various diseases including various cancer-related diseases. In the case of tumor metastasis to the brain, antibodies or nanoparticle vaccines can also be administered in conjunction with other agents that increase passage of the agents of the invention across the blood-brain barrier (BBB).
FORMULATION
[0157] Antibody compositions of the present invention, e.g., anti-glycan antibodies produced by the methods of the present invention, or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan-containing molecules, for the treatment of cancer- related conditions, e.g., metastatic cancer, or for treatment of infectious disease as described herein are often administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components. (See Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically- acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
[0158] Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functional ized Sepharose™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).
[0159] For parenteral administration, compositions of antibodies or nanoparticle vaccine of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodies can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient. An exemplary composition comprises monoclonal antibody at 5 mg/mL, formulated in aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.
[0160] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-1 19, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
[0161] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.
[0162] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%- 95% of active ingredient, preferably 25%-70%.
[0163] Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins. Glenn et al., Nature 391: 851, 1998. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.
[0164] Alternatively, transdermal delivery can be achieved using a skin patch or using transferosomes. Paul et al., Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.
[0165] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
DIAGNOSTIC USES
[0166] Characteristics of Antibody Compositions and Plant Viral Particle Compositions for Use as Diagnostic Reagents. Human antibody compositions of the present invention, e.g., anti-glycan antibodies produced by the methods of the present invention, or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan-containing molecules, as described herein for use in diagnostic methods to identify metastatic tumor cells, e.g., cells from metastatic epithelial cancer, colorectal carcinoma, gastric carcinoma, oral carcinoma, pancreatic carcinoma, ovarian carcinoma, or renal cell carcinoma, or for diagnosis of infectious disease, are preferably produced using the methods described above. The methods result in virtually unlimited numbers of antibodies and antibody compositions of the invention of any epitope binding specificity and very high binding affinity to any desired antigen. In general, the higher the binding affinity of an antibody for its target, the more stringent wash conditions can be performed in an immunoassay to remove nonspecifically bound material without removing target antigen. Accordingly, antibodies and antibody compositions of the invention used in the above assays usually have binding affinities of at least 108, 109, 1010, 1011 or 1012 M" '. Further, it is desirable that antibodies used as diagnostic reagents have a sufficient on-rate to reach equilibrium under standard conditions in at least 12 hours, preferably at least five hours and more preferably at least one hour.
[0167] Antibodies and antibody compositions of the invention used in the claimed methods preferably have a high immunoreactivity, that is, percentages of antibodies molecules that are correctly folded so that they can specifically bind their target antigen. Such can be achieved by expression of sequences encoding the antibodies in E. coli as described above. Such expression usually results in immunoreactivity of at least 80%, 90%, 95% or 99%.
[0168] Some methods of the invention employ polyclonal preparations of antibodies and antibody compositions of the invention as diagnostic reagents. The use of polyclonal mixtures has a number of advantages with respect to compositions made of one monoclonal antibody. By binding to multiple sites on a target, polyclonal antibodies or other polypeptides can generate a stronger signal (for diagnostics) than a monoclonal that binds to a single site. Further, a polyclonal preparation can bind to numerous variants of a prototypical target sequence {e.g., allelic variants, species variants, strain variants, drug-induced escape variants) whereas a monoclonal antibody may bind only to the prototypical sequence or a narrower range of variants thereto.
[0169] In methods employing polyclonal human antibodies prepared in accordance with the methods described above, the preparation typically contains an assortment of antibodies with different epitope specificities to the intended target antigen. A difference in epitope binding specificities can be determined by a competition assay.
[0170] Samples and Target. Although human or avian antibodies can be used as diagnostic reagents for any kind of sample, and are useful as diagnostic reagents for human samples. Samples can be obtained from any tissue or body fluid of a patient. Preferred sources of samples include, whole blood, plasma, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. Samples can also be obtained from biopsies of internal organs or from cancers. Samples can be obtained from clinical patients for diagnosis or research or can be obtained from undiseased individuals, as controls or for basic research.
[0171] The methods can be used for detecting any type of target antigen. Exemplary target antigens including tumor antigens, for example, tumor antigens for metastatic epithelial cancer, colorectal carcinoma, gastric carcinoma, oral carcinoma, pancreatic carcinoma, ovarian carcinoma, or renal cell carcinoma, or target antigens or infectious bacteria, virus, fungi or parasites. Other target antigens are human proteins whose expression levels or compositions have been correlated with human disease or other phenotype. Examples of such antigens include adhesion proteins, hormones, growth factors, cellular receptors, autoantigens, autoantibodies, and amyloid deposits. Other targets of interest include tumor cell antigens, such as carcinoembryonic antigen. Other antigens of interest are class I and class II MHC antigens.
[0172] Formats for Diagnostic Assays. Human antibodies can be used to detect a given target in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. See Harlow & Lane, supra; U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074; 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876, each incorporated herein by reference in their entirety and for all purposes.
[0173] Immunometric or sandwich assays are a preferred format. See U.S. Pat. Nos. 4,376,110; 4,486,530; 5,914,241 ; and 5,965,375, each incorporated herein by reference in their entirety and for all purposes. Such assays use one antibody or population of antibodies immobilized to a solid phase, and another antibody or population of antibodies in solution. Typically, the solution antibody or population of antibodies is labelled. If an antibody population is used, the population typically contains antibodies binding to different epitope specificities within the target antigen. Accordingly, the same population can be used for both solid phase and solution antibody. Solid phase and solution antibodies can be contacted with target antigen in either order or simultaneously. If the solid phase antibody is contacted first, the assay is referred to as being a forward assay. Conversely, if the solution antibody is contacted first, the assay is referred to as being a reverse assay. If target is contacted with both antibodies simultaneously, the assay is referred to as a simultaneous assay. After contacting the target with antibody, a sample is incubated for a period that usually varies from about 10 min to about 24 hr and is usually about 1 hr. A wash step is then performed to remove components of the sample not specifically bound to the antibody being used as a diagnostic reagent. When solid phase and solution antibodies are bound in separate steps, a wash can be performed after either or both binding steps. After washing, binding is quantified, typically by detecting label linked to the solid phase through binding of labelled solution antibody. Usually for a given pair of antibodies or populations of antibodies and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of target antigen. Concentrations of antigen in samples being tested are then read by interpolation from the calibration curve. Analyte can be measured either from the amount of labelled solution antibody bound at equilibrium or by kinetic measurements of bound labelled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of target in a sample.
[0174] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, and also particles, such as agarose, a dextran-based gel, dipsticks, particulates, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX™. (Amersham Pharmacia Biotech, Piscataway N.J.) Immobilization can be by absorption or by covalent attachment. Optionally, antibodies can be joined to a linker molecule, such as biotin for attachment to a surface bound linker, such as avidin.
LABELS
[0175] The particular label or detectable group used in the assay is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H5 14C, 35S, 125I, 121I, 112In, "mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18F, 11C, 15O, (for Positron emission tomography), 99mTC, 111In (for Single photon emission tomography), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, and the like) beads. Patents that described the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated herein by reference in their entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals, 6th Ed., Molecular Probes, Inc., Eugene OR.).
[0176] The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
[0177] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti- ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti- ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
[0178] The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbel liferone, and the like Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems which may be used, see, U.S. Pat. No. 4,391,904, incorporated herein by reference in its entirety and for all purposes.
[0179] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple calorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
[0180] Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection. [0181] Frequently, the antibody compositions of the present invention, e.g., anti-glycan antibodies produced by the methods of the present invention, or vaccines comprising a plant viral particle displaying a plurality of glycan-containing molecules, for the treatment of cancer-related conditions, e.g., metastic cancer, or for treatment of infectious disease as described herein, will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
TOXICITY
[0182] Preferably, a therapeutically effective dose of the antibody compositions of the present invention, e.g., anti-glycan antibodies produced by the methods of the present invention, or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan- containing molecules, for the treatment of cancer-related conditions, e.g., metastic cancer, or for treatment of infectious disease as described herein will provide therapeutic benefit without causing substantial toxicity.
[0183] Toxicity of the antibodies or nanoparticle vaccine described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LDioo (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the antibodies or plant viral particles described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et ai, 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1,
KITS
[0184] Also within the scope of the invention are kits comprising the compositions {e.g., polyclonal anti-glycan antibodies or nanoparticle vaccines, at least some of the nanoparticles covalently bound to glycan-containing molecules) of the invention and instructions for use. The kit can further contain a least one additional reagent, or one or more additional human antibodies of the invention {e.g., a human antibody having a complementary activity which binds to an epitope in the antigen distinct from the first human antibody). Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit. [0185] Other embodiments and uses will be apparent to one skilled in the art in light of the present disclosures.
EXEMPLARY EMBODIMENTS
EXAMPLE 1
Convenient ligation of carbohydrates to a viral surface
[0186] Carbohydrates 1-4 (Scheme 1) - each significant in biology and human pathology (see Supporting Information) - were prepared as 2-azidoethanol adducts by enzymatic and chemical methods as previously described. D. Vasiliu et al., Carb. Res. 2006, 341, 1447- 1457; C-Y. Huang et al., Proc. Natl. Acad. ScL USA 2006, 103, 15-20. The 30-nm wild-type cowpea mosaic virus (CPMV) particle was acylated on exterior lysine side chains with N- hydroxysuccinimide linkers 5 or 6, containing azide or alkyne groups, respectively (Scheme 1, bottom). Q. Wang et al., Chem. Biol. 2002, 9, 805-81 1. The resulting particles were then addressed by the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction using ligands 9. Q. Wang et al., J. Am. Chem. Soc. 2003, 125, 3192-3193. The use of tris(carboxyethyl)phosphine (TCEP) as reducing agent is no longer recommend. The use of low concentrations of ascorbate or a Cu(I) precursor under inert atmosphere usually provides better results. S. Sen Gupta et al., Bioconj. Chem. 2005, 16, 1572-1579. In order to facilitate the measurement of the number of carbohydrates attached to the protein scaffold, 1, 2, and 4 were joined to fluorescein dialkyne 7 by the CuAAC reaction. S. Sen Gupta et al., Chem. Commun. 2005, 4315-4317; G. W. Warr et al., Immunol Today 1995, 16, 392-398. The resulting carbohydrate-alkyne adducts were readily purified away from excess 7 by gel permeation chromatography. Sialyl Lewis" derivative 3 was used directly as the azide to ensure the fluorescein dye had no deleterious or inhibitory effects on the immune response. The CuAAC method allows for the convenient covalent deposition of minimal quantities of these highly valuable compounds on proteins, allowing us to achieve high loadings on the viral surface with the use of relatively modest amounts of material. S. Sen Gupta et al., Bioconj. Chem. 2005, 16, 1572-1579.
Figure imgf000054_0001
blood group A antigen tn-LacNAc
Figure imgf000054_0002
3 4 sialyl Lewis* globo-H
Figure imgf000054_0003
17 BSA 29 ± 5
Scheme 1. (Top) Glycans attached to carrier proteins by azide-alkyne cycloaddition. (Bottom) Preparation of virus-carbohydrate conjugates, (i) 20% DMSO in 0.1 M phosphate buffer, pH 7.0, RT, 5 or 6 [13 mM, 300 equiv. with respect to viral subunits), CPMV (4 mg/mL (42.8 μM in subunits)]; (ii) see reference (S. Sen Gupta et al., Bioconj. Chem. 2005, 16, 1572- 1579); (iii) 15% DMF in 0.1 M Tris buffer, pH 8.0, CuSO4 (2 mM), 9 (4 mM), 8 (5 mM), CPMV-azide (2 mg/mL, 21.4 μM in protein subunits), tris(carboxyethyl)phosphine (4 mM), RT, 18 h.; (iv) improved conditions: 0.1 M Tris buffer, pH 8.0, CuOTf (I mM), 10 (2 mM), 8 or carbohydrate-azide (0.3 mM), CPMV-azide or CPMV-alkyne (2 mg/mL), N2 atmosphere, RT, 12-18 h. NHS denotes the N-hydroxysuccinimide ester. [0187] The virus-carbohydrate conjugates were used to inoculate chickens by intramuscular injection, in order to evaluate their capacities for inducing an IgY immune response. The avian IgY isotype is the functional equivalent of mammalian IgG and is considered to be its evolutionary precursor. (G. W. Warr et al., Immunol. Today 1995, 16, 392- 398.) IgY's have several practical advantages over IgG's for use as polyclonal reagents, including large isolated yields from the eggs of immunized hens and reduced cross-reactivity with native mammalian proteins. (A. Larsson et al.l, J. Immunol. Meth. 1992, 156, 79-83.) Total IgY was isolated by poly(ethyleneglycol) precipitation from eggs collected from the immunized birds, providing approximately 1 gram of polyclonal antibody per experiment from a total of 12 eggs per bird. Unless otherwise indicated, the antibodies obtained in this manner were used without further purification. Each inoculation was performed on two chickens in parallel and the results analyzed independently.
[0188] In addition to chicken, the attachment of oligosaccharides to the exterior surfaces of capsids derived from bacteriophage Q-beta and cowpea mosaic virus has also resulted in effective anti-glycan immune response in mice. As an example, the ganglioside GM3 carbohydrate (Neu5Acα2-3Galβl-4Glcβ-l inker) was conjugated to Q-beta and administered in the standard manner. A strong IgM response targeting the Neu5Aca2-3Gal epitope arose at day 14 and persisted to day 35 and beyond, and a strong IgG response arose slightly later, giving an antibody population targeting the same epitope. Binding specificities were determined by analysis of the total serum antibody on an extensive glycan array, covering all major glycan types. Similar experiments were also performed with CPMV and the Tn antigen, which produced similar strong immune response (see, Miermont et al., Chem. Eur. J. 2008, 14, 4939).
EXAMPLE 2
Antibody specificities are assayed on a glycan microarray
[0189] The specificity of glycan binding by each IgY was determined using printed microarrays containing 200 (vl.O) or 264 (v2.0) glycans, as previously described (Blixt et al., Proc. Nat. Acad Sci. USA 101, 17033-17038, 2004). The displayed sugars span a wide range of motifs and provide an excellent way to rapidly profile both the selectivity and overall affinity of glycan-binding proteins. For all chickens, antibodies present in the eggs prior to immunization presented a characteristic recognition pattern (rhamnose, Fuc-α2/4-GlcNAc, Glcβl-6Glc; See Figure 7). In contrast, the antibodies isolated from the virus-displayed blood group A antigen (11) were remarkably specific, binding only to the parent structure and closely-related analogues
(Figure Ia). Weak binding was observed to the H-type related structures (Fuc((l-2)-Gal-); interestingly, the fucose determinant was required for binding. While members of this polyclonal preparation (and all crude IgY samples described here) contained antibodies that recognized other components of the antigen (CPMV protein and fluorescein, determined by ELISA, not shown), the glycan specificity exhibited by the polyclonal anti-11 is remarkable and justifies the use of this IgY material, without further purification, as a reagent for the detection of blood group A and B antigens.
[0190] Immunization with sialyl-Lewis* conjugate 13 gave rise to IgY isolates that were somewhat less selective, although they were highly potent and showed good response to sialyl-containing carbohydrates, with additional binding to other carbohydrates present generally in the serum (Figure Ib). Note, however, that anti-13 polyclonal IgY did not distinguish between type 1 (Galβ(l,3)GlcNAc) and type 2 (Galβ(l,4)GlcNAc) disaccharides and thus binds to both Lea (Gal((l-3)[Fuc((l-4)]GlcNAc) and Lex (Gal((l-4)[Fuc((l-3)]GlcNAc).
[0191] Figure 1 shows glycan array binding analysis of crude IgY isolated after (a) no immunization (array vl .O); (b) immunization with 11 (array v2.0); and (c) immunization with 13 (array vl .O). In c, the dominant motif recognized by these antibodies is shown in the box; a key to the symbols used is boxed in panel b. In addition, a few strong peaks are provided by non- sialyl carbohydrates that are likely to be present in serum due to bacterial exposure, such as 127 (a peptidoglycan from gram negative bacterial cell wall) and 200 (rhamnose, a common bacterial sugar).
EXAMPLE 3
Polyclonal IgY antibodies can be enriched through affinity purification to increase specificity
[0192] The use of tri-LacNAc conjugate 12 produced a mono-LacNAc selective response: Figure 2a shows that a high proportion of the glycans bound by the polyclonal IgY antibody contained mono-LacNAc repeats, either alone or as part of a larger structure. The large number of hits compared to Figure Ib, for example, is also a consequence of the fact that LacNAc-containing structures are well represented on the array used for analysis. To further improve the specificity of the anti-12 IgY material, two functional ized agarose supports were constructed for affinity purification, one bearing the full tri-LacNAc structure 2 and the other the mono-LacNAc analogue 18 (Figure 2), again using the convenient CuAAC methodology (Punna, et al., Bioconj. Chem. 16, 1536-1541 , 2005). The agarose-2 support was found to be too potent, in that LacNAc-specific members of the polyclonal population could not be eluted without exposure to conditions causing irreversible denaturation of the protein. In contrast, agarose-18 allowed the removal of antibodies that recognize non-LacNAc glycans (Figure 2b) and CPMV protein (Figure 2d) in the flowthrough solution and the elution of antibodies that recognize the minimal LacNAc epitope (Figure 2c). The same general results were obtained from an independent immunization experiment, but some variation was observed in the ability of the polyclonal mixture to bind fucosylated LacNAc.
[0193] Figure 2 shows glycan array binding analysis using array version vl .O of anti-2 antibodies purified by affinity filtration through agarose-18: (a) total IgY from immunization with 12; in the box is the cartoon representation of the structure of the virus-displayed carbohydrate; (b) flow-through after washing; (c) binding profile of antibodies eluted with pH 2 buffer followed by neutralization to pH 7. (d) ELISA assay for anti-CPMV antibodies of the material obtained at each step of affinity purification of polyclonal anti-12, at identical protein concentrations. These data show that CPMV-binding antibodies are removed in the flow-through step, and are therefore not present in the glycan-binding "elution" fraction. To the right of (d) is the LacNAc structure attached to agarose-alkyne to create the affinity purification column.
EXAMPLE 4
CPMV and KLH elicit a similarly potent antibody response
[0194] Since comparative glycan array analysis of antibody or lectin specificity is a relatively new endeavor, independent duplicate experiments were performed using the same batch of antigen. CPMV (15) and KLH (16) conjugates of tri-LacNAc 2 were prepared, omitting the fluorescein labeling step to avoid potential degradation in the performance or solution-phase stability of the KLH carrier protein by the hydrophobic dye. In general, excellent reproducibility in the glycan array analyses was observed (Supporting Information), considering all of the variables that could come into play in inoculation, immune response, and sample handling.
[0195] The overall polyclonal binding pattern of anti-15 and anti-16 antibodies closely mirrored that observed for anti-12 antibodies, showing that both the fluorescein label in 12, and the different carrier proteins of 15 and 16, had little effect on the manner in which the carbohydrate epitopes were recognized, in contrast to other cases discussed in the literature (Kagan et al., Cancer Immunol. Immunother. 2005, 54, 524-430; and Kiessling et al., Chem. Biol. 1996, 3, 71-77). The polyclonal population of anti-15 did show stronger binding to many more glycans compared to anti-16 antibodies (Supporting Information), but more tests are needed with other attached carbohydrates to determine if the virus reliably provides for a more intense immune response. EXAMPLE 5
Globo-H decorated CPMV elicits polyclonal antibodies with comparable specificity to a monoclonal antibody
[0196] Danishefsky and coworkers have administered a KLH-globo-H conjugate to cancer patients and characterized the polyclonal immune response in comparison to bacterial polysaccharide vaccines (Wang et al., Proc. Natl. Acad. Sci. USA 2000, 97, 2719-2724; and Slovin et al., Proc. Natl. Acad. Sci. USA 1999, 96, 5710-5715). We prepared the CPMV-globo- H antigen 14 from the corresponding fluorescein-labeled conjugate and submitted it to the standard chicken inoculation protocol. The avian IgY response to this antigen was highly potent and selective, comparing favorably with, if not quite matching, the specificity of a commercially available monoclonal IgM antibody against globo-H (Figure 3a). The latter (Figure 3b) displayed strongest binding to globo-H itself (glycan 56 on the array) and the H-type-3 trisaccharide Fuc((l-2)Gal((l-3)GalNAc(- (58), but also recognized the related ganglioside fucosyl-GMl (59, 60) and GlcNAc((l-2)Gal((l-3)GalNAc(- (158). The avian IgY showed the strongest affinity for the same globo-H related antigens (56, 58), but also recognized a somewhat wider range of Fuc((l-2)Gal((l-3) structures including the H-type-1 (Fuc((l-2)Gal((l-3)GlcNAc sugars 63 and 64, and, more weakly, H-type-2 (Fuc((l-2)Gal((l-4)GlcNAc) and 2'-fucosyllactose (Fuc((l- 2)Gal((l-4)Glc) structures (69,72-74). Compound 55 (Fuc((l-2)Gal((l-3)GalNAc((l-3)Gal(-, a terminal fragment of globo-H, was recognized much less well by both monoclonal IgM and polyclonal IgY antibodies than the full structure 56. Both antibodies distinguish between globo- H related antigens and Leb and Ley sugars that bear additional fucosylation adjacent to the terminal fucose unit (57, 65-68).
[0197] Figure 3 shows glycan array binding analysis using array version v2.0 of (a) anti-14 IgY (0.15 mg/mL), and (b) commercially available monoclonal IgG against globo-H (0.035 mg/mL, Axorra, Inc.). In each case, symbolic representations of the structures of the glycans most strongly bound are shown; a key to the symbols used is boxed in panel b. On the left, the identification number and average signal intensities are listed for each glycan structurel.
[0198] Similar signal intensities were obtained with polyclonal anti-14 only 4.3 times more concentrated than the monoclonal antibody, in spite of the expectation that only a small fraction of the polyclonal mixture should respond to the glycan antigen. Factors that may contribute to this potent binding behavior include enrichment in the percentage of glycan-binding members of the polyclonal ensemble, higher avidity of some members of that mixture, and/or a difference in secondary antibody performance in the ELISA-type glycan array assay. The last is unlikely to be more than a minor contributor. While we hope to gain further insight into the reasons, the practical benefit is clear: we can obtain many hundreds of milligrams of potent and selective polyclonal IgY antibody far less expensively than the cost of producing much smaller quantities of monoclonal IgG or IgM. This has important implications for the development of new diagnostic tools relying on carbohydrate detection.
[0199] The glycan array technology used here is a convenient and effective way to define anti-carbohydrate specificities, and will have two important uses in our future studies. First, the binding profiles provided by the arrays represent a wealth of information that we hope will allow us to correlate antibody properties with the structures and conditions of immunization. Secondly, glycan array profiling is a highly useful method of quality control for the development of carbohydrate-targeted reagents and immunotherapies.
EXAMPLE 6
Efficient Production of Anti-Glycan Polyclonal Antibodies
[0200] The present study provides the efficient production (and purification when necessary) of anti-glycan polyclonal antibodies using a combination of bioorthogonal ligation chemistry and polyvalent display on an immunogenic virus scaffold. To our knowledge, few other cases of anti-glycan IgY antibodies have been obtained, and purification of anti- carbohydrate antibodies by affinity chromatography is rare. Immunization of mice has yielded several monoclonal antibodies specific for some carbohydrates; most, but not all, are of the IgM class (Gilewski et al., Proc. Nat. Acad. ScL USA 98, 3270-3275, 2001). In one notable example (GaI-13), a double selection procedure was used to screen more than 700 hybridoma clones, of which only one produced antibodies binding to the target Galαl-3Gal structure. We suggest that the expense and effort required to create a monoclonal antibody against a carbohydrate may be unnecessary in certain cases if a sufficiently selective polyclonal mixture can be readily obtained. The availability of robust and selective attachment chemistry, precisely controlled attachment positions on structurally discrete viral scaffolds, and control of tether length and flexibility, will also allow us to test parameters of structure and polyvalent display that may contribute to effective anti-carbohydrate antigenicity. Studies toward this end are in progress.
EXAMPLE 7 Materials and Methods
[0201] Carbohydrate synthesis. Compounds 2-4 were previously described, as was the immediate precursor to 1 [H-type-2 (Fucαl,2-Galβl,4-GlcNAc)] (Vasiliu et al., Carb. Res. 2006, 341, 1447-1457; and Huang et al., Proc. Natl. Acad. Sci. USA 2006, 103, 15-20). The conversion of this molecule to 1 was performed as follows. The trisaccharide (150 mg, 0.25 mmol) and UDP-GIcNAc (350mg, 0.57 mmol) were dissolved in aqueous buffer (25 mL) containing MnCl2 (40 mM) and NAD+, (0.3 mM). UDPGal-4'-epimerase (350 U) and GTA (10 U) were added and the pH was adjusted to 7.5. The reaction was stirred at 37°C for 24 hours to give 85-90% product formation as judged by thin-layer chromatography. The mixture was centrifuged (7500 rpm, 15 min) and the supernatant was loaded onto a Sephadex G15 column (5 x 170 cm) equilibrated and eluted with 5% H-BuOH in water. All fractions containing product were collected and lyophilized. The residue (250 mg) was dissolved in water (3 mL), loaded on a 5 micron Altima NH2 prep HPLC column (2.5 x 25cm), and eluted at 5 mL/min with an isocratic mixture Of CH3CNiH2O containing 0.1% trifluoroacetic acid). The desired compound emerged from the column at 80-120 mL. The fractions were pooled and neutralized with concentrated NH4OH, evaporated at reduced pressure, and lyophilized to give 1 as a dry white powder (130 mg, 0.16 mmol, 64%).
[0202] Acylation of CPMV. Unless otherwise noted, the final solvent mixture for all reactions with CPMV contained 20% DMSO in 0.1 M phosphate buffer (pH 7.0). NHS-azide 5 or NHS-alkyne 6 (13 mM, 300 equiv. with respect to the concentration of viral asymmetric subunit) was mixed with wild-type CPMV (4 mg/mL, 43 μM protein subunit) overnight at room temperature. Purification of derivatized virus on >1 mg scale was performed by ultracentrifugation over a 10-40% sucrose gradient, pelleting of the recovered virus, and dissolution of the resulting material in Tris buffer (0.1 M, pH 8). Mass recoveries of derivatized viruses were typically 60-80%; all such samples were composed of >95% intact particles as determined by analytical size-exclusion FPLC on a Superose-6 column.
[0203] Carbohydrate-fluorescein conjugates (8). To a solution of 1 (10 mg, 12.4 μmol) in H2O (1 mL) was added 7 (70 mg, 0.125 mmol) in THF (1 mL). J-BuOH (1 mL) was added, followed by sodium ascorbate (0.5 M in H2O, 72 μL, 36 μmol) and CuSO4 (0.5 M in H2O, 24 μL, 12 μmol). The reaction mixture was stirred in a closed vial for 48 h at room temperature, and was monitored by TLC (Rf= 0.6) in 8:3:3:2 (EtOAc/MeOH/AcOH/H;,©), as well as by disappearance of the azide peak (2100 cm"1) in the infrared spectrum. Upon completion, the volatile solvents were removed by rotary evaporation and an additional 5 mL of H2O was added. Most of the excess 7 was removed by extraction with ethyl acetate. The aqueous phase was concentrated by evaporation and residual 7 was removed by column chromatography (Sephadex G-15, 95:5 H2O/BuOH), giving the desired fluorescein conjugate 8 as a yellow solid (11 mg, 65% yield) upon lyophilization of the collected fraction. MALDI-TOF: [M+H]+ = 1361, [M+Na]+ = 1383, [M+K]+ = 1399. The analogous derivatives of 2 and 4 were prepared in approximately 55% yield using the same procedure.
[0204] Virus-carbohydrate conjugates 11, 12, and 14. CPMV-azide (0.5 mL of 2 mg/mL solution) was incubated with complementary alkyne (8, 5 mM) in Tris buffer (0.1 M, pH 8, 0.5 mL) containing 9 (3 mM), CuSO4 (2 mM) and tris(carboxyethyl)phosphine (4 mM), for 18 h at room temperature. The resulting virus conjugates were purified by ultracentrifugation over a 10-40% sucrose gradient, pelleting of the recovered virus, and dissolution of the resulting material in potassium phosphate buffer (0.1 M, pH 7). Mass recoveries of derivatized viruses were typically 60-80%; all such samples were composed of >95% intact particles as determined by analytical size-exclusion FPLC. The carbohydrate loading on these conjugates was determined by quantitative measurement of fluorescein absorbance against calibrated protein-dye standards. At 260 nm, CPMV at 0.10 mg/mL gives a standard absorbance of 0.80, and fluorescein exhibits a molar absorbtivity of 70,000 M"1 cm"1 at 495 nm. The average molecular weight of the CPMV virion is 5.6 x 106 g/mol.
[0205] Virus-carbohydrate conjugates 13 and 15. CPMV -alkyne derived from the reaction of wild-type virus with NHS ester 6 was pelleted by ultracentrifugation and resuspended in degassed 0.1M Tris buffer (pH 8.0) in a nitrogen-filled glove box. A solution at 2 mg/ml (concentration determined by uv-vis spectroscopy) was mixed with 2 or 3 (0.3 mM) in the presence of CuOTf (1 mM), 10 (2 mM) and degassed 0.1M Tris buffer (pH 8.0), all under inert atmosphere. Gentle agitation on a slow rotor for 12-18 h under nitrogen was followed by purification of the virus-triazole conjugates as described above. The loading of carbohydrate- azide compounds onto conjugates 13 and 15 was estimated by analogy to otherwise identical reactions using fluorescein-azide (Supporting Information) with the CPMV -alkyne scaffold.
EXAMPLE 8
Synthetic procedures
[0206] Materials. The following were obtained from the indicated sources: white leghorn hens, 3-6 months old (Mclntyre Farms, San Diego, CA); monoclonal antibody ALX- 804-550, ot-globo-H (Axorra, LLC, San Diego); goat-α-IgY-FITC and total IgY purification service (Genway, Biotech., San Diego); rabbit-α-mouse IgG/IgM/IgA-FITC (Abeam, Inc., Cambridge, MA); CarboxyLink resin, 4% (Pierce Biotechnology, Inc., Rockford, IL). All aspects of the care and handling of the chickens were performed in accordance with national and local guildelines, under the supervision of the TSRI Institutional Animal Care and Use Committee. [0207] Carbohydrate syntheses. Carbohydrates 1-4 were prepared as 2-azidoethanol β- anomeric adducts by enzymatic and chemical synthesis as previously described (Vasiliu et al., Carb. Res. 2006, 341, 1447-1457; and Huang et al., Proc. Natl. Acad. Sci. USA 2006, 103, 15- 20). A representative example (compound 1) of the last step and purification is given in the Experimental section. The 1H NMR (500 MHz, D2O) spectrum of 1 is shown in Figure 4.
[0208] Procedure for immobilization oftri- and mono-LacNAc for affinity purification. Stock solutions (50 mM) of the following reagents were prepared: 2,6-lutidine (DMF), 2,2'- bipyridine (DMF), CuBr (DMF), and sodium ascorbate (water). A slurry of agarose-alkyne beads64 (1.0 equiv) in DMF in a disposable frit was treated with carbohydrate-azide (4.0 equiv), followed by 2,6-lutidine (8.0 equiv), 2,2'-bipyridine (8.0 equiv), cuprous bromide (4.0 equiv), and finally sodium ascorbate (8.0 equiv). The resulting suspension was bubbled with a gentle flow of nitrogen for 1 min, capped, and rotated at room temperature for 12-18 hours. The reaction mixture was drained and washed sequentially with approximately 5 column volumes each of DMF, H2O, MeOH, 0.1 M aq EDTA, H2O and DMF to obtain carbohydrate-agarose beads. A parallel experiment with a dye-azide under otherwise identical conditions provided highly colored beads confirming a positive reaction.
[0209] KLH and BSA conjugates 16 and 17. Bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) at 4 mg/mL in 0.1 M phosphate buffer (pH 7.0) were treated with 335 equiv. of NHS-alkyne linker 6. The reactions were agitated by gentle rocking overnight at room temperature and the products purified by two rounds of dialysis in 1 liter of distilled H2O. The resulting conjugates were degassed by gentle N2 sparging before storing in under nitrogen atmosphere. Fluorescein-azide and tri-LacNAc-azide (2) were joined with these alkyne- derivatized proteins under the same conditions as used for 12 and 13 (2 mg/mL protein, 0.3 mM azide, 1 mM CuOTf , 2 mM ligand 10, in 0.1 M Tris buffer, pH 8). Each conjugate was allowed to proceed under nitrogen for 18 hours at room temperature and purified by dialysis as described above. Both KLH-alkyne and BSA-alkyne reactions with fluorescein-azide produced bright yellow protein conjugates; the BSA reaction was performed in order to verify the success of attachment methodology by MALDI-TOF mass spectrometry: BSA, observed m/z = 66,000; BSA-fluorescein, observed m/z = 93700 (fluorescein-liner = 640 Da, 43 fluoresceins per BSA); BSA-2 (conjugate 17), observed m/z = 104,400 (glycan-1 inker = 1335 Da, 29 glycans per BSA).
[0210] Chicken Immunization. White Leghorn hens (3-6 months old) were immunized with 200 μg of the appropriate virus-glycan conjugate; Freund's complete adjuvant (CFA) was co-injected with the initial dose. Hens were boosted with an additional 100 μg of CPMV conjugate and incomplete Freund's adjuvant (IFA) on days 14, 28, and 49. Eggs were collected and cataloged from days 24 to 70. The final 12 eggs from each bird were pooled; isolation of total IgY by Genway Biotech (San Diego, CA) provided approximately one gram of polyclonal antibody per chicken. As a control, wild type CPMV was incubated with CFA and IFA overnight at the same concentrations used for immunization. These samples were dialyzed and found by size-exclusion chromatography to contain exclusively fully intact virions and to have lost no material to precipitation (data not shown).
[0211] Polyclonal IgY affinity purification. Agarose beads functionalized with either tri- or mono-LacNAc were prepared by azide-alkyne cycloaddition as previous described (Punna et al., Bioconj. Chem. 16, 1536-1541, 2005). For each IgY sample to be purified, 50 mg was incubated for 12 hours with 3 mL of beaded agarose bearing compound 18 attached at approximately 60% of the total number of sites on the support. The mixture was poured into a small column and the "flowthrough" fraction collected. The column was then washed with approximately 100 mL of standard buffer (PBS, pH 7.4) and then with 0.1 M glycine buffer (pH 2, 5 mL, "elution" fraction) which was immediately neutralized in 1.0 M Tris-HCl buffer (pH 8.0). The flowthrough fraction was re-incubated in the mono-LacNAc agarose column for 1 hour and eluted in the same fashion twice more to ensure maximum yield of tri-LacNAc specific antibodies. After three such rounds, the elution fractions were pooled and concentrated to 1 mg/mL for analysis on the glycan array. The concentrated "wash" fractions contained no protein as determined by uv-vis spectroscopy.
[0212] Loading of carbohyrate 2 on CPMV and KLH. The attachment of 2 to CPMV (conjugate 15, 80 ± 8 attached molecules per virion) was quantified by comparison to otherwise identical reactions using a fluorescein-azide derivative. Because KLH is a heterogeneous aggregate, the number of attached molecules of 2 in conjugate 16 could not be determined, but we presume that the loading was high, based on identical side-by-side reactions with bovine serum albumin to give 17, which was analyzed by MALDI-TOF mass spectrometry (Figure 5). In addition, reaction of KLH with fluorescein-azide under otherwise identical conditions gave brightly colored protein after two rounds of dialysis. Figure IA shows glycan array binding analysis (array vl .O) of crude IgY isolated from eggs prior to immunization.
[0213] Reproducibility. Conjugates 15 and 16 were separately administered to two chickens, and the IgY isolated from each was analyzed separately. Figure 6 shows the ratios of absolute signal intensities for these independent experiments involving each antigen; Tables Sl and S2 contain the numerical data for these plots. For glycans showing significant antibody binding (>20% of the maximum value in each analysis), the absolute signal intensities for the independent trials were generally within a factor of 4 of each other, and often within a factor of 2. None of the glycans showing less reproducible recognition contained the unsubstituted LacNAc epitope that is the major recognition motif of the antigen. Note that the anomalously large signals for anti-15 were all generated in one of the two experiments, suggesting not random error, but rather an error in one analysis that did not occur in the other. Possibilities include a few spots in one of the array slides receiving a higher than expected concentration of glycan, or a stronger immune response of one chicken compared to the other, somehow distributed over only a few antibodies. The single anomalous response observed in the analysis of anti-16 derived from an unusually large reading in one of the two array analyses, and so it is impossible to speculate on its cause.
[0214] Figure 6 shows plots of ratios of significant signals (>20% of the maximum) from glycan array (v2.0) analyses of total IgY generated by different chickens (designated A and B) against (left) CPMV conjugate 15 and (right) KLH conjugate 16. Numerical data for these plots can be found in Supporting Information. Glycans exhibiting a greater than 10-fold difference in absolute signal intensity from one experiment to the other are labeled with the following numbers: 13 = α-L-Rha-1 inker; 33 = [3-OSO3]Galβl-3GlcNAcβ-linker; 76 = Fucαl- 3GlcNAcβ-linker; 81 = GalNAcαl-3(Fucαl-2)Galβl-4GlcNAcβ-linker; 133 = Galβl- 3GlcNAcβ-linker; 226 = Neu5Acα2-3Galβl-3GlcNAcβ-linker; 227 = Neu5Acα2-3Galβl-4[6- OSO3]GlcNAcβ-linker.
[0215] Figure 7 shows a repeat of the generation and analysis of anti-14 (globo-H on CPMV). Very good reproducibility was observed. Figure 7 shows independent repeat of the experiment described in Figure 3, showing glycan array (v2.0) binding analysis of anti-14 IgY. The only significant difference between these data and the analysis shown in Figure 3 is the strong response to globoside Galqtl-4)Galβl-4)Glc (glycan 1 1 1).
[0216] A comparison of the average strong signal intensities for anti-15 vs. anti-16 total IgY is shown in Figure 8 (numerical data in Table 3); comparison of the weaker signals is shown in Figure 9 (numerical data in Table 4). For five strongly-recognized glycans and approximately a dozen weakly-recognized glycans, the detection signal was more than 50-fold better with antibody derived from immunization with CPMV 15, and only one (probably anomalous; see Fig. S5) signal from KLH 16 was correspondingly more intense than its CPMV counterpart. We do not know if the greater potency of anti-15 vs. anti-16 IgY observed for certain glycans is the result of stronger binding affinities or greater relative concentrations of the specific glycan- binding members of the polyclonal library. While these results suggest that CPMV can elicit at least some more potent IgY molecules than does KLH, a firm conclusion must await additional experiments. [0217] Figure 8 shows plots of ratios of averaged signals from glycan array (v2.0) analyses of total IgY generated by two different chickens against CPMV conjugate 15 vs. KLH conjugate 16; all IgY samples were used at 0.1 mg/mL. To the right are the structures of the glycans exhibiting a greater than 100-fold difference in absolute signal intensity comparing anti- 15 to anti-16 or vice versa. For clarity, not all of the glycan numbers are shown on the x-axis.
[0218] Figure 9 shows plots of ratios of low-intensity signals (<20% of the maximum) from glycan array (v2.0) analyses of total IgY generated by two different chickens against CPMV conjugate 15 and KLH conjugate 16. Numerical data for this plot can be found in Table 4. To the right are the structures of the glycans exhibiting a greater than 50-fold difference in absolute signal intensity comparing anti-15 to anti-16 or vice versa.
[0219] Table 1. Test of reproducibility in IgY generation and glycan array analysis (v2.0) for total IgY derived from immunization with 15 (CPMV display; Figure 6). Samples derived from two different chickens are designated "exp. A" and "exp. B."
Glycans giving average signals >20% of maximum intensity, ordered by glycan number.
ican # signal for signal for ratio A/B ratio B/A Glycan structure exp. Λ exp. B
13 25643 2509 10 2 0 1 α-L-Rhα-Sp8
21 52440 53039 1 0 1 0 β-GlcNAc-SpO
22 46609 30789 1 5 0 7 β-GlcNAc-Sp8
26 54005 1 1797 4 6 0 2 [3OSO3][6OSO3]Galβ 1 -4[6OSO3]GlcNAcβ-Sp0
27 43507 36067 1 2 0 8 [3OSO3][6OSO3]Galβ 1 -4GlcNAcβ-SpO
31 22178 2451 9 0 0 1 [3OSO3]Galβl-3(Fucαl-4)GlcNAcβ-Sp8
33 45774 2939 15 6 0 1 [3OSO3]Galβl-3GlcNAcβ-Sp8
35 1 1497 10533 1 1 0 9 [3OSO3]Galβl -4[6OSO3]GlcNAcβ-Sp8
36 56195 50143 1 1 0 9 [3OSO3]Galβ 1 -4GlcNAcβ-SpO
37 47287 39840 1 2 0 8 [3OSO3]Galβ 1 -4GlcNAcβ-Sp8
39 40922 62802 0 7 1 5 [4OSO3][6OSO3]Galβ 1 -4GlcNAcβ-SpO
40 39757 33315 1 2 0 8 [4OSO3]Galβ 1 -4GlcNAcβ-Sp8
43 12108 10738 1 1 0 9 [6OSO3]Galβl-4Glcβ-Sp8
44 45228 34412 1 3 0 8 [6OSO3]Galβl-4GlcNAcβ-Sp8
49 41913 39750 1 1 0 9 9NAcNeu5Acα2-6Galβ 1 -4GlcNAcβ-Sp8
56 30937 7321 4 2 0 2 Fucα 1 -2Galβ 1 -3GalNAcβ 1 -3Galα 1 -4Galβ 1 -4Glcβ-Sp9
58 28129 12520 2 2 0 4 Fucαl -2Galβ 1 -3GalNAcα-Sp8
69 42962 56185 0 8 1 3 Fucα 1 -2Galβ 1 -4GlcNAcβ 1 -3Galβ 1 -4GIcNAc-SpO
Fucαl -2Galβ 1 -4GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ 1 -3Galβ 1
70 32943 35788 0 9 1 1
4GlcNAcβ-SpO
71 3221 1 10428 3 1 0 3 Fucαl -2Galβ 1 -4GlcNAcβ-SpO
72 16844 15963 1 1 0 9 Fucα 1 -2Galβ 1 -4GlcNAcβ-Sp8
73 9257 16837 0 5 1 8 Fucαl-2Galβl-4Glcβ-SpO
74 19334 10429 1 9 0 5 Fucαl-2Galβ-Sp8
75 49567 35712 1 4 0 7 Fucαl-2GlcNAcβ-Sp8
76 39446 2481 15 9 0 1 Fucαl-3GlcNAcβ-Sp8
77 17727 2805 6 3 0 2 Fucαl-4GlcNAcβ-Sp8
81 37084 2847 13 0 0 1 GalNAcα 1 -3(Fucαl -2)Galβ 1 -4GlcNAcβ-SpO
89 14242 10452 1 4 0 7 GalNAcβ 1 -3(Fucα 1 -2)Galβ-Sp8
90 34682 38375 0 9 1 1 GalNAcβ 1 -3Galα 1 -4Galβ 1 -4GlcNAcβ-SpO Glycan U signal for signal for exp. A exp. B ratio A/B ratio B/A Glycan structure
91 11310 11430 10 10 GalNAcβ 1 -4(Fucα 1 -3)GlcNAcβ-Sp0
92 42253 44091 10 10 GalNAcβ 1 -4GlcNAcβ-SpO
93 43524 34757 13 08 GalNAcβ 1 -4GlcNAcβ-Sp8
100 29939 42157 07 14 Gala 1 -3(GaIa 1 -4)Galβ 1 -4GlcNAcβ-Sp8
104 13288 10743 12 08 Galαl-3Galβl-3GlcNAcβ-SpO
105 39729 37678 1 1 09 Gala 1 -3Galβ 1 -4GlcNAcβ-Sp8
108 20806 5184 40 02 Gala 1 -4(Fuca 1 -2)Galβ 1 -4GlcNAcβ-Sp8
109 47906 28757 17 06 Gala 1 -4Galβ 1 -4GlcNAcβ-SpO
110 49454 40535 12 08 Gala 1 -4Galβ 1 -4GlcNAcβ-Sp8
111 33390 26843 12 08 Galαl -4Galβl-4Glcβ-SpO
112 36600 28128 13 08 Galαl -4GlcNAcβ-Sp8
131 40329 36229 I 1 09 Galβ 1 -3GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ-SpO
133 26070 1632 160 01 Galβl-3GlcNAcβ-Sp0
Galβ 1 -4GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ 1 -3Galβ 1 -
146 55491 50234 1 1 09
4GlcNAcβ-SpO
147 35510 47113 08 13 Galβ 1 -4GlcNAcβ 1 -3Galβ 1 -4GIcNAc(J-SpO
152 39802 47454 08 12 Galβl-4GlcNAcβ-SpO
153 42091 41712 10 10 Galβl-4GlcNAcβ-Sp8
156 44344 44298 10 10 GlcNAcαl -3Galβ 1 -4GlcNAcβ-Sp8
157 36591 24478 15 07 GlcNAcα 1 -6Galβ 1 -4GlcNAcβ-Sp8
166 34086 50710 07 15 GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ-SpO
170 43019 33623 13 08 GlcNAcβ 1 -4Galβ 1 -4GlcNAcβ-Sp8
176 32934 42085 08 13 GlcNAcβl-6Galβl-4GlcNAcβ-Sp8
186 33529 13066 26 04 GlcAβl-6Galβ-Sp8
187 39994 18410 22 05 KDNα2-3Galβl-3GlcNAcβ-Sp0
188 37430 40623 09 11 KDNα2-3Galβ 1 -4GlcNAcβ-SpO
209 51542 46240 1 1 09 Neu5Acα2-3(GalNAcβ 1 -4)Galβl -4GlcNAcβ-SpO
210 29398 35884 08 12 Neu5 Acα2-3(GalNAcβ 1 -4)Galβ 1 -4GlcNAcβ-Sp8
215 32798 45686 07 14 Neu5 Acα2-3GalNAcβ 1 -4GlcNAcβ-Sp0
224 37570 40245 09 1 1 NeuAcα2-3Galβ 1 -3GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ-SpO
225 17338 4723 37 03 Neu5Acα2-3Galβ 1 -3GlcNAcβ-Sp0
226 26344 1178 224 00 Neu5Acα2-3Galβl-3GlcNAcβ-Sp8
Neu5 Acα2-3Galβ 1 -4(Fucα 1 -3)GlcNAcβ 1 -3Galβ 1 -
233 40739 36948 0 9
4GlcNAcβ-Sp8
Neu5Acα2-3Galβl -4GlcNAcβ 1 -3Galβl -4GlcNAcβ 1 -
235 40677 49031 0 8 1 2
3Galβl-4GlcNAcβ-Sp0
236 46775 34349 14 07 Neu5Acα2-3Galβl-4GlcNAcβ-SpO
237 29180 49161 06 17 Neu5Acα2-3Galβl-4GlcNAcβ-Sp8
238 46625 46104 10 10 Neu5Acα2-3Galβl -4GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ-Sp0
243 29110 47050 06 16 Neu5 Acα2-6GalNAcβ 1 -4GlcNAcβ-SpO
245 43960 53581 08 12 Neu5Acα2-6Galβl-4GlcNAcβ-SpO
246 31461 33220 09 1 1 Neu5Acα2-6Galβl -4GlcNAcβ-Sp8
248 27400 30043 09 1 1 Neu5Acα2-6Galβl -4GlcNAcβl-3Galβl-4GlcNAcβ-Sp0
255 47102 40448 12 09 Neu5Acβ2-6Galβ 1 -4GlcNAcβ-Sp8
258 33331 11736 28 04 Neu5Gcα2-3Galβl -3GlcNAcβ-SpO
260 35885 38815 09 1 1 Neu5Gcα2-3Galβl -4GlcNAcβ-SpO
263 42659 36762 12 09 Neu5Gcα2-6Galβl-4GlcNAcβ-SpO [0220] Table 2: Test of reproducibility in IgY generation and glycan array analysis (v2.0) for total IgY derived from immunization with 16 (KLH display; Figure 6). Samples derived from two different chickens are designated "exp. A" and "exp. B."
Glycans giving average signals >20% of maximum intensity, ordered by glycan number
signal for signal for yean U ratio Λ/B ratio 1 Glycan structure exp. A exp. B
26 39480 24340 16 06 [3OSO3][6OSO3]Galβ 1 -4[6OSO3]GlcNAcβ-Sp0
27 29227 40593 07 14 [3OSO3][OOSO3]GaIpMGIcNAcP-SpO
35 43268 16070 27 04 [3OSO3]GaIP 1 -4[6OSO3]GICNACP-SPS
36 37988 45518 08 12 [3OSO3]GaIP l-4GlcNAcp-SpO
37 20934 6746 31 03 [3OSO3]GaIP 1 -4GlcNAcP-Sp8
39 36764 45964 08 13 [4OSO3][OOSO3]GaIPMGIcNAcP-SpO
40 39335 32309 12 08 [4OSO3]GaIP 1 -4GlcNAcβ-Sp8
44 27418 35969 08 13 [6OSO3]GaIP 1 -4GlcNAcβ-Sp8
49 19043 9655 20 05 9NAcNeu5Acα2-6Galβl -4GlcNAcP-Sp8
56 30584 44429 07 15 Fucαl-2Galβl-3GalNAcp i-3Galαl-4Gaipi-4Glcp-Sp9
58 43176 41569 10 10 Fucct 1 -2Galβ 1 -3GalNAcα-Sp8
61 23786 43605 05 18 Fucα 1 -2Gaip 1 -3GlcNAcβ 1 -3Galβ 1 -4GIcP-Sp 10
62 29511 33572 09 1 1 Fucαl-2Gaipi-3GlcNAcβl -3Gaipi-4Glcp-Sp8
63 35149 37576 09 11 Fucαl-2Gaipi-3GlcNAcβ-SpO
64 22465 29216 08 13 Fucα 1 -2GaIP 1 -3GlcNAcβ-Sp8
69 37349 52975 07 14 Fucαl-2Gaip i-4GlcNAcβl -3Galβl-4GlcNAc-SpO
Fucαl-2Galβl-4GlcNAcβl -3Galβl-4GlcNAcβ l-
70 42472 35833 12 08
3Galβl-4GlcNAcβ-SpO
71 22391 7822 29 03 Fucα 1 -2Galβ 1 -4GlcNAcβ-SpO
72 53364 10703 50 02 Fucαl-2Galβl-4GlcNAcβ-Sp8
73 41923 35888 12 09 Fucα 1 -2Galβ 1 -4Glcβ-SpO
74 34850 23163 15 07 Fucαl-2Galβ-Sp8
90 41087 52761 08 13 GalNAcβ 1 -3Galα 1 -4Galβ 1 -4GlcNAcβ-SpO
92 51136 49951 10 10 GaINAcP 1 -4GlcNAcP-SpO
93 40941 41131 10 10 GaINAcP 1 -4GlcNAcβ-Sp8
100 38993 41840 09 11 Galαl -3(GaIaI -4)Gaip 1 -4GlcNAcβ-Sp8
105 37703 31997 12 08 Gala 1 -3Galβ 1 -4GlcNAcβ-Sp8
108 10357 19176 05 19 Gala 1 -4(Fuca 1 -2)Gaip 1 -4GlcNAcP-Sp8
109 58168 41451 14 07 Gala 1 -4Gaip 1 -4GlcNAcP-SpO
110 52197 42691 12 08 Gala 1 -4GaIP 1 -4GlcNAcp-Sp8
112 23971 19855 12 08 Galαl-4GlcNAcp-Sp8
131 17844 19039 09 1 1 Gaip 1 -3GIcNAcP 1 -3Galβ 1 -4GlcNAcβ-SpO
Gaip 1 -4GIcNAcP 1 -3Galβ 1 -4GlcNAcβ 1 -3Gaip 1 -
146 30959 56431 05 18
4GlcNAcP-SpO
147 35100 36570 10 10 Gaip 1 -4GIcNAcP l-3Galβl -4GICNACP-SPO
152 49714 23960 21 05 GalβMGlcNAcβ-SpO
153 46557 27037 17 06 Galβl -4GlcNAcβ-Sp8
156 51110 50473 10 10 GlcNAcα 1 -3Galβ 1 -4GlcNAcβ-Sp8
157 39996 30209 13 08 GlcNAcα 1 -6Galβ 1 -4GlcNAcβ-Sp8
166 44997 40640 11 09 GlcNAcβl-3Galβl -4GlcNAcβl-3Galβl-4GlcNAcβ-SpO
170 46674 40654 1 1 09 GlcNAcβ 1 -4Galβ 1 -4GlcNAcβ-Sp8
176 41812 33723 12 08 GlcNAcβ 1 -6Galβ 1 -4GlcNAcβ-Sp8
188 39257 31168 13 08 KDNα2-3Galβ 1 -4GICNACP-SPO
209 48090 32827 15 07 Neu5Acα2-3(GalNAcβ 1 -4)Galβ 1 -4GICNACP-SPO
210 37474 41762 09 1 1 Neu5Acα2-3(GalNAcpi-4)Gaipi -4GICNACP-SPS
215 25628 22622 1 1 09 Neu5 Acα2-3GalNAcβ 1 -4GICNACP-SPO
224 40629 34403 12 08 NeuAcα2-3Galβ 1 -3GlcNAcβ 1 -3Galβ l-4GlcNAcβ-Sp0
227 49542 1933 256 00 Neu5Acα2-3Galβl-4[6OSO3]GlcNAcβ-Sp8
Neu5 Acα2-3Galβ 1 -4(Fucα 1 -3)GlcNAcβ 1 -3Galβ 1 -
233 40186 41367 10 10
4GlcNAcβ-Sp8
235 41814 29294 14 07 Neu5Acα2-3Galβ 1 -4GIcNAcP 1 -3Galβl -4GlcNAcβ 1 - signal for signal for
Glycan # ratio A/B ratio B/A Glycan structure exp. A exp. B
3Galβl-4GlcNAcβ-SpO
236 33548 9448 36 03 Neu5Acα2-3Galβ 1 -4GlcNAcβ-SpO
237 17925 5391 33 03 Neu5Acα2-3Galβl-4GlcNAcβ-Sp8
238 47277 33162 14 07 Neu5 Acα2-3Galβ 1 -4GlcNAcβl -3Galβ 1 -4GlcNAcβ-SpO
243 40768 21386 19 05 Neu5Acα2-6GalNAcβ 1 -4GlcNAcβ-SpO
245 53016 39072 14 07 Neu5Acα2-6Galβ 1 -4GlcNAcβ-SpO
246 23550 18202 13 08 Neu5Acα2-6Galβl-4GlcNAcβ-Sp8
248 25860 29446 09 1 1 Neu5Acα2-6Galβ 1 -4GlcNAcβl -3Galβ 1 -4GlcNAcβ-Sp0
255 20122 20064 10 10 Neu5 Acβ2-6Galβ 1 -4GlcNAcβ-Sp8
260 35576 13963 25 04 Neu5Gcα2-3Galβl-4GlcNAcβ-SpO
263 32193 30390 1 1 09 Neu5Gcα2-6Galβl-4GlcNAcβ-SpO
[0221] Table 3: (data for Figure 8; comparison of CPMV and KLH platforms). Glycan array analysis (v2.0) for total IgY derived from immunization with 15 vs. 16. Average signals are derived from the independent experiments listed in Tables Sl and S2.
Glycans giving average signals >20% of maximum intensity for either antι-15 or antι-16, ordered by glycan number
av av
G ra r g. signal for g. signal for Glycan structure lycan # tio 15/16 atio 16/15 anti-15 anti-16
1 14 15 0
88 α-L-Rhα-Sp8 076 9ii 0
21 52740 950 555 00 β-GlcNAc-SpO
22 38699 2177 178 01 β-GlcNAc-Sp8
26 32901 31910 10 10 [3OSO3][6OSO3]Galβ 1 -4[6OSO3]GlcNAcβ-Sp0
27 39787 34910 1 1 09 [3OSO3][6OSO3]Galβl-4GlcNAcβ-Sp0
31 12315 35 3568 00 [3OSO3]Galβl-3(Fucαl-4)GlcNAcβ-Sp8
33 24357 178 1371 00 [3OSO3]Galβl-3GlcNAcβ-Sp8
35 11015 29669 04 27 [3OSO3]GaIβl-4[6OSO3]GlcNAcβ-Sp8
36 53169 41753 13 08 [3OSO3]Galβl-4GlcNAcβ-Sp0
37 43564 13840 31 03 [3OSO3]Galβl-4GlcNAcβ-Sp8
39 51862 41364 13 08 [4OSO3][6OSO3]Galβ 1 -4GlcNAcβ-Sp0
40 36536 35822 10 10 [4OSO3]Galβl -4GlcNAcβ-Sp8
43 11423 4663 24 04 [6OSO3]Galβl -4Glcβ-Sp8
44 39820 31693 13 08 [6OSO3]Galβl -4GlcNAcβ-Sp8
49 40831 14349 28 04 9NAcNeu5 Acα2-6Galβ 1 -4GlcNAcβ-Sp8
56 19129 37506 05 20 Fucα 1 -2Galβ 1 -3GalNAcβ 1 -3Galα 1 -4GaI β 1 -4Glcβ-Sp9
58 20324 42373 05 21 Fucα 1 -2GaI β 1 -3 GalNAcα-Sp8
61 4685 33695 01 72 Fucαl -2Galβ 1 -3GlcNAcβ 1 -3Galβ 1 -4Glcβ-Sp 10
62 2202 31541 01 143 Fucα 1 -2Galβ 1 -3GlcNAcβ 1 -3Galβ 1 -4Glcβ-Sp8
63 5227 36363 01 70 Fucα 1 -2Galβ 1 -3GlcNAcβ-Sp0
64 2135 25840 01 121 Fucαl -2Galβ 1 -3GlcNAcβ-Sp8
69 49573 45162 1 1 09 Fucαl -2Galβl -4GlcNAcβl-3Galβl -4GlcNAc-SpO
Fucαl-2Galβl-4GlcNAcβ l-3Galβl -4GlcNAcβl -3Galβl-
70 34366 39153 09 1 1 4GlcNAcβ-SpO
71 21320 15106 14 07 Fucα 1 -2Galβ 1 -4GlcNAcβ-SpO
72 16404 32034 05 20 Fucαl -2Galβ 1 -4GlcNAcβ-Sp8
73 13047 38905 03 30 Fucα 1 -2Galβ 1 -4Glcβ-SpO
74 14881 29007 05 19 Fucαl-2Galβ-Sp8
75 42639 4602 93 01 Fucαl-2GlcNAcβ-Sp8
76 20963 6268 33 03 Fucαl-3GlcNAcβ-Sp8 av av
G ra r g. signal for g. signal for Glycan structure lycan U tio 15/16 atio 16/15 anti-15 anti-16
77 10266 300 342 00 Fucαl-4GlcNAcβ-Sp8
81 19966 49 4102 00 GalNAcα 1 -3(Fucα 1 -2)Galβ 1 -4GlcNAcβ-SpO
89 12347 5187 24 04 GalNAcβ 1 -3(Fucα 1 -2)Galβ-Sp8
90 36529 46924 08 13 GalNAcβ 1 -3Galα 1 -4Galβ 1 -4GlcNAcβ-SpO
91 11370 10678 1 1 09 GalNAcβ 1 -4(Fucα 1 -3)GlcNAcβ-SpO
92 43172 50543 09 12 GalNAcβ 1 -4GlcNAcβ-SpO
93 39141 41036 10 10 GalNAcβ 1 -4GlcNAcβ-Sp8
100 36048 40417 09 1 1 Gala 1 -3(GaIa 1 -4)Galβ 1 -4GlcNAcβ-Sp8
104 12015 7493 16 06 Gala 1 -3Galβ 1 -3GlcNAcβ-Sp0
105 38703 34850 1 1 09 Gala 1 -3Galβ 1 -4GlcNAcβ-Sp8
108 12995 14766 09 11 Gala 1 -4(Fuca 1 -2)Galβ 1 -4GlcNAcβ-Sp8
109 38332 49810 08 13 Gala 1 -4Galβ 1 -4GlcNAcβ-SpO
110 44995 47444 09 1 1 Galαl-4Galβ l-4GlcNAcβ-Sp8
111 30117 5599 54 02 Galαl-4Galβ l-4Glcβ-SpO
112 32364 21913 15 07 GaIαl-4GlcNAcβ-Sp8
131 38279 18441 21 05 Galβ 1 -3GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ-SpO
133 13851 479 289 00 Galβl-3GlcNAcβ-Sp0
Galβl-4GlcNAcβl-3Galβl-4GlcNAcβl-3Galβ l -
146 52863 43695 12
08 4GlcNAcβ-Sp0
147 41311 35835 12 09 Galβ 1 -4GlcNAcβ 1 -3Galβl -4GlcNAcβ-SpO
152 43628 36837 12 08 Galβl-4GlcNAcβ-SpO
153 41902 36797 1 1 09 Galβl-4GlcNAcβ-Sp8
156 44321 50792 09 1 1 GlcNAcαl-3Galβl -4GlcNAcβ-Sp8
157 30535 35102 09 1 1 GlcNAcα 1 -6Galβl -4GlcNAcβ-Sp8
166 42398 42819 10 10 GlcNAcβl -3Galβl-4GlcNAcβl -3Galβl -4GlcNAcβ-SpO
170 38321 43664 09 1 1 GlcNAcβ 1 -4Galβ 1 -4GlcNAcβ-Sp8
176 37510 37767 10 10 GlcNAcβ 1 -6Galβ 1 -4GlcNAcβ-Sp8
186 23297 245 951 00 GlcAβl-6Galβ-Sp8
187 29202 8504 34 03 KDNα2-3Galβl -3GlcNAcβ-Sp0
188 39026 35212 1 1 09 KDNα2-3Galβl -4GlcNAcβ-SpO
209 48891 40459 12 08 Neu5Acα2-3(GalNAcβ l-4)Galβl-4GlcNAcβ-Sp0
210 32641 39618 08 12 Neu5 Acα2-3(GalNAcβ 1 -4)Galβ 1 -4GlcNAcβ-Sp8
215 39242 24125 16 06 Neu5Acα2-3GalNAcβ 1 -4GlcNAcβ-Sp0
224 38908 37516 10 10 NeuAcα2-3Galβ 1 -3GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ-SpO
225 11031 831 133 01 Neu5Acα2-3Galβl -3GlcNAcβ-SpO
226 13761 5659 24 04 Neu5Acα2-3Galβl-3GlcNAcβ-Sp8
227 53 25737 00 486 Neu5Acα2-3Galβl -4[6OSO3]GlcNAcβ-Sp8
Neu5 Acα2-3Galβ 1 -4(Fucα 1 -3)GlcNAcβ 1 -3Galβ 1 -
233 38844 40776 10
10 4GlcNAcβ-Sp8
Neu5 Acα2-3Galβ 1 -4GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ 1 -
235 44854 35554 13
08 3Galβl-4GlcNAcβ-SpO
236 40562 21498 19 05 Neu5Acα2-3Galβl-4GlcNAcβ-Sp0
237 39170 11658 34 03 Neu5Acα2-3Galβl -4GlcNAcβ-Sp8
238 46364 40220 2 09 Neu5Acα2-3Galβl-4GlcNAcβl-3Galβl -4GlcNAcβ-Sp0
243 38080 31077 2 08 Neu5 Acα2-6GalNAcβ 1 -4GlcNAcβ-Sp0
245 48770 46044 1 09 Neu5 Acα2-6Galβ 1 -4GlcNAcβ-SpO
246 32340 20876 5 06 Neu5 Acα2-6Galβ 1 -4GlcNAcβ-Sp8
248 28721 27653 0 10 Neu5 Acα2-6Galβ 1 -4GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ-SpO
255 43775 20093 22 05 Neu5 Acβ2-6Galβ 1 -4GlcNAcβ-Sp8
258 22533 1147 196 01 Neu5Gcα2-3Galβl-3GlcNAcβ-SpO
260 37350 24770 15 07 Neu5Gcα2-3Galβl-4GlcNAcβ-SpO
263 39710 31291 3 08 Neu5Gcα2-6Galβl -4GlcNAcβ-SpO
[0222] Table 4. (data for Figure 9; comparison of CPMV and KLH platforms). Glycan array analysis (v2.0) for total IgY derived from immunization with 15 vs. 16. These are the weak signals (average signal intensity <20% of maximum for both; values are derived from the independent experiments listed in Tables Sl and S2.
Signal strength in the bottom 20% for both antι-15 and antι-16 avg. signal avg. signal ratio ratio
Glycan # Glycan structure for anti-lS for anti-16 15/16 16/15
96 35 2 7 0 4 AGP
2 32 26 1 2 0 8 AGP-A
3 32 42 0 8 1 3 AGP-B
4 107 42 2 6 04 Ceruloplasmine
5 34 53 0 6 16 Fibrinogen
6 1576 7190 0 2 46 Transferrin
7 5392 2115 2 5 04 α-D-Gal-Sp8
8 27 16 1 7 06 α-D-Glc-Sp8
9 62 59 1 0 10 a-D-Man-Sp8
10 32 31 1 0 10 α-GalNAc-Sp8
11 25 3091 0 0 1262 α-L-Fuc-Sp8
12 10522 5250 2 0 05 α-L-Fuc-Sp9
14 34 598 0 1 178 α-Neu5Ac-Sp8
15 138 58 2 4 04 Neu5Acαl-2-Sp82
16 44 27 1 6 06 β-Neu5Ac-Sp8
17 51 22 2 3 04 β-D-Gal-Sp8
18 73 34 2 1 05 β-D-Glc-Sp8
19 25 60 0 4 24 β-D-Man-Sp8
20 3195 49 65 6 00 β-GalNAc-Sp8
23 144 8 17 6 01 β-GlcN(Gc)-Sp8
24 160 38 4 2 02 (Galβ 1 -4GlcNAcβ)2-3,6-GalNAcα-Sp8
25 19 39 0 5 20 (GlcNAcβ 1 -3(GlcNAcβ 1 -6) GlcNAcβl -4)GlcNAc-Sp8
28 35 24 1 5 07 [3OSO3]Galβl-4Glcβ-Sp8
29 1 10 31 3 6 03 [3OSO3]Galβl -4(6OSO3)Glcβ-Sp0
30 4611 7700 0 6 17 [3OSO3]Galβl -4(6OSO3)Glcβ-Sp8
32 4623 33 141 1 00 [3OSO3]GaIβl -3GalNAcα-Sp8
34 180 3988 0 0 222 [3OSO3]Galβl -4(Fucαl-3)GlcNAcβ-Sp8
38 9287 7623 1 2 08 [3OSO3]Galβ-Sp8
41 84 129 0 7 15 6-H2PO3Manα-Sp8
42 848 51 16 7 01 [6OSO3]Galβl-4Glcβ-Sp0
45 24 28 0 9 1 1 [6OSO3]Galβl-4[6OSO3]Glcβ-Sp8
46 551 21 26 7 00 NeuAcα2-3[6OSO3]Galβl -4GlcNAcβ-Sp8
47 1483 27 55 1 00 [6OSO3]GlcNAcβ-Sp8
48 10528 6326 1 7 06 9NAcNeu5Acα-Sp8
50 41 14 2 9 03 5-OS-G
51 29 22 1 3 08 7-OS-G
52 24 175 0 1 74 9-OS-G
53 40 19 2 1 05 1 1 -OS-G
54 167 49 3 4 03 l l-OS-Sp8
55 5200 9692 0 5 19 Fucα 1 -2Galβ 1 -3GalNAcβ 1 -3Galα-Sp9
57 4850 3705 1 3 08 Fucα 1 -2Galβ 1 -3(Fucα 1 -4)GlcNAcβ-Sp8
59 1378 448 3 1 03 Fucα 1 -2Galβ 1 -3GalNAcβ 1 -4(Neu5 Acα2-3)Galβ 1 -4Glcβ-SpO
60 5414 8519 0 6 16 Fucα 1 -2Galβ 1 -3GalNAcβ 1 -4(Neu5 Acα2-3)Galβ 1 -4Glcβ-Sp9
Fucα 1 -2Galβ 1 -4(Fucα 1 -3)GlcNAcβ 1 -3Galβ 1 -4(Fucα 1 -
65 310 1 1 27 5 00
3)GlcNAcβ-SpO
Fucαl -2Galβ 1 -4(Fucα 1 -3)GlcNAcβ 1 -3Galβ 1 -4(Fucα 1 -
66 543 34 15 7 01
3)GlcNAcβ 1 -3Galβ 1 -4(Fucα 1 -3)GlcNAcβ-SpO
67 18 34 05 19 Fucα 1 -2Galβ 1 -4(Fucα 1 -3)GlcNAcβ-SpO
68 5216 7695 07 15 Fucα 1 -2Galβ 1 -4(Fucα 1 -3)GlcNAcβ-Sp8
78 1415 933 15 07 Fucβ l -3GlcNAcβ-Sp8
79 29 26 1 1 09 GalNAcα 1 -3(Fucα 1 -2)Galβ 1 -3GlcNAcβ-SpO
80 1886 24 797 00 GalNAcαl -3(Fucα 1 -2)Galβ 1 -4(Fucαl -3)GlcNAcβ-SpO
82 2548 41 618 00 GalNAcαl -3(Fucαl -2)Galβ 1 -4GlcNAcβ-Sp8
83 34 29 12 09 GalNAcα 1 -3(Fucα 1 -2)Galβ 1 -4Glcβ-SpO Signal strength in the bottom 20% for both antι-lS andantι-16 avg. signal avg. signal ratio ratio Glycan structure for anti-lS for anti-16 15/16 16/15
84 21 26 08 12 GalNAcαl-3(Fucαl-2)Galβ-Sp8
85 43 6148 00 1418 GalNAcAl-3GalNAcβ-Sp8
86 2168 597 36 03 GalNAcαl-3Galβ-Sp8
87 4315 1979 22 05 GalNAcαl -4(Fucα 1 -2)Galβ 1 -4GlcNAcβ-Sp8
88 466 319 15 07 GalNAcβ 1 -3GalNAcα-Sp8
94 249 210 12 08 Galαl-2Galβ-Sp8
95 28 37 08 13 Gala 1 -3(Fuca 1 -2)Galβ 1 -3GlcNAcβ-Sp0
96 543 93 58 02 Gala 1 -3(Fuca 1 -2)Galβ l-4(Fuca 1 -3)GlcNAcβ-Sp0
97 2332 42 561 00 Gala 1 -3(Fuca 1 -2)Galβ 1 -4GIcNAc-SpO
98 24 54 04 22 Gala 1 -3(Fuca l-2)Galβ 1 -4Glcβ-SpO
99 7738 4946 16 06 Galαl-3(Fucαl-2)Galβ-Sp8
101 30 30 10 10 Galαl-3GalNAcα-Sp8
102 82 62 13 08 Galαl-3GalNAcβ-Sp8
103 252 80 31 03 Galαl-3Galβl-4(Fucαl -3)GlcNAcβ-Sp8
106 109 251 04 23 Galαl-3Galβl-4Glcβ-SpO
107 2568 4249 06 17 Galαl-3Galβ-Sp8
113 1415 497 28 04 Galαl-6Glcβ-Sp8
114 281 35 79 01 Galβl-2Galβ-Sp8
40 Galβ 1 -3(Fucα 1 -4)GlcNAcβ 1 -3Galβ 1 -4(Fucα 1 -3)GlcNAcβ-
115 198 49 02
SpO
116 581 14 417 00 Galβ 1 -3(Fucα 1 -4)GlcNAcβ 1 -3Galβ 1 -4GlcNAcβ-SpO
117 198 23 86 01 Galβ 1 -3(Fucα 1 -4)GlcNAc-SpO
118 100 16 62 02 Galβ 1 -3(Fucα 1 -4)GlcNAc-Sp8
119 108 38 28 04 Galβ 1 -3(Fucα 1 -4)GlcNAcβ-Sp8
120 6379 6108 10 10 Galβ 1 -3 (Galβ 1 -4GlcNAcβl-6)GalNAcα-Sp8
121 32 26 12 08 Galβl -3(GlcNAcβl-6)GalNAcα-Sp8
122 20 36 05 18 Galβl-3(Neu5Acα2-6)GalNAcα-Sp8
123 23 29 08 13 Galβl -3(Neu5Acβ2-6)GalNAcα-Sp8
124 23 33 07 14 Galβ 1 -3(Neu5Acα2-6)GlcNAcβ 1 -4GaIβ 1 -4Glcβ-Sp 10
125 29 22 13 08 Galβl -3GalNAcα-Sp8
126 192 1104 02 58 Galβl -3GalNAcβ-Sp8
127 166 3198 01 192 Galβ 1 -3GalNAcβ 1 -3GaIaI -4Galβ 1 -4Glcβ-SpO
128 49 37 13 08 Galβl-3GalNAcβl-4(Neu5Acα2-3)Galβl -4Glcβ-Sp0
129 20 717 00 355 Galβ 1 -3GalNAcβ 1 -4Galβ 1 -4Glcβ-Sp8
130 10431 6978 15 07 Galβl-3Galβ-Sp8
132 2375 183 129 01 Galβ 1 -3GlcNAcβ 1 -3Galβ 1 -4Glcβ-Sp 10
134 2332 58 400 00 Galβl-3GlcNAcβ-Sp8
135 123 39 32 03 Galβ 1 -4(Fucα 1 -3)GlcNAcβ-SpO
136 106 19 55 02 Galβ 1 -4(Fucα 1 -3)GlcNAcβ-Sp8
137 Galβ 1 -4(Fucα 1 -3)GlcNAcβ 1 -4Galβ 1 -4(Fucα 1 -3)GlcNAcβ-
862 34 25 6 0 0
SpO
Galβ 1 -4(Fucαl -3)GlcNAcβ 1 -4Galβ 1 -4(Fucα 1 -3)GlcNAcβ 1 -
138 1298 28 46 2 0 0
4Galβ 1 -4(Fucα 1 -3)GlcNAcβ-Sp0
139 34 150 02 44 Galβl-4[6OSO3]Glcβ-Sp0
140 1127 70 161 01 Galβl-4[6OSO3]Glcβ-Sp8
141 3688 590 63 02 Galβ 1 -4GalNAcα 1 -3(Fucα 1 -2)Galβ 1 -4GlcNAcβ-Sp8
142 5641 2329 24 04 Galβ 1 -4GalNAcβ 1 -3(Fucα 1 -2)Galβ 1 -4GlcNAcβ-Sp8
143 26 15 17 06 Galβ 1 -4GlcNAcβ 1 -3(Galβ 1 -4GlcNAcβ 1 -6)GalNAcα-Sp8
144 80 41 20 05 Galβl-4GlcNAcβl-3GalNAcα-Sp8
Galβl -4GlcNAcβl-3Galβl -4(Fucαl-3)GlcNAcβl -3Galβ l-
145 9278 9553 10 10
4(Fucαl-3)GlcNAcβ-SpO
148 35 44 08 13 Galβl -4GlcNAcβl-3Galβl -4Glcβ-SpO
149 43 48 09 1 1 Galβ 1 -4GlcNAcβ 1 -3Galβ 1 -4Glcα-Sp8
150 18 21 09 1 1 Galβ 1 -4GlcNAcβ 1 -6(Galβ 1 -3)GalNAcα-Sp8
151 3620 55 652 00 Galβ 1 -4GlcNAcβ 1 -6GalNAcα-Sp8
154 23 120 02 51 Galβl-4Glcβ-SpO
155 6373 4409 14 07 Galβl-4Glcβ-Sp8
158 44 14 31 03 GlcNAcβ 1 -2Galβ 1 -3GalNAcα-Sp8 Signal strength in the bottom 20% for both anti- JS and ant I- 16 avg. signal avg. signal
Glycan # ratio ratio Glycan structure for anti-lS for anti-16 15/16 16/15
159 28 29 10 1 1 GlcNAcβ 1 -3(GlcNAcβ 1 -6)GalNAcα-Sp8 160 27 19 14 0 7 GlcNAcβ 1 -3(GlcNAcβ 1 -6)Galβ 1 -4GlcNAcβ-Sp8 161 24 65 04 27 GlcNAcβ 1 -3GalNAcα-Sp8 162 25 29 08 12 GlcNAcβ l-3Galβ-Sp8 163 14 36 04 25 GlcNAcβl-3Galβl-3GalNAcα-Sp8 164 36 23 16 06 GlcNAcβl-3Galβl-4GIcNAcβ-Sp0 165 10646 7390 14 07 GlcNAcβ 1 -3Galβ 1 -4GIcNAcβ-Sp8 167 1680 63 269 00 GlcNAcβ 1 -3Galβ 1 -4Glcβ-SpO 168 4357 88 494 00 GlcNAcβ l-4MDPLys 169 9797 5438 18 06 GlcNAcβ 1 -4(GlcNAcβ I -6)GalNAcα-Sp8 171 15 36 04 24 (GlcNAcβ M)6β-Sp8 172 15 13 12 09 (GlcNAcβ l-4)5β-Sp8 173 21 11 19 05 GlcNAcβ 1 -4GlcNAcβ 1 -4GlcNAcβ-Sp8 174 43 21 21 05 GlcNAcβ 1 -6(Galβ 1 -3)GalNAcα-Sp8 175 7619 7633 10 10 GlcNAcβ 1 -6GalNAcα-Sp8 177 44 20 22 05 Glcαl-4Glcβ-Sp8 178 529 76 69 01 Glcαl-4Glcα-Sp8 179 88 159 06 18 Glcαl-6Glcαl-6Glcβ-Sp8 180 282 43 66 02 Glcβl-4Glcβ-Sp8 181 311 51 61 02 Glcβl-6Glcβ-Sp8 182 30 39 08 13 G-ol-amine 183 26 21 13 08 GlcAα-Sp8 184 58 12 47 02 GlcAβ-Sp8 185 5990 39 1549 00 GlcAβl-3Galβ-Sp8 189 27 24 1 1 09 Manα 1 -2Manαl -2Manα 1 -3Manα-Sp9 190 40 23 17 06 Manα 1 -2Manαl -3(M ana 1 -2Mana 1 -6)Mano-Sp9 191 12 24 05 20 Manα 1 -2Manα 1 -3Manα-Sp9
Manα 1 -6(M ana 1 -2Mana 1 -3)Mana 1 -6(Mana2Manal -
192 22 34 06 16
3)Manβ 1 -4GlcNAcβ 1 -4GlcNAcβ-N
Manα 1 -2Manα 1 -6(Manα 1 -3)Manα 1 - 193 46 01 87
6(Manα2Manα2Manα 1 -3)Manβ 1 -4GlcNAcβ 1 -4GlcNAcβ-N
Manαl -2Manα 1 -2Manα 1 -3(Manα 1 -2Manα 1 -3(Manα 1 - 194 20 33 06 17
2Manα 1 -6)Manαl -6)Manβ 1 -4GlcNAcβ 1 -4GlcNAcβ-N
195 26 12 22 04 Manα 1 -3 (Manα 1 -6)Manα-Sp9
196 782 29 274 00 Manα 1 -3 (Manα 1 -2Manα 1 -2Manα 1 -6)Manα-Sp9
Manα 1 -6(Manα 1 -3)Manα 1 -6(Manα2Manα 1 -3)Manβ 1 -
197 40 01 67
4GlcNAcβl -4GlcNAcβ-N
Manα 1 -6(Manα 1 -3)Manα 1 -6(Manαl -3)Manβ 1 -4GlcNAcβ 1 -
198 67 50 13 07
4 GlcNAcβ-N
199 280 131 21 05 Man5_9mix N
200 23 31 07 14 Manβ l-4GlcNAcβ-SpO
201 42 24 17 06 Neu5Acα2-3(Galβl-3GalNAcβl-4)Galβl-4Glcβ-Sp0
202 24 33 07 14 Neu5Acα2-3Galβ 1 -3GalNAcα-Sp8
NeuAcα2-8NeuAcα2-8NeuAcα2-8NeuAcα2-3(GalNAcβ 1 -
203 23 31 07 14
4)Galβl -4Glcβ-SpO
112 Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3(GalNAcβ 1 -4)Galβ 1 -
204 13 01
4Glcβ-SpO
205 26 39 07 15 Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ 1 -4GIcβ-SpO
206 39 42 09 1 1 Neu5 Acα2-8Neu5 Acα2-3(GalNAcβ 1 -4)Galβ 1 -4Glcβ-SpO
207 74 35 21 05 Neu5Acα2-8Neu5Acα2-8Neu5Acα-Sp8
208 5790 3551 16 06 Neu5Acα2-3(6-O-Su)Galβl-4(Fucαl -3)GlcNAcβ-Sp8
211 14 40 04 28 Neu5Acα2-3(GalNAcβ 1 -4)Galβ 1 -4Glcβ-SpO
212 20 24 NeuAcα2-3(NeuAcα2-3Galβl -3GalNAcβl-4)Galβl -4Glcβ-
09 12
SpO
213 69 265 03 39 Neu5Acα2-3(Neu5Acα2-6)GalNAcα-Sp8
214 5361 4376 12 08 Neu5Acα2-3GalNAcα-Sp8
216 43 30 15 07 Neu5Acα2-3Galβl-3(6OSO3)GlcNAc-Sp8
217 87 13 65 02 Neu5 Acα2-3Galβ 1 -3(Fucα 1 -4)GlcNAcβ-Sp8 Signal strength in the bottom 20% for both anti- IS and antι-16 avg. signal avg. signal ratio ratio
Glycan U Glycan structure for anti-15 for anti- 16 15/16 16/15
NeuAcα2-3Galβ 1 -3(Fucα 1 -4)GIcNAcβ 1 -3Galβ 1 -4(Fucα 1 -
218 6194 7819 0 8 1 3
3)GlcNAcβ SpO
219 205 32 64 02 Neu5Acα2-3Galβl-3(Neu5Acα2-3Galβl-4)GlcNAcβ-Sp8
220 49 16 31 03 Neu5Acα2-3Galβl-3[6OSO3]GalNAcα-Sp8
221 16 27 06 16 Neu5Acα2-3Galβl-3(Neu5Acα2-6)GalNAcA-Sp8
222 41 132 03 32 Neu5Acα2-3Galβ-Sp8
223 5431 5518 10 10 NeuAcα2-3Galβ 1 -3GalNAcβ 1 -3GaIaI -4Galβ 1 -4GIcβ-SpO
228 165 29 57 02 Neu5Acα2-3Galβ 1 -4(Fucα 1 -3)(6OSO3)GlcNAcβ-Sp8
Neu5Acα2-3Galβl-4(Fucαl-3)GlcNAcβl-3Galβl-4(Fucαl-
229 2075 387 54 02
3)GlcNAcβ 1 -3Galβ 1 -4(Fucαl -3)GlcNAcβ-Sp0
230 2397 1181 20 05 Neu5Acα2-3Galβl -4(Fucα 1 -3)GlcNAcβ-SpO
231 60 24 25 04 Neu5Acα2-3Galβl -4(Fucα 1 -3)GlcNAcβ-Sp8
232 10654 4626 23 04 Neu5Acα2-3Galβl-4(Fucαl-3)GlcNAcβl -3Galβ-Sp8
234 32 28 Neu5Acα2-3Galβl-4GlcNAcβl-3Galβl -4(Fucαl-3)GlcNAc-
1 1 09
SpO
239 35 15 23 04 Neu5Acα2-3Galβl -4Glcβ-SpO
240 45 42 1 1 09 Neu5Acα2-3Galβl-4Glcβ-Sp8
241 39 23 17 06 Neu5Acα2-6(Galβl-3)GalNAcα-Sp8
242 7561 7714 10 10 Neu5Acα2-6GalNAcα-Sp8
244 9250 4527 20 05 Neu5Acα2-6Ga!βl-4[6OSO3]GIcNAcβ-Sp8
Neu5 Acα2-6Gal β 1 -4GlcNAcβ 1 -3Galβ 1 -4(Fucα 1 -
247 951 50 189 01
3)GlcNAcβ 1 -3Galβ 1 -4(Fucα 1 -3)GlcNAcβ-Sp0
249 27 22 12 08 Neu5 Acα2-6Gal β 1 -4Glcβ-SpO
250 61 66 09 1 1 Neu5 Acα2-6Gal β 1 -4Glcβ-Sp8
251 51 24 21 05 Neu5Acα2-6Galβ-Sp8
252 28 26 1 1 09 Neu5Acα2-8Neu5Acα-Sp8
253 28 28 10 10 Neu5Acα2-8Neu5Acα2-3Galβl-4Glcβ-Sp0
254 8228 7061 12 09 Neu5 Acβ 1 -6GalNAcα-Sp8
256 621 34 182 01 Neu5Acβ2-6(Galβl-3)GalNAcα-Sp8
257 5559 365 152 01 Neu5Gcα2-3Galβl-3(Fucαl -4)GlcNAcβ-SpO
259 7974 6958 11 09 Neu5Gcα2-3Galβ 1 -4(Fucαl -3)GlcNAcβ-Sp0
261 36 42 09 12 Neu5Gcα2-3Galβl-4Glcβ-SpO
262 10685 10460 10 10 Neu5 G cα2-6GalNAcα-SpO
264 2523 17 1479 00 Neu5Gcα-Sp8
[3OSO3]Galβl- Fucαl-2Galβl-
32
3GalNAcα-Sp8 59 3GalNAcβl-
Table 5.Glycan array v.2.0
[3OSO3]Galβl- 4(Neu5Acα2-3)Galβl-
3GlcNAcβ-Sp8 4Glcβ-SpO
[3OSO3]Galβl-4(Fucαl- Fucαl-2Galβl-
# Glycan structure
3)GlcNAcβ-Sp8 60 3GalNAcβl-
1 AGP
[3OSO3]Galβl- 4(Neu5Acα2-3)Galβl-
2 AGP-A J 4[6OSO3]GlcNAcβ-Sp8 4Glcβ-Sp9
3 AGP-B [3OSO3]Galβl- Fucαl-2Galβl-
4 Ceruloplasmine 4GlcNAcβ-SpO 61 3GlcNAcβl-3Galβl-
5 Fibrinogen [3OSO3]Galβl- 4Glcβ-SplO
4GlcNAcβ-Sp8 Fucαl-2Galβl-
6 Transferrin
62 3GlcNAcβl-3Galβl-
7 α-D-Gal-Sp8 38 [3OSO3]Galβ-Sp8
[4OSO3] [6OSO3]Galβl- 4Glcβ-Sp8
8 α-D-Glc-Sp8
4GlcNAcβ-SpO 63 Fucαl-2Galβl-
9 a-D-Man-Sp8
[4OSO3]Galβl- 3GlcNAcβ-SpO
10 α-GalNAc-Sp8 4GlcNAcβ-Sp8 ,. Fucαl-2Galβl-
11 α-L-Fuc-Sp8 3GlcNAcβ-Sp8
41 6-H2PO3Manα-Sp8
12 α-L-Fuc-Sp9 Fucαl-2Galβl-4(Fucαl-
40 [6OSO3]Galβl-4Glcβ-
65 3)GlcNAcβl-3Galβl-
13 α-L-Rhα-Sp8 42 SpO 4(Fucα 1 -3)GlcNAcβ-SpO
14 α-Neu5Ac-Sp8 .„ [6OSO3]Galβl-4Glcβ- Fucα 1 -2Galβ 1 -4(Fucα 1 -
15 Neu5Acαl-2-Sp82 Sp8 3)GlcNAcβl-3Galβl- [6OSO3]Galβl-
16 β-Neu5Ac-Sp8 66 4(Fucαl-3)GlcNAcβl-
4GlcNAcβ-Sp8
17 β-D-Gal-Sp8 3Galβl-4(Fucαl- [6OSO3]Galβl-
18 β-D-Glc-Sp8 3)GlcNAcβ-SpO 4[6OSO3]Glcβ-Sp8
19 β-D-Man-Sp8 , Fucαl-2Galβl-4(Fucαl- NeuAcα2- 3)GlcNAcβ-SpO
20 β-GalNAc-Sp8 46 3[6OSO3]Galβl-
68 Fucαl-2Galβl-4(Fucαl-
21 β-GlcNAc-SpO 4GlcNAcβ-Sp8 3)GlcNAcβ-Sp8
22 β-GlcNAc-Sp8 47 [6OSO3]GlcNAcβ-Sp8 Fucαl-2Galβl-
23 β-GlcN(Gc)-Sp8 48 9NAcNeu5Acα-Sp8 69 4GIcN Acβ 1-3 Galβ 1- (Galβ 1 -4GIcN Acβ)2-3 ,6- 9NAcNeu5 Acα2-6Galβ 1 -
49 4GIcNAc-SpO GalNAcα-Sp8 4GlcNAcβ-Sp8 Fucαl-2Galβl- (GlcNAcβl- 50 5-OS-G 4GlcNAcβl-3Galβl-
70
25 3(GlcNAcβl-6) 51 7-OS-G 4GlcNAcβl-3Galβl-
GlcNAcβl-4)GlcNAc- 52 9-OS-G 4GlcNAcβ-SpO
Sp8 53 11 -OS-G Fucαl-2Galβl-
71
[3OSO3][6OSO3]Galβl-
54 ll-OS-Sp8 4GlcNAcβ-SpO
4[6OSO3]GlcNAcβ-Sp0 Fucαl-2Galβl- Fucαl-2Galβl-
[3OSO3] [6OSO3]Galβl- 55 4GlcNAcβ-Sp8 3GalNAcβl-3Galα-Sp9
4GlcNAcβ-SpO --. Fucαl-2Galβl-4Glcβ- Fucαl-2GaIβl-
00 [3OSO3]Galβl-4Glcβ- SpO
56 3GaIN Acβ 1-3GaIaI -
28 Sp8 74 Fucαl-2Galβ-Sp8 4Galβl-4Glcβ-Sp9
[3OSO3]Galβl- 75 Fucαl-2GlcNAcβ-Sp8
4(6OSO3)Glcβ-Sp0 Fucαl-2Galβl-3(Fucαl-
57 4)GlcNAcβ-Sp8 76 Fucαl-3GlcNAcβ-Sp8
[3OSO3]Galβl-
30
4(6OSO3)Glcβ-Sp8 Fucαl-2Galβl- 77 Fucαl-4GlcNAcβ-Sp8
58
3GalNAcα-Sp8 78 Fucβl-3GlcNAcβ-Sp8
[3OSO3]Galβl-3(Fucαl-
31 7Q GalNAcαl-3(Fucαl-
4)GlcNAcβ-Sp8 2)Galβl-3GlcNAcβ-SpO /ilU-Mllt^ UULNtI 11U. UUO. ITO
GalNAcαl-3(Fucαl- 106 Galαl-3Galβl-4Glcβ- Galβl-3GlcNAcβl- 80 2)Galβl-4(Fucαl- SpO 3Galβl -4GlcNAcβ-SpO
3)GlcNAcβ-SpO 107 Galαl-3Galβ-Sp8 132 Galβ 1-3 GIcN Acβl- gl GalNAcαl-3(Fucαl- Galαl-4(Fucαl-2)Galβl- 3Galβl-4Glcβ-SplO
108
2)Galβl -4GICNACP-SPO 4GlcNAcβ-Sp8 133 Galβl-3GlcNAcβ-SpO
GalNAcαl-3(Fucαl- oς> Galαl-4Galβl- 134 Galβl-3GlcNAcβ-Sp8
2)Galβl-4GlcNAcβ-Sp8 4GlcNAcβ-SpO Galβl-4(Fucαl-
135
GalNAcαl-3(Fucαl- Galαl-4Galβl- 3)GlcNAcβ-SpO
2)Galβl-4Glcβ-SpO 4GlcNAcβ-Sp8 .., Galβl-4(Fucαl- g4 GalNAcαl-3(Fucαl- ... Galαl-4Galβl-4Glcβ- 3)GlcNAcβ-Sp8
2)Galβ-Sp8 SpO Galβl-4(Fucαl- R, GaINAcAl -3GaIN Acβ- 112 Galαl-4GlcNAcβ-Sp8 137 3)GlcNAcβl-4Galβl-
Sp8 113 Galαl-6Glcβ-Sp8 4(Fucαl-3)GlcNAcβ-SpO 86 GalNAcαl-3Galβ-Sp8 114 Galβl-2Galβ-Sp8 Galβl-4(Fucαl-
GalNAcαl-4(Fucαl- Galβl-3(Fucαl- 3)GlcNAcβl-4Galβl-
2)Galβl-4GlcNAcβ-Sp8 115 4)GlcNAcβl-3Galβl- 138 4(Fucαl-3)GlcNAcβl- QC GalNAcβ l-3GalNAcα- 4(Fucαl-3)GlcNAcβ-SpO 4Galβl-4(Fucαl- 88 Sp8 Galβl-3(Fucαl- 3)GlcNAcβ-SpO gQ GalNAcβ 1 -3 (Fucαl- 116 4)GlcNAcβl-3Galβl- nQ Galβl-4[6OSO3]Glcβ-
2)Galβ-Sp8 4GlcNAcβ-SpO SpO 90 GalNAcβ 1 -3GaIaI- ] )7 Galβl-3(Fucαl- ... Galβl-4[6OSO3]Glcβ-
4Galβl-4GlcNAcβ-SpO 4)GlcNAc-SpO Sp8
GalNAcβ l-4(Fucαl- ..„ Galβl-3(Fucαl- Galβl-4GalNAcαl-
3)GlcNAcβ-SpO 4)GlcNAc-Sp8 141 3(Fucαl-2)Galβl- q? GalNAcβ l-4GlcNAcβ- Galβl-3(Fucαl- 4GlcNAcβ-Sp8
SpO 4)GlcNAcβ-Sp8 Galβl-4GalNAcβl- q3 GalNAcβ l-4GlcNAcβ- Galβl-3(Galβl- 142 3(Fucαl-2)Galβl-
Sp8 120 4GlcNAcβl-6)GalNAcα- 4GlcNAcβ-Sp8 94 Galαl-2Galβ-Sp8 Sp8 Galβl-4GlcNAcβl-
Galαl-3(Fucαl-2)Galβl- Galβl-3(GlcNAcβl- 143 3(Galβl-4GlcNAcβl-
3GlcNAcβ-SpO 6)GalNAcα-Sp8 6)GalNAcα-Sp8 % Galαl-3(Fucαl-2)Galβl- ]22 Galβl-3(Neu5Acα2- Galβ 1 -4GIcN Acβl -
4(Fucαl -3)GlcNAcβ-SpO 6)GalNAcα-Sp8 3GalNAcα-Sp8 97 Galαl-3(Fucαl-2)Galβl- _3 Galβl-3(Neu5Acβ2- Galβ 1-4GIcN Acβl -
4GIcNAc-SpO 6)GalNAcα-Sp8 3Galβl-4(Fucαl-
Galαl-3(Fucαl-2)Galβl- Galβl-3(Neu5Acα2- 3)GlcNAcβl-3Galβl-
4Glcβ-SpO 124 6)GlcNAcβl-4Galβl- 4(Fucαl-3)GlcNAcβ-SpO QQ Gala 1-3 (Fucαl-2)Galβ- 4Glcβ-SplO Galβ 1 -4GIcN Acβl -
Sp8 125 Galβl-3GalNAcα-Sp8 146 3Galβl-4GlcNAcβl- 00 Gala 1 -3(GaIaI -4)Galβl- 126 Galβl-3GalNAcβ-Sp8 3Galβl-4GlcNAcβ-SpO
4GlcNAcβ-Sp8 Galβ 1-3 GalNAcβ 1- ._ Galβl-4GlcNAcβl-01 Galαl-3GalNAcα-Sp8 127 3Galαl-4Galβl-4Glcβ- 3Galβl-4GlcNAcβ-SpO02 Galαl-3GalNAcβ-Sp8 SpO Galβl-4GlcNAcβl- Galαl-3Galβl-4(Fucαl- Galβl-3GalNAcβl- 3Galβl-4Glcβ-SpO 3)GlcNAcβ-Sp8 128 4(Neu5Acα2-3)Galβl- Galβl-4GlcNAcβl- Galαl-3Galβl- 4Glcβ-SpO 3Galβl-4Glcα-Sp8 3GlcNAcβ-SpO Galβl-3GalNAcβl- 0 Galβl-4GlcNAcβl- Galαl-3Galβl- 4Galβl-4GIcβ-Sp8 6(Galβl-3)GalNAcα-Sp8 4GlcNAcβ-Sp8 130 Galβl-3Galβ-Sp8 Galβ 1-4GIcN Acβl - 6GalNAcα-Sp8 152 Galβl-4GlcNAcβ-SpO 182 G-ol-amine Neu5Acα2-3(Galβl-
153 Galβl-4GlcNAcβ-Sp8 183 GlcAα-Sp8 201 3GalNAcβl-4)Galβl-
154 Galβl-4Glcβ-SpO 4Glcβ-SpO
184 GlcAβ-Sp8
155 Galβl-4Glcβ-Sp8 Neu5Acα2-3Galβl-
185 GlcAβl-3Galβ-Sp8 56 GlcNAcαl-3Galβl- 3GalNAcα-Sp8
186 GlcAβl-6Galβ-Sp8
4GlcNAcβ-Sp8 NeuAcα2-8NeuAcα2-
187 KDNα2-3Galβl-
GlcNAcαl-όGalβl- 8NeuAcα2-8NeuAcα2-
157 3GlcNAcβ-SpO 203
4GlcNAcβ-Sp8 3(GalNAcβl-4)Galβl-
188 KDNα2-3Galβl- „ GlcNAcβl -2Galβl- 4Glcβ-SpO 4GlcNAcβ-SpO
3GalNAcα-Sp8 Neu5Acα2-8Neu5Acα2- Manαl-2Manαl- 8Neu5Acα2-
GlcNAcβl -3(GlcNAcβl- 189
159 2Manαl-3Manα-Sp9 204 3(GalNAcβl-4)Galβl-
6)GalNAcα-Sp8 Manαl-2Manαl- 4Glcβ-SpO
GlcNAcβl-3(GlcNAcβl- 190 3(Manαl-2Manαl- Neu5Acα2-8Neu5Acα2-
6)Galβl-4GlcNAcβ-Sp8 6)Manα-Sp9 205 8Neu5Acα2-3Galβl- .,. GlcNAcβl -3GalNAcα- Manαl-2Manαl-3Manα- 4Glcβ-SpO
Sp8 191 Sp9 Neu5Acα2-8Neu5Acα2- 162 GlcNAcβl-3Galβ-Sp8 Manα 1 -6(Manα 1 - 206 3(GalNAcβl-4)Galβl-
GlcNAcβl -3Galβl- 2Manαl-3)Manαl- 4Glcβ-SpO
3GalNAcα-Sp8 192 6(Manα2Manαl-
Neu5Acα2-8Neu5Acα2-
GlcNAcβl -3Galβl- 3)Manβl-4GlcNAcβl- 207 8Neu5Acα-Sp8
4GlcNAcβ-SpO 4GlcNAcβ-N Neu5Acα2-3(6-O- , GlcNAcβl -3Galβl- Manαl-2Manαl-
208 Su)Galβl-4(Fucαl-
4GlcNAcβ-Sp8 6(Manα 1 -3 )Manα 1 -
3)GlcNAcβ-Sp8
GlcNAcβl -3Galβl- 193 6(Manα2Manα2Manαl- 9 Neu5Acα2-3(GalNAcβl- 166 4GlcNAcβl-3Galβl- 3)Manβl-4GlcNAcβl-
4)Galβl-4GlcNAcβ-SpO
4GlcNAcβ-SpO 4GlcNAcβ-N 0 Neu5Acα2-3(GalNAcβl-
GlcNAcβl-3Galβl- Manαl-2Manαl-
4)Galβl-4GlcNAcβ-Sp8
4Glcβ-SpO 2Manαl-3(Manαl- 168 GlcNAcβl -4MDPLys 194 2Manαl-3(Manαl- Neu5Acα2-3(GalNAcβl-
GlcNAcβl -4(GlcNAcβl- 2Manαl-6)Manαl- 4)Galβl-4Glcβ-SpO
6)GalNAcα-Sp8 6)Manβl-4GlcNAcβl- NeuAcα2-3 (NeuAcα2-
GlcNAcβl -4Galβl- 4GlcNAcβ-N 212 3Galβl-3GalNAcβl-
4GlcNAcβ-Sp8 ]Qi. Manα 1-3 (Manα 1- 4)Galβl-4Glcβ-SpO
171 (GlcNAcβl -4)6β-Sp8 6)Manα-Sp9 - Neu5Acα2-3(Neu5Acα2-
172 (GlcNAcβl -4)5β-Sp8 Manα 1-3 (Manα 1- 6)GalNAcα-Sp8 GlcNAcβl -4GlcNAcβl- 196 2Manαl-2Manαl- Neu5Acα2-3GalNAcα-
214 4GlcNAcβ-Sp8 6)Manα-Sp9 Sp8
. GlcNAcβl -6(Galβl- Manαl-6(Manαl- Neu5Acα2-3GalNAcβl-
3)GalNAcα-Sp8 3)Manαl- 4GlcNAcβ-SpO ._, GlcNAcβl -6GalNAcα- 197 6(Manα2Manαl- 7]f. Neu5Acα2-3Galβl-
Sp8 3)Manβl-4GlcNAcβl- 3(6OSO3)GlcNAc-Sp8
, GlcNAcβl-6Galβl- 4GlcNAcβ-N Neu5Acα2-3Galβl- 4GlcNAcβ-Sp8 Manα 1 -6(Manα 1 - 217 3(Fucαl-4)GlcNAcβ-
Sp8
177 Glcαl-4Glcβ-Sp8 „ 3)Manαl-6(Manαl-
178 Glcαl-4Glcα-Sp8 3)Manβl-4GlcNAcβl-4 NeuAcα2-3Galβl- GlcNAcβ-N 3(Fucαl-4)GlcNAcβl- ]70 Glcαl -6Glcαl -6Glcβ- 3Galβl-4(Fucαl-
Sp8 199 Man5_9mixN
3)GlcNAcβ SpO
180 Glcβl-4Glcβ-Sp8 200 Manβl-4GlcNAcβ-SpO
181 Glcβl-6Glcβ-Sp8 Neu5Acα2-3Galβl- 23fi Neu5Acα2-3Galβl- 259 Neu5Gcα2-3Galβl-
219 3(Neu5Acα2-3Galβl- 4GlcNAcβ-SpO 4(Fucαl-3)GlcNAcβ-SpO 4)GlcNAcβ-Sp8 Neu5Acα2-3Galβl- Neu5Gcα2-3Galβl-
237 260
220 Neu5Acα2-3Galβl- 4GlcNAcβ-Sp8 4GlcNAcβ-SpO 3[6OSO3]GalNAcα-Sp8 Neu5Acα2-3Galβl- , Neu5Gcα2-3Galβl- Neu5Acα2-3Galβl- 238 4GlcNAcβl-3Galβl- 4Glcβ-SpO
221 3(Neu5Acα2- 4GlcNAcβ-SpO Neu5Gcα2-6GalNAcα-
262 6)GalNAcA-Sp8 2- Neu5Acα2-3Galβl- SpO
222 Neu5Acα2-3Galβ-Sp8 4Glcβ-SpO 26_ Neu5Gcα2-6Galβl- NeuAcα2-3Galβl- 240 Neu5Acα2-3Galβl- 4GlcNAcβ-SpO
223 3GalNAcβl-3Galαl- 4Glcβ-Sp8 264 Neu5Gcα-Sp8 4Galβl-4Glcβ-SpO Neu5Acα2-6(Galβl- NeuAcα2-3Galβl- 3)GalNAcα-Sp8
224 3GlcNAcβl-3Galβl- - .„ Neu5Acα2-6GalNAcα- 4GlcNAcβ-SpO qZ Sp8 Neu5Acα2-3Galβl- 243 Neu5Acα2-6GalNAcβl-
225 3GlcNAcβ-SpO 4GlcNAcβ-SpO Neu5Acα2-3Galβl- Neu5Acα2-6Galβl-
226 244 3GlcNAcβ-Sp8 4[6OSO3]GlcNAcβ-Sp8 Neu5Acα2-3Galβl- 245 Neu5Acα2-6Galβl- 4[6OSO3]GlcNAcβ-Sp8 4GlcNAcβ-SpO Neu5Acα2-3Galβl- Neu5Acα2-6Galβl-
246
228 4(Fucαl- 4GlcNAcβ-Sp8 3)(6OSO3)GlcNAcβ-Sp8 Neu5Acα2-6Galβl- Neu5Acα2-3Galβl- 4GlcNAcβl-3Galβl- 4(Fucαl-3)GlcNAcβl- 247 4(Fucαl-3)GlcNAcβl-
229 3Galβl-4(Fucαl- 3Galβl-4(Fucαl- 3)GlcNAcβl-3Galβl- 3)GlcNAcβ-SpO 4(Fucαl-3)GlcNAcβ- Neu5Acα2-6Galβl- SpO 248 4GlcNAcβl-3GaIβl- Neu5Acα2-3Galβl- 4GlcNAcβ-SpO
2304(Fucαl-3)GlcNAcβ- Neu5Acα2-6Galβl- SpO 249 4Glcβ-SpO Neu5Acα2-3Galβl- Neu5Acα2-6Galβl-
231 4(Fucαl-3)GlcNAcβ- 250 4Glcβ-Sp8 Sp8 251 Neu5Acα2-6Galβ-Sp8 Neu5Acα2-3Galβl- Neu5Acα2-8Neu5Acα-
232 4(Fucαl-3)GlcNAcβl- 252 Sp8 3Galβ-Sp8 Neu5Acα2-8Neu5Acα2- Neu5Acα2-3Galβl- 253 3Galβl-4Glcβ-SpO
233 4(Fucαl-3)GlcNAcβl- 0<;. Neu5Acβl-6GalNAcα- 3Galβl-4GlcNAcβ-Sp8 2M Sp8 Neu5Acα2-3Galβl- Neu5Acβ2-6Galβl-
234 4GlcNAcβl-3Galβl- 255
4GlcNAcβ-Sp8 4(Fucαl-3)GlcNAc-SpO Neu5Acβ2-6(Galβl- Neu5Acα2-3Galβl- 256
3)GalNAcα-Sp8 4GlcNAcβl-3Galβl-
235 Neu5Gcα2-3Galβl- 4GlcNAcβl-3Galβl- 3(Fucαl-4)GlcNAcβ-SpO 4GlcNAcβ-SpO Neu5Gcα2-3Galβl-
258
3GlcNAcβ-SpO References
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Claims

What is Claimed:
1. A vaccine comprising, protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent.
2. The vaccine of claim 1 wherein at least some of the protein nanoparticles are bound to a plurality of different glycan-containing molecules.
3. The vaccine of claim 1 wherein the glycan-containing molecule is a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or
1 i popoly saccharide .
4. The vaccine of claim 1 wherein the protein nanoparticle is a virus, bacteriophage, nucleoprotein nanoparticle, viral nanoparticle, viral capsid particle, vault protein, dendrimer or chaperonin.
5. The vaccine of claim 1 wherein the protein nanoparticle is a plant viral particle.
6. The vaccine of claim 5 wherein the plant viral particle is a Comovirus, Tombusvirus, Sobemovirus, or Nepovirus.
7. The vaccine of claim 5 wherein the plant viral particle is a Comovirus.
8. The vaccine of claim 5 wherein the plant viral particle is a cowpea mosaic virus.
9. The vaccine of claim 1 wherein the protein nanoparticles are covalently bound to glycan- containing molecules through alkyne azide linkage.
10. The vaccine of claim 1 wherein the protein nanoparticles are covalently bound to glycan- containing molecules through a plurality of amino acid residues on the protein nanoparticle.
11. The vaccine of claim 10 wherein the protein nanoparticles are covalently bound to glycan-containing molecules through N-hydroxysuccinimide ester linkage.
12. The vaccine of claim 1, comprising at least 100 glycan-containing molecules per protein nanoparticle.
13. The vaccine of claim 1, comprising at least 150 glycan-containing molecules per protein nanoparticle.
14. The vaccine of claim 1, comprising at least 200 glycan-containing molecules per protein nanoparticle.
15. A method for producing anti-glycan antibodies in a vertebrate subject comprising, administering to the vertebrate subject a vaccine comprising protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent, and isolating the anti-glycan antibodies from a biological sample of the vertebrate subject.
16. The method of claim 15 wherein at least some of the viral subunits are bound to a plurality of different glycan-containing molecules.
17. The method of claim 15 wherein the glycan-containing molecule is a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide.
18. The method of claim 15 comprising isolating the anti-glycan antibodies from total immunoglobulin of the vertebrate subject.
19. The method of claim 18 comprising purifying IgG or IgY antibodies from the total immunoglobulin.
20. The method of claim 15 wherein the protein nanoparticle is a virus, bacteriophage, nucleoprotein nanoparticle, viral nanoparticle, viral capsid particle, vault protein, dendrimer or chaperonin.
21. The method of claim 15 wherein the protein nanoparticle is a plant viral particle.
22. The method of claim 21 wherein the plant viral particle is a Comovirus, Tombusvirus, Sobemovirus, or Nepovirus.
23. The method of claim 21 wherein the plant viral particle is a Comovirus.
24. The method of claim 21 wherein the plant viral particle is a cowpea mosaic virus.
25. The method of claim 15 wherein the protein nanoparticles are covalently bound to glycan-containing molecules through alkyne azide linkage.
26. The method of claim 15 wherein the protein nanoparticles are covalently bound to glycan-containing molecules through a plurality of amino acid residues on the protein nanoparticle.
27. The method of claim 26 wherein the protein nanoparticles are covalently bound to glycan-containing molecules through N-hydroxysuccinimide ester linkage.
28. The method of claim 15 wherein the vertebrate subject is a mammalian subject or an avian subject.
29. The method of claim 28 wherein the biological sample is one or more eggs of the avian subject.
30. The method of claim 28 wherein the biological sample is serum of the vertebrate subject.
31. The method of claim 15, comprising covalently coupling at least 100 glycan-containing molecules per protein nanoparticle.
32. The method of claim 15, comprising covalently coupling at least 150 glycan-containing molecules per protein nanoparticle.
33. The method of claim 15, comprising covalently coupling at least 200 glycan-containing molecules per protein nanoparticle.
34. An isolated anti-glycan antibody of claim 15.
35. A method for treating cancer in a vertebrate subject comprising, administering to the vertebrate subject a vaccine comprising protein nanoparticles, at least some of the protein nanoparticles covalently bound to glycan-containing molecules, and a pharmaceutically acceptable carrier or diluent, wherein the vaccine elicits an immune response to the plurality of glycan molecules to reduce or eliminate cancer in the vertebrate subject.
36. The method of claim 35 wherein at least some of the viral subunits are bound to a plurality of different glycan-containing molecules.
37. The method of claim 35 wherein the glycan-containing molecule is a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycomimetic, or glycophospholipid.
38. The method of claim 35 wherein the protein nanoparticle is a virus, bacteriophage, nucleoprotein nanoparticle, viral nanoparticle, viral capsid particle, vault protein, dendrimer or chaperonin.
39. The method of claim 35 wherein the protein nanoparticle is a plant viral particle.
40. The method of claim 39 wherein the plant viral particle is a Comovirus, Tombusvirus, Sobemovirus, or Nepovirus.
41. The method of claim 39 wherein the plant viral particle is a Comovirus.
42. The method of claim 39 wherein the plant viral particle is a cowpea mosaic virus.
43. The method of claim 35 wherein the protein nanoparticles are covalently bound to glycan-containing molecules through alkyne azide linkage.
44. The method of claim 35 wherein the protein nanoparticles are covalently bound to glycan-containing molecules through a plurality of amino acid residues on the protein nanoparticle.
45. The method of claim 44 wherein the protein nanoparticles are covalently bound to glycan-containing molecules through N-hydroxysuccinimide ester linkage.
46. The method of claim 35, further comprising covalently coupling at least 100 glycan- containing molecules per protein nanoparticle.
47. The method of claim 35, further comprising covalently coupling at least 150 glycan- containing molecules per protein nanoparticle.
48. The method of claim 35, further comprising covalently coupling at least 200 glycan- containing molecules per protein nanoparticle.
49. The method of claim 35, further comprising administering the vaccine to the vertebrate subject via an oral, pulmonary, oropharyngeal, nasopharyngeal, topical, intravenous, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular route
50. A method for preventing or treating infectious disease in a vertebrate subject comprising, administering to the vertebrate subject the anti-glycan antibody of claim 34, in an amount effective to reduce or eliminate infectious disease in the vertebrate subject.
51. The method of claim 50, further comprising administering the anti-glycan antibody orally to the vertebrate subject.
52. The method of claim 50, further comprising administering the anti-glycan antibody to the vertebrate subject via an oral, pulmonary, oropharyngeal, nasopharyngeal, topical, intravenous, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular route
53. The method of claim 50 wherein the anti-glycan antibody binds to a plurality of different glycan-containing molecules.
54. The method of claim 50 wherein the glycan-containing molecule is a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide.
55. The method of claim 50 wherein the vertebrate subject is a mammalian subject or an avian subject.
56. The method of claim 50 wherein the infectious disease is a bacterial infectious disease, viral infectious disease, fungal infectious disease, or infectious parasitic disease.
57. The method of claim 50 wherein the infectious disease is an antibiotic-resistant bacterial infectious disease.
58. A method for diagnosing disease in a vertebrate subject comprising, contacting a tissue sample from the vertebrate subject with the anti-glycan antibody of claim 34, and detecting binding of the antibody to the tissue sample indicating presence of glycan- containing molecules relating to the disease in the vertebrate subject.
59. The method of claim 58 wherein the disease is inflammatory disease, infectious disease, cancer, or metabolic disease.
60. The method of claim 58 wherein the anti-glycan antibody binds to a plurality of different glycan-containing molecules.
61. The method of claim 58 wherein the glycan-containing molecule is a carbohydrate, glycoaminoacid, glycopeptide, glycolipid, glycoaminoglycan, glycoprotein, glycoconjugate, glycosyl phosphatidylinositol-linked glycoconjugate, glycomimetic, glycophospholipid, or lipopolysaccharide.
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